Science.gov

Sample records for acid binding capacity

  1. Decreased plasma arachidonic acid binding capacity in neonates.

    PubMed

    Sadowitz, P D; Walenga, R W; Clark, D; Stuart, M J

    1987-01-01

    Arachidonic acid (AA) metabolites have been implicated in neonatal pathologic states such as respiratory distress syndrome (RDS). Since free (nonprotein bound) AA is the substrate for synthesis of these compounds, a decreased capacity to bind AA in neonatal plasma could contribute to these disorders. AA binding was assayed by equilibrium dialysis in plasma samples from healthy adults and various infant groups. Plasma from these infant groups bound significantly less AA than adult plasma. Premature infants with RDS and premature infants receiving intralipid had the lowest capacity to bind AA. The increased availability of free AA may be important in neonatal pathophysiologic states involving arachidonate metabolites.

  2. Bile acid binding capacity of fish protein hydrolysates from discard species of the West Mediterranean Sea.

    PubMed

    Pérez-Gálvez, Raúl; García-Moreno, Pedro J; Morales-Medina, Rocío; Guadix, Antonio; Guadix, Emilia M

    2015-04-01

    Fish protein hydrolysates (FPH), produced from the six main discard species from the West Mediterranean Sea (sardine, horse mackerel, axillary seabream, bogue, small-spotted catshark and blue whiting) were tested for their bile acid binding capacity. This capacity is directly linked to the ability to inhibit bile reabsorption in the ileum and therefore to lower cholesterol levels in the bloodstream. From each species, FPH were obtained by three different enzymatic treatments employing two serine endoproteases (subtilisin and trypsin) sequentially or in combination. The results show statistically significant differences among the fish species, attaining interesting average values of bile acid binding capacity for blue whiting (27.32% relative to cholestyramine on an equal protein basis) and horse mackerel (27.42% relative to cholestyramine on an equal protein basis). The enzymatic treatments did not significantly affect the ability of a given species to bind bile acids. These results are similar to other protein sources, such as soy protein or casein, of proven hypocholesterolemic effect. It can be concluded that fish protein hydrolysates from these discard species are suitable as ingredients in the formulation of cholesterol-lowering supplements.

  3. Modulation of FadR Binding Capacity for Acyl-CoA Fatty Acids Through Structure-Guided Mutagenesis

    DOE PAGES

    Bacik, John-Paul; Yeager, Chris M.; Twary, Scott N.; ...

    2015-09-18

    FadR is a versatile global regulator in Escherichia coli that controls fatty acid metabolism and thereby modulates the ability of this bacterium to grow using fatty acids or acetate as the sole carbon source. FadR regulates fatty acid metabolism in response to intra-cellular concentrations of acyl-CoA lipids. The ability of FadR to bind acyl-CoA fatty acids is hence of significant interest for the engineering of biosynthetic pathways for the production of lipid-based biofuels and commodity chemicals. Based on the available crystal structure of E. coli bound to myristoyl- CoA, we predicted amino acid positions within the effector binding pocket thatmore » would alter the ability of FadR to bind acyl-CoA fatty acids without affecting DNA binding. We utilized fluorescence polarization to characterize the in-vitro binding properties of wild type and mutant FadR. We found that a Leu102Ala mutant enhanced binding of the effector, likely by increasing the size of the binding pocket for the acyl moiety of the molecule. Conversely, the elimination of the guanidine side chain (Arg213Ala and Arg213Met mutants) of the CoA moiety binding site severely diminished the ability of FadR to bind the acyl-CoA effector. These results demonstrate the ability to fine tune FadR binding capacity. The validation of an efficient method to fully characterize all the binding events involved in the specific activity (effector and DNA operator binding) of FadR has allowed us to increase our understanding of the role of specific amino acids in the binding and recognition of acyl-CoA fatty acids and will greatly facilitate efforts aimed at engineering tunable FadR regulators for synthetic biology.« less

  4. Modulation of FadR Binding Capacity for Acyl-CoA Fatty Acids Through Structure-Guided Mutagenesis

    SciTech Connect

    Bacik, John-Paul; Yeager, Chris M.; Twary, Scott N.; Martí-Arbona, Ricardo

    2015-09-18

    FadR is a versatile global regulator in Escherichia coli that controls fatty acid metabolism and thereby modulates the ability of this bacterium to grow using fatty acids or acetate as the sole carbon source. FadR regulates fatty acid metabolism in response to intra-cellular concentrations of acyl-CoA lipids. The ability of FadR to bind acyl-CoA fatty acids is hence of significant interest for the engineering of biosynthetic pathways for the production of lipid-based biofuels and commodity chemicals. Based on the available crystal structure of E. coli bound to myristoyl- CoA, we predicted amino acid positions within the effector binding pocket that would alter the ability of FadR to bind acyl-CoA fatty acids without affecting DNA binding. We utilized fluorescence polarization to characterize the in-vitro binding properties of wild type and mutant FadR. We found that a Leu102Ala mutant enhanced binding of the effector, likely by increasing the size of the binding pocket for the acyl moiety of the molecule. Conversely, the elimination of the guanidine side chain (Arg213Ala and Arg213Met mutants) of the CoA moiety binding site severely diminished the ability of FadR to bind the acyl-CoA effector. These results demonstrate the ability to fine tune FadR binding capacity. The validation of an efficient method to fully characterize all the binding events involved in the specific activity (effector and DNA operator binding) of FadR has allowed us to increase our understanding of the role of specific amino acids in the binding and recognition of acyl-CoA fatty acids and will greatly facilitate efforts aimed at engineering tunable FadR regulators for synthetic biology.

  5. Techno-functional properties and in vitro bile acid-binding capacities of tamarillo (Solanum betaceum Cav.) hydrocolloids.

    PubMed

    Gannasin, Sri Puvanesvari; Adzahan, Noranizan Mohd; Mustafa, Shuhaimi; Muhammad, Kharidah

    2016-04-01

    Hydrocolloids were extracted from seed mucilage and the pulp fractions from red tamarillo (Solanum betaceum Cav.) mesocarp, and characterisation of their techno-functional properties and in vitro bile acid-binding capacities was performed. The seed mucilage hydrocolloids that were extracted, using either 1% citric acid (THC) or water (THW), had a good foaming capacity (32-36%), whereas the pulp hydrocolloids that were extracted, using 72% ethanol (THE) or 20mM HEPES buffer (THH), had no foaming capacity. The pulp hydrocolloid, however, possessed high oil-holding and water-holding capacities in the range of 3.3-3.6 g oil/g dry sample and 25-27 g water/g dry sample, respectively. This enabled the pulp hydrocolloid to entrap more bile acids (35-38% at a hydrocolloid concentration of 2%) in its gelatinous network in comparison to commercial oat fibre and other hydrocolloids studied. The exceptional emulsifying properties (80-96%) of both hydrocolloids suggest their potential applications as food emulsifiers and bile acid binders.

  6. Purification and characterisation of a glutamic acid-containing peptide with calcium-binding capacity from whey protein hydrolysate.

    PubMed

    Huang, Shun-Li; Zhao, Li-Na; Cai, Xixi; Wang, Shao-Yun; Huang, Yi-Fan; Hong, Jing; Rao, Ping-Fan

    2015-02-01

    The bioavailability of dietary ionised calcium is affected by intestinal basic environment. Calcium-binding peptides can form complexes with calcium to improve its absorption and bioavailability. The aim of this study was focused on isolation and characterisation of a calcium-binding peptide from whey protein hydrolysates. Whey protein was hydrolysed using Flavourzyme and Protamex with substrate to enzyme ratio of 25:1 (w/w) at 49 °C for 7 h. The calcium-binding peptide was isolated by DEAE anion-exchange chromatography, Sephadex G-25 gel filtration and reversed phase high-performance liquid chromatography (RP-HPLC). A purified peptide of molecular mass 204 Da with strong calcium binding ability was identified on chromatography/electrospray ionisation (LC/ESI) tandem mass spectrum to be Glu-Gly (EG) after analysis and alignment in database. The calcium binding capacity of EG reached 67·81 μg/mg, and the amount increased by 95% compared with whey protein hydrolysate complex. The UV and infrared spectrometer analysis demonstrated that the principal sites of calcium-binding corresponded to the carboxyl groups and carbonyl groups of glutamic acid. In addition, the amino group and peptide amino are also the related groups in the interaction between EG and calcium ion. Meanwhile, the sequestered calcium percentage experiment has proved that EG-Ca is significantly more stable than CaCl2 in human gastrointestinal tract in vitro. The findings suggest that the purified dipeptide has the potential to be used as ion-binding ingredient in dietary supplements.

  7. Tree species affect cation exchange capacity (CEC) and cation binding properties of organic matter in acid forest soils.

    PubMed

    Gruba, Piotr; Mulder, Jan

    2015-04-01

    Soil organic matter (SOM) in forest soil is of major importance for cation binding and acid buffering, but its characteristics may differ among soils under different tree species. We investigated acidity, cation exchange properties and Al bonding to SOM in stands of Scots pine, pedunculate oak, Norway spruce, European beech and common hornbeam in southern Poland. The content of total carbon (Ct) was by far the major contributor to total cation exchange capacity (CECt) even in loamy soils and a strong relationship between Ct and CECt was found. The slope of the regression of CECt to Ct increased in the order hornbeam≈oakacid pH range was smallest for hornbeam and oak, and largest for spruce and pine soils. This was supported by the apparent dissociation constant (pKapp) values of SOM, which were largest in soils under oak. The maximum values of Al saturation were similar between the stands. However, maximum Al bonding to SOM occurred at higher pH values in soils under pine and spruce than under oak. Therefore, at any value in the acid pH range, the SOM in pine soil has less Al complexed and more adsorbed H+ than SOM from oak soils. Such differences in Al and H bonding are not only important for pH buffering and metal solubility controls, but also for stabilization of SOM via saturation of functional groups by Al and H.

  8. Dependence of protein binding capacity of dimethylamino-γ-butyric-acid (DMGABA)-immobilized porous membrane on composition of solvent used for DMGABA immobilization

    NASA Astrophysics Data System (ADS)

    Iwanade, Akio; Umeno, Daisuke; Saito, Kyoichi; Sugo, Takanobu

    2013-06-01

    Dimethylamino-γ-butyric acid (DMGABA) as an ampholite was reacted with the epoxy group of the poly-glycidyl methacrylate chain grafted onto the pore surface of a porous hollow-fiber polyethylene membrane by radiation-induced graft polymerization. DMGABA was dissolved in a mixture of dioxane and water at various dioxane volume fractions, defined by dividing the dioxane volume by the total volume. The equilibrium binding capacity (EBC) of the DMGABA-immobilized porous hollow-fiber membrane for lysozyme was evaluated in the permeation mode. The EBC was varied from a 1/50-fold monolayer binding capacity to a 10-fold monolayer binding capacity by controlling the composition of the solvent used for DMGABA immobilization and the molar conversion of the epoxy group into the DMGABA group.

  9. Fatty Acid Saturation of Albumin Used in Resuscitation Fluids Modulates Cell Damage in Shock: In Vitro Results Using a Novel Technique to Measure Fatty Acid Binding Capacity.

    PubMed

    Penn, Alexander H; Dubick, Michael A; Torres Filho, Ivo P

    2017-03-21

    The use of albumin for resuscitation has not proven as beneficial in human trials as expected from numerous animal studies. One explanation could be the practice of adding fatty acid (FA) during manufacture of pharmaceutical albumin. During ischemia, unbound free FAs (FFA) in the circulation could potentially induce cellular damage. We hypothesized that albumins with higher available binding capacities (ABC) for FFAs may prevent that damage. Therefore, we developed a technique to measure ABC, determined if pharmaceutical human serum albumin (HSA) has decreased ABC compared to FA-free bovine serum albumin (BSA), and if binding capacity would affect hemolysis when blood is mixed with exogenous FFA at levels similar to those observed in shock. The new assay used exogenous oleic acid (OA), glass fiber filtration, and a FFA assay kit. RBC hemolysis was determined by mixing 0-5 mM OA with PBS, HSA, FA-free BSA, or FA-saturated BSA and measuring plasma hemoglobin after incubation with human blood. 5% HSA contained 4.7±0.2 mM FFA, leaving an ABC of 5.0 ± 0.6 mM, compared to FA-free BSA's ABC of 7.0 ± 1.3 mM (P < 0.024). Hemolysis after OA was reduced with FA-free BSA but increased with FA-saturated BSA. HSA provided intermediate results. 25% solutions of FA-free BSA and HSA were more protective, while 25% FA-saturated BSA was more damaging than 5% solutions. These findings suggest that increased FA saturation may reverse albumin's potential benefit to lessen cellular damage and may explain, at least in part, its failure in human trauma studies.

  10. Lipid binding capacity of spider hemocyanin.

    PubMed

    Cunningham, M; Gómez, C; Pollero, R

    1999-09-01

    The spider hemocyanin capacity to bind different lipid classes was evaluated by measuring some binding kinetic parameters. A very high lipoprotein (VHDL) which contains hemocyanin, was isolated from Polybetes pythagoricus hemolymph plasma and delipidated. Hemocyanin was bound separately to labelled palmitic acid, phosphatidylcholine, cholesterol, and triolein resulting in several artificial lipoprotein structures. It was possible to corroborate in vitro the lipid-hemocyanin interactions which had been previously observed and, consequently, the apolipoprotein role played by the respiratory pigment of spiders. Lipoproteins were analysed by gel filtration chromatography, and three subfractions with different hemocyanin structures were obtained. The four lipid classes were only bound to the hexameric structure (420 Kda), possibly to low polarity sites. Upon radioactivity measurements of the protein-associated lipids, maximal binding ratios (Mr), dissociation constants (Kd), and the maximal binding effectiveness at low lipid concentrations (Eo) were calculated. Lipid/protein ratios were increased proportionally to each available lipid concentration, following a hyperbolic binding model. Values of saturation, affinity, and maximal binding efficiency to hemocyanin were found to be different for each lipid class assayed. The highest lipid/protein ratio (41.5) was obtained with the free fatty acid and the lowest (7.2) with triolein. Phosphatidylcholine and cholesterol showed the highest relative affinities for hemocyanin (Kd = 63 x 10(-5) M and 74 x 10(-5) M, respectively). Phosphatidylcholine at low concentrations, similar to the physiological ones, presented the highest Eo value. Maximal lipid/protein ratios reached in vitro, were greater than those in P. pythagoricus VHDL, pointing out that hemocyanin could play the apolipoprotein role even under physiological conditions with a very high plasma lipid concentration. J. Exp. Zool. 284:368-373, 1999.

  11. Specific triplex binding capacity of mixed base sequence duplex nucleic acids used for single-nucleotide polymorphism detection.

    PubMed

    Daksis, Jasmine I; Erikson, Glen H

    2005-01-01

    Specific base recognition and binding between native double-stranded DNA (dsDNA) and complementary single-stranded DNA (ssDNA) of mixed base sequence is presented. Third-strand binding, facilitated and stabilized by a DNA intercalator, YOYO-1, occurs within 5 min at room temperature. This triplex binding capability has been used to develop a homogeneous assay that accurately detects 1-, 2-, or 3-bp mutations or deletions in the dsDNA target. Every type of 1-bp mismatch can be identified. The assay can reliably distinguish homozygous from heterozygous polymerase chain reaction (PCR)-amplified genomic dsDNA, thus providing a highly sensitive clinical diagnostic assay.

  12. Neocosmospora sp.-derived resorcylic acid lactones with in vitro binding capacity for human opioid and cannabinoid receptors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bioassay-guided fractionation of a fungus Neocosmospora sp. (UM-031509) resulted in the isolation of three new resorcyclic acid lactones, neocosmosin A (2), neocosmosin B (3) and neocosmosin C (4). Three known resorcylic acid lactones, monocillin IV (1), monocillin II (5) and monorden (6) were also ...

  13. Fermentation of liquid coproducts and liquid compound diets: Part 2. Effects on pH, acid-binding capacity, organic acids and ethanol during a 6-day storage period.

    PubMed

    Scholten, R H; Rijnen, M M; Schrama, J W; Boer, H; van der Peet-Schwering, C M; Den Hartog, L A; Vesseur, P C; Verstegen, M W

    2001-06-01

    The effects of a 6-day storage period on changes in pH, acid-binding capacity, level of organic acids and ethanol of three liquid coproducts [liquid wheat starch (LWS), mashed potato steam peel (PSP) and cheese whey (CW)] and two liquid compound diets [liquid grower diet (LGD) and liquid finisher diet (LFD)] were studied. All products, except LWS, showed a significant decrease in pH and acid-binding capacity during storage. At the end of the storage period, all products reached a pH of between 3.5 and 3.9. In general, it can be concluded that the lactic acid content, and to a lesser extent the acetic acid content, increased dramatically during storage. In contrast, the ethanol content increased significantly in the liquid compound diets only. The pattern of changes in pH and organic acids during the 6-day storage period was different between the liquid coproducts and the liquid compound diets. At the start of storage, liquid coproducts are already in the 'middle' of the fermentation process, while liquid compound diets need approximately 24-36 h before fermentation begins. Consequently, in practice a different approach to obtain fermented diets is needed for liquid coproducts and liquid compound diets.

  14. The productive cellulase binding capacity of cellulosic substrates.

    PubMed

    Karuna, Nardrapee; Jeoh, Tina

    2017-03-01

    Cellulosic biomass is the most promising feedstock for renewable biofuel production; however, the mechanisms of the heterogeneous cellulose saccharification reaction are still unsolved. As cellulases need to bind isolated molecules of cellulose at the surface of insoluble cellulose fibrils or larger aggregated cellulose structures in order to hydrolyze glycosidic bonds, the "accessibility of cellulose to cellulases" is considered to be a reaction limiting property of cellulose. We have defined the accessibility of cellulose to cellulases as the productive binding capacity of cellulose, that is, the concentration of productive binding sites on cellulose that are accessible for binding and hydrolysis by cellulases. Productive cellulase binding to cellulose results in hydrolysis and can be quantified by measuring hydrolysis rates. In this study, we measured the productive Trichoderma reesei Cel7A (TrCel7A) binding capacity of five cellulosic substrates from different sources and processing histories. Swollen filter paper and bacterial cellulose had higher productive binding capacities of ∼6 µmol/g while filter paper, microcrystalline cellulose, and algal cellulose had lower productive binding capacities of ∼3 µmol/g. Swelling and regenerating filter paper using phosphoric acid increased the initial accessibility of the reducing ends to TrCel7A from 4 to 6 µmol/g. Moreover, this increase in initial productive binding capacity accounted in large part for the difference in the overall digestibility between filter paper and swollen filter paper. We further demonstrated that an understanding of how the productive binding capacity declines over the course of the hydrolysis reaction has the potential to predict overall saccharification time courses. Biotechnol. Bioeng. 2017;114: 533-542. © 2016 Wiley Periodicals, Inc.

  15. Binding capacity of molecularly imprinted polymers and their nonimprinted analogs.

    PubMed

    Dorkó, Zsanett; Szakolczai, Anett; Verbić, Tatjana; Horvai, George

    2015-12-01

    Molecularly imprinted polymers bind their target compounds at binding sites. The binding sites are typically based on some type of functional group, such as carboxyl group. The total amount of such functional groups and their distribution into available and unavailable groups is not well known. The total binding capacity is usually indirectly determined from adsorption isotherms, which are measured much below the theoretical binding capacity. This work shows that in a variety of differently prepared, methacrylic acid based molecularly imprinted and nonimprinted polymers, all carboxylic groups used for the polymer synthesis are retained in the polymer, 80-90% of them can be accessed by strong bases and essentially the same amount can be used for adsorption of weak bases. This high level of adsorption can only be achieved, however, if the adsorbed weak base is strong enough, if the polymer is sufficiently elastic and if the solvent does not compete too strongly for the binding sites. These results may explain why the maximum binding capacities obtained from isotherm measurements are usually not equal to the total amount of available binding sites. This study confirms the usefulness of nonimprinted polymers at high loadings.

  16. [Larval stages of Ascaris lumbricoides: hyaluronan-binding capacity].

    PubMed

    Ponce-León, Patricia; Foresto, Patricia; Valverde, Juana

    2009-03-01

    Hyaluronic acid has important functions in inflammatory and tissue reparation processes. Owing to the varied strategies of the parasites to evade the host's immune response, as well as the multiple functions and physiological importance of hyaluronic acid, the aim was to study the hyaluronan binding capacity by Ascaris lumbricoides larval stages. Larval concentrates were prepared by hatching A. lumbricoides eggs. The larvae were collected by the Baermann method. The test of serum soluble CD44 detection by Agregation Inhibition was modified. All the larval concentrates presented hyaluronan binding capacity. The obtained results allow to suppose the existence of an hyaluronic acid specific receptor in A. lumbricoides. This receptor eventually might compete with the usual receptors of the host. The parasite might use this mechanism to evade the immune response.

  17. Label-Free LC-MS Profiling of Skeletal Muscle Reveals Heart-Type Fatty Acid Binding Protein as a Candidate Biomarker of Aerobic Capacity.

    PubMed

    Malik, Zulezwan Ab; Cobley, James N; Morton, James P; Close, Graeme L; Edwards, Ben J; Koch, Lauren G; Britton, Steven L; Burniston, Jatin G

    2013-12-01

    Two-dimensional gel electrophoresis provides robust comparative analysis of skeletal muscle, but this technique is laborious and limited by its inability to resolve all proteins. In contrast, orthogonal separation by SDS-PAGE and reverse-phase liquid chromatography (RPLC) coupled to mass spectrometry (MS) affords deep mining of the muscle proteome, but differential analysis between samples is challenging due to the greater level of fractionation and the complexities of quantifying proteins based on the abundances of their tryptic peptides. Here we report simple, semi-automated and time efficient (i.e., 3 h per sample) proteome profiling of skeletal muscle by 1-dimensional RPLC electrospray ionisation tandem MS. Solei were analysed from rats (n = 5, in each group) bred as either high- or low-capacity runners (HCR and LCR, respectively) that exhibited a 6.4-fold difference (1,625 ± 112 m vs. 252 ± 43 m, p < 0.0001) in running capacity during a standardized treadmill test. Soluble muscle proteins were extracted, digested with trypsin and individual biological replicates (50 ng of tryptic peptides) subjected to LC-MS profiling. Proteins were identified by triplicate LC-MS/MS analysis of a pooled sample of each biological replicate. Differential expression profiling was performed on relative abundances (RA) of parent ions, which spanned three orders of magnitude. In total, 207 proteins were analysed, which encompassed almost all enzymes of the major metabolic pathways in skeletal muscle. The most abundant protein detected was type I myosin heavy chain (RA = 5,843 ± 897) and the least abundant protein detected was heat shock 70 kDa protein (RA = 2 ± 0.5). Sixteen proteins were significantly (p < 0.05) more abundant in HCR muscle and hierarchal clustering of the profiling data highlighted two protein subgroups, which encompassed proteins associated with either the respiratory chain or fatty acid oxidation. Heart-type fatty acid binding protein (FABPH) was 1.54-fold (p

  18. Effects of organic ligands on fractionation of rare earth elements (REEs) in hydroponic plants: an application to the determination of binding capacities by humic acid for modeling.

    PubMed

    Ding, ShiMing; Liang, Tao; Zhang, ChaoSheng; Yan, JunCai; Zhang, ZiLi

    2006-12-01

    Previous studies have revealed the fractionation processes of rare earth elements (REEs) in hydroponic plants, with a heavy REE (HREE, the elements from Gd to Lu) enrichment in leaves. In this study, effects on the HREE enrichment in soybean leaves with additions of carboxylic acids (acetate, malate, citrate, NTA, EDTA and DTPA) and two soil humic acids (HAs) were investigated. REE speciation in carboxylic acid and HA solutions was simulated using Visual MINTEQ and Model V, respectively. The results showed that the effects caused by carboxylic acids were strongly dependent on the differences between their binding strengths for light REEs (LREEs, the elements from La to Eu) and those for HREEs. A good correlation existed between these effects and the changes of free REE ions in solutions. This relationship was also observed for the HA treatments, provided that the intrinsic equilibrium constants of REEs for cation-proton exchange with HA (i.e., pK(MHA)) in Model V were estimated using a free-energy relationship with the stability constants for REE complexation with lactic acid. It is suggested that this set of pK(MHA) values is more suitable for use in Model V for the simulation of REE complexation with HA.

  19. Determination of binding capacity and adsorption enthalpy between Human Glutamate Receptor (GluR1) peptide fragments and kynurenic acid by surface plasmon resonance experiments.

    PubMed

    Csapó, E; Majláth, Z; Juhász, Á; Roósz, B; Hetényi, A; Tóth, G K; Tajti, J; Vécsei, L; Dékány, I

    2014-11-01

    The interaction between kynurenic acid (KYNA) and two peptide fragments (ca. 30 residues) of Human Glutamate Receptor 201-300 (GluR1) using surface plasmon resonance (SPR) spectroscopy was investigated. Because of the medical interest in the neuroscience, GluR1 is one of the important subunits of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR). AMPARs are ionotoropic glutamate receptors, which are mediating fast synaptic transmission and are crucial for plasticity in the brain. On the other hand, KYNA has been suggested to have neuroprotective activity and it has been considered for apply in therapy in certain neurobiological disorders. In this article the adsorption of the GluR1201-230 and GluR1231-259 peptides were studied on gold biosensor chip. The peptides were chemically bonded onto the gold surface via thiol group of L-cysteine resulted in the formation of peptide monolayer on the SPR chip surface. Because the GluR1231-259 peptide does not contain L-cysteine the Val256 was replaced by Cys256. The cross sectional area and the surface orientation of the studied peptides were determined by SPR and theoretical calculations (LOMETS) as well. The binding capability of KYNA on the peptide monolayer was studied in the concentration range of 0.1-5.0 mM using 150 mM NaCl ionic strength at pH 7.4 (±0.02) in phosphate buffer solutions. In order to determine the binding enthalpy the experiments were carried out between +10°C and +40°C. The heat of adsorption was calculated by using adsorption isotherms at different surface loading of KYNA on the SPR chip.

  20. Americium binding to humic acid.

    PubMed

    Peters, A J; Hamilton-Taylor, J; Tipping, E

    2001-09-01

    The binding of americium (Am) by peat humic acid (PHA) has been investigated at Am concentrations between 10(-1) and 10(-7) M at pH approximately 2.6 in the presence and absence of Cu as a competing ion. Cu-PHA binding was also investigated in order to derive independent binding constants for use in modeling the competitive binding studies. Humic ion-binding model VI was used to compare the acquired data with previously published binding data and to investigate the importance of high-affinity binding sites in metal-PHA binding. Am was not observed to bind to high-affinity, low-concentration binding sites. The model VI parameter deltaLK2 takes into accountthe small number of strong sites in PHA and was found to be important for Cu-PHA binding but not for Am-PHA binding, regardless of whether Cu was present. Analysis of the PHA sample revealed that it contained a considerable quantity of Fe not removed by the extraction procedure, much of which is believed to be present as Fe(III). Model VI was then used to investigate the possible importance of the presence of Fe(III) in the Am-PHA binding experiments. When Fe(III) was assumed to be present, improved descriptions of the data by model VI were obtained by assuming that all of the metals [Am, Cu, and Fe(III)] undergo strong binding. This highlights the importance of Fe(III) competition in metal-PHA binding studies and possible shortcomings in the extraction procedure used to extract PHA.

  1. The role of amino acid electron-donor/acceptor atoms in host-cell binding peptides is associated with their 3D structure and HLA-binding capacity in sterile malarial immunity induction

    SciTech Connect

    Patarroyo, Manuel E.; Almonacid, Hannia; Moreno-Vranich, Armando

    2012-01-20

    Highlights: Black-Right-Pointing-Pointer Fundamental residues located in some HABPs are associated with their 3D structure. Black-Right-Pointing-Pointer Electron-donor atoms present in {beta}-turn, random, distorted {alpha}-helix structures. Black-Right-Pointing-Pointer Electron-donor atoms bound to HLA-DR53. Black-Right-Pointing-Pointer Electron-acceptor atoms present in regular {alpha}-helix structure bound to HLA-DR52. -- Abstract: Plasmodium falciparum malaria continues being one of the parasitic diseases causing the highest worldwide mortality due to the parasite's multiple evasion mechanisms, such as immunological silence. Membrane and organelle proteins are used during invasion for interactions mediated by high binding ability peptides (HABPs); these have amino acids which establish hydrogen bonds between them in some of their critical binding residues. Immunisation assays in the Aotus model using HABPs whose critical residues had been modified have revealed a conformational change thereby enabling a protection-inducing response. This has improved fitting within HLA-DR{beta}1{sup Asterisk-Operator} molecules where amino acid electron-donor atoms present in {beta}-turn, random or distorted {alpha}-helix structures preferentially bound to HLA-DR53 molecules, whilst HABPs having amino acid electron-acceptor atoms present in regular {alpha}-helix structure bound to HLA-DR52. This data has great implications for vaccine development.

  2. Bilirubin Binding Capacity in the Preterm Neonate.

    PubMed

    Amin, Sanjiv B

    2016-06-01

    Total serum/plasma bilirubin (TB), the biochemical measure currently used to evaluate and manage hyperbilirubinemia, is not a useful predictor of bilirubin-induced neurotoxicity in premature infants. Altered bilirubin-albumin binding in premature infants limits the usefulness of TB in premature infants. In this article, bilirubin-albumin binding, a modifying factor for bilirubin-induced neurotoxicity, in premature infants is reviewed.

  3. 21 CFR 862.1415 - Iron-binding capacity test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Iron-binding capacity test system. 862.1415... Systems § 862.1415 Iron-binding capacity test system. (a) Identification. An iron-binding capacity test system is a device intended to measure iron-binding capacity in serum. Iron-binding capacity...

  4. 21 CFR 862.1415 - Iron-binding capacity test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Iron-binding capacity test system. 862.1415... Systems § 862.1415 Iron-binding capacity test system. (a) Identification. An iron-binding capacity test system is a device intended to measure iron-binding capacity in serum. Iron-binding capacity...

  5. 21 CFR 862.1415 - Iron-binding capacity test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Iron-binding capacity test system. 862.1415... Systems § 862.1415 Iron-binding capacity test system. (a) Identification. An iron-binding capacity test system is a device intended to measure iron-binding capacity in serum. Iron-binding capacity...

  6. 21 CFR 862.1415 - Iron-binding capacity test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Iron-binding capacity test system. 862.1415... Systems § 862.1415 Iron-binding capacity test system. (a) Identification. An iron-binding capacity test system is a device intended to measure iron-binding capacity in serum. Iron-binding capacity...

  7. 21 CFR 862.1415 - Iron-binding capacity test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Iron-binding capacity test system. 862.1415... Systems § 862.1415 Iron-binding capacity test system. (a) Identification. An iron-binding capacity test system is a device intended to measure iron-binding capacity in serum. Iron-binding capacity...

  8. Biological variability of transferrin saturation and unsaturated iron binding capacity

    PubMed Central

    Adams, PC; Reboussin, DM; Press, RD; Barton, JC; Acton, RT; Moses, GC; Leiendecker-Foster, C; McLaren, GD; Dawkins, FW; Gordeuk, VR; Lovato, L; Eckfeldt, JH

    2007-01-01

    Background Transferrin saturation is widely considered the preferred screening test for hemochromatosis. Unsaturated iron binding capacity has similar performance at lower cost. However, the within-person biological variability of both these tests may limit their ability at commonly used cut points to detect HFE C282Y homozygous patients. Methods The Hemochromatosis and Iron Overload Screening (HEIRS) Study screened 101,168 primary care participants for iron overload using tansferrin saturation, unsaturated iron binding capacity, ferritin and HFE C282Y and H63D genotyping. Transferrin saturation and unsaturated iron binding capacity were performed at initial screening and again when selected participants and controls returned for a clinical examination several months later. A missed case was defined as a C282Y homozygote who had transferrin saturation below cut point (45 % women, 50 % men) or unsaturated iron binding capacity above cut point (150 μmol/L women, 125 μmol/L men) at either the initial screening or clinical examination, or both, regardless of serum ferritin. Results There were 209 C282Y previously undiagnosed homozygotes with transferrin saturation and unsaturated iron binding capacity testing done at initial screening and clinical examination. Sixty-eight C282Y homozygotes (33%) would have been missed at these transferrin saturation cut points (19 men, 49 women, median SF 170 μg/L, first and third quartiles 50 and 474 μg/L), and 58 homozygotes (28 %) would have been missed at the unsaturated iron binding capacity cut points (20 men, 38 women, median SF 168 μg/L, quartiles 38 and 454 μg/L). There was no advantage to using fasting samples. Conclusions The within-person biological variability of transferrin saturation and unsaturated iron binding capacity limit their usefulness as an initial screening test for expressing C282Y homozygotes. PMID:17976429

  9. Predicting protein dynamic binding capacity from batch adsorption tests.

    PubMed

    Carta, Giorgio

    2012-10-01

    The dynamic binding capacity (DBC) and its dependence on residence time influence the design and productivity of adsorption columns used in protein capture applications. This paper offers a very simple approach to predict the DBC of an adsorption column based on a measurement of the equilibrium binding capacity (EBC) and of the time needed to achieve one-half of the EBC in a batch adsorption test. The approach is based on a mass transfer kinetics model that assumes pore diffusion with a rectangular isotherm; however, the same approach is also shown to work for other systems where solute transport inside the particle occurs through other transport mechanisms.

  10. Ion binding capacity of enzyme modified citrus peel and pectin

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A wide ranging group of enzyme modified pectin and polygalacturonic acid (PGA) samples were tested for their biosorption potential, specifically their ability to bind Pb. Two sample sets were the pH 4.5 and 7.5 demethylated series obtained from demethylation of a model homogalacturonan produced usi...

  11. Influence of binding pH and protein solubility on the dynamic binding capacity in hydrophobic interaction chromatography.

    PubMed

    Baumann, Pascal; Baumgartner, Kai; Hubbuch, Jürgen

    2015-05-29

    Hydrophobic interaction chromatography (HIC) is one of the most frequently used purification methods in biopharmaceutical industry. A major drawback of HIC, however, is the rather low dynamic binding capacity (DBC) obtained when compared to e.g. ion exchange chromatography (IEX). The typical purification procedure for HIC includes binding at neutral pH, independently of the proteins nature and isoelectric point. Most approaches to process intensification are based on resin and salt screenings. In this paper a combination of protein solubility data and varying binding pH leads to a clear enhancement of dynamic binding capacity. This is shown for three proteins of acidic, neutral, and alkaline isoelectric points. High-throughput solubility screenings as well as miniaturized and parallelized breakthrough curves on Media Scout RoboColumns (Atoll, Germany) were conducted at pH 3-10 on a fully automated robotic workstation. The screening results show a correlation between the DBC and the operational pH, the protein's isoelectric point and the overall solubility. Also, an inverse relationship of DBC in HIC and the binding kinetics was observed. By changing the operational pH, the DBC could be increased up to 30% compared to the standard purification procedure performed at neutral pH. As structural changes of the protein are reported during HIC processes, the applied samples and the elution fractions were proven not to be irreversibly unfolded.

  12. Binding of coumarins to site I of human serum albumin. Effect of the fatty acids.

    PubMed

    Zatón, A M; Ferrer, J M; Ruiz de Gordoa, J C; Marquínez, M A

    1995-07-14

    It is known that binding site I on human serum albumin (HSA) consists of a zone of two overlapping regions: the specific binding region represented by warfarin binding and the specific binding region represented by azapropazone and phenylbutazone binding. In this paper binding parameters to defatted HSA and to HSA with fatty acids (molar ratio of fatty acid/HSA = 4) were compared. High-affinity binding sites for warfarin, 4-chromanol, 4-hydroxycoumarin, coumarin, 3-acetylcoumarin and phenylbutazone (759,549 M-1 > Ka > 67,024 M-1) constitute binding site I on HSA. In this binding area defatted HSA can bind two molecules of warfarin, but the presence of fatty acids diminish the binding capacity of warfarin to HSA (2 > n > 1).

  13. FCA does not bind abscisic acid.

    PubMed

    Risk, Joanna M; Macknight, Richard C; Day, Catherine L

    2008-12-11

    The RNA-binding protein FCA promotes flowering in Arabidopsis. Razem et al. reported that FCA is also a receptor for the phytohormone abscisic acid (ABA). However, we find that FCA does not bind ABA, suggesting that the quality of the proteins assayed and the sensitivity of the ABA-binding assay have led Razem et al. to erroneous conclusions. Because similar assays have been used to characterize other ABA receptors, our results indicate that the ABA-binding properties of these proteins should be carefully re-evaluated and that alternative ABA receptors are likely to be discovered.

  14. Magnetophoretic cell sorting is a function of antibody binding capacity.

    PubMed

    McCloskey, Kara E; Moore, Lee R; Hoyos, Mauricio; Rodriguez, Alex; Chalmers, Jeffrey J; Zborowski, Maciej

    2003-01-01

    Antibody binding capacity (ABC) is a term representing a cell's ability to bind antibodies, correlating with the number of specific cellular antigens expressed on that cell. ABC allows magnetically conjugated antibodies to bind to the targeted cells, imparting a magnetophoretic mobility on each targeted cell. This enables sorting based on differences in the cell magnetophoretic mobility and, potentially, a magnetic separation based on the differences in the cell ABC values. A cell's ABC value is a particularly important factor in continuous magnetic cell separation. This work investigates the relationship between ABC and magnetic cell separation efficiency by injection of a suspension of immunomagnetically labeled quantum simply cellular calibration microbeads of known ABC values into fluid flowing through a quadrupole magnetic sorter. The elution profiles of the outlet streams were evaluated using UV detectors. Optimal separation flow rate was shown to correlate with the ABC of these microbeads. Comparing experimental and theoretical results, the theory correctly predicted maximum separation flow rates but overestimated the separation fractional recoveries.

  15. Nonenzymatic glycation of transferrin: decrease of iron-binding capacity and increase of oxygen radical production.

    PubMed

    Fujimoto, S; Kawakami, N; Ohara, A

    1995-03-01

    The total iron-binding capacity (TIBC) and iron contents of diabetic rat serum, as well as the iron-binding capacity of glycated transferrin and oxygen radical production by the glycated proteins were examined. The TIBC and iron content of diabetic rat sera were found to be much lower than those of control rat sera. Incubation of human serum with glucose in vitro resulted in a significant fall of its unsaturated iron-binding capacity (UIBC) with time. When apotransferrin was incubated with glucose, its UIBC significantly decreased. The iron content of holotransferrin was markedly reduced by incubation with bathophenanthroline sulphonic acid (BPSA) in the presence of glucose, although the content was not altered by incubation with BPSA alone. The generation of superoxide radical (O2-) and hydroxyl radical (OH.) by the glycated holotransferrin was much greater than that by glycated apotransferrin. Glycated holotransferrin showed significantly accelerated hydroxyl radical production by the hypoxanthine-xanthine oxidase system, while intact holotransferrin did not. Treatment of holotransferrin with glucose caused the fragmentation of the protein, while the same treatment of apotransferrin did not. These results suggest that iron ions in the glycated transferrin molecule are bound loosely to the protein and are redox-active and the glycated holotransferrin produces oxygen radicals including O2- and OH. efficiently, and that the glycated transferrin does not function as an iron-binding protein.

  16. Leukocyte protease binding to nucleic acids promotes nuclear localization and cleavage of nucleic acid binding proteins.

    PubMed

    Thomas, Marshall P; Whangbo, Jennifer; McCrossan, Geoffrey; Deutsch, Aaron J; Martinod, Kimberly; Walch, Michael; Lieberman, Judy

    2014-06-01

    Killer lymphocyte granzyme (Gzm) serine proteases induce apoptosis of pathogen-infected cells and tumor cells. Many known Gzm substrates are nucleic acid binding proteins, and the Gzms accumulate in the target cell nucleus by an unknown mechanism. In this study, we show that human Gzms bind to DNA and RNA with nanomolar affinity. Gzms cleave their substrates most efficiently when both are bound to nucleic acids. RNase treatment of cell lysates reduces Gzm cleavage of RNA binding protein targets, whereas adding RNA to recombinant RNA binding protein substrates increases in vitro cleavage. Binding to nucleic acids also influences Gzm trafficking within target cells. Preincubation with competitor DNA and DNase treatment both reduce Gzm nuclear localization. The Gzms are closely related to neutrophil proteases, including neutrophil elastase (NE) and cathepsin G. During neutrophil activation, NE translocates to the nucleus to initiate DNA extrusion into neutrophil extracellular traps, which bind NE and cathepsin G. These myeloid cell proteases, but not digestive serine proteases, also bind DNA strongly and localize to nuclei and neutrophil extracellular traps in a DNA-dependent manner. Thus, high-affinity nucleic acid binding is a conserved and functionally important property specific to leukocyte serine proteases. Furthermore, nucleic acid binding provides an elegant and simple mechanism to confer specificity of these proteases for cleavage of nucleic acid binding protein substrates that play essential roles in cellular gene expression and cell proliferation.

  17. Kinetics of ligand binding to nucleic acids.

    PubMed

    Arakelyan, V B; Babayan, S Y; Tairyan, V I; Arakelyan, A V; Parsadanyan, M A; Vardevanyan, P O

    2006-02-01

    Ligand binding to nucleic acids (NA) is considered as a stationary Markov process. It is shown that the probabilistic description of ligand-NA binding allows one to describe not only the kinetics of the change of number of bound ligands at arbitrary fillings but also to calculate stationary values of the number of bound ligands and its dispersion. The general analysis of absorption isotherms and kinetics of ligand binding to NA make it possible to determine of rate constants of ligand-NA complex formation and dissociation.

  18. Determination of binding capacity and adsorption enthalpy between Human Glutamate Receptor (GluR1) peptide fragments and kynurenic acid by surface plasmon resonance experiments. Part 2: Interaction of GluR1270-300 with KYNA.

    PubMed

    Csapó, E; Bogár, F; Juhász, Á; Sebők, D; Szolomájer, J; Tóth, G K; Majláth, Z; Vécsei, L; Dékány, I

    2015-09-01

    In the course of our previous work, the interactions of two peptide fragments (GluR1201-230 and GluR1231-259) of human glutamate receptor (GluR1201-300) polypeptide with kynurenic acid (KYNA) were investigated by surface plasmon resonance (SPR) spectroscopy. Besides quantitation of the interactions, the enthalpies of binding of KYNA on certain peptide fragment-modified gold surfaces were also reported. In the present work, a third peptide fragment (GluR1270-300) of the glutamate receptor was synthesized and its interaction with KYNA was investigated by an SPR technique. This 31-membered peptide was chemically bonded onto a gold-coated SPR chip via a cysteine residue. The peptide-functionalized biosensor chip was analyzed by atomic force microscopy (AFM) and theoretical calculations were performed on the structure and dimensions of the peptide on the gold surface. In order to determine the isosteric heat of adsorption of the binding of KYNA on the peptide-functionalized gold thin film, SPR experiments were carried out between +10°C and +40°C. The results on the GluR1270-300-KYNA system were compared with the previously published binding parameters of the interactions of GluR1201-230 and GluR1231-259 with KYNA. The binding abilities of KYNA with all three peptide fragments immobilized on the gold surface were estimated by a molecular docking procedure and the binding free energies of these AMPA receptor subunits with KYNA were determined.

  19. Iodine binding to humic acid.

    PubMed

    Bowley, H E; Young, S D; Ander, E L; Crout, N M J; Watts, M J; Bailey, E H

    2016-08-01

    The rate of reactions between humic acid (HA) and iodide (I(-)) and iodate (IO3(-)) have been investigated in suspensions spiked with (129)I at concentrations of 22, 44 and 88 μg L(-1) and stored at 10 °C. Changes in the speciation of (129)I(-), (129)IO3(-) and mixed ((129)I(-) + (129)IO3(-)) spikes were monitored over 77 days using liquid chromatography inductively coupled plasma mass spectrometry (LC-ICP-MS). In suspensions spiked with (129)I(-) 25% of the added I(-) was transformed into organic iodine (Org-(129)I) within 77 days and there was no evidence of (129)IO3(-) formation. By contrast, rapid loss of (129)IO3(-) and increase in both (129)I(-) and Org-(129)I was observed in (129)IO3(-)-spiked suspensions. However, the rate of Org-(129)I production was greater in mixed systems compared to (129)IO3(-)-spiked suspensions with the same total (129)I concentration, possibly indicating IO3(-)I(-) redox coupling. Size exclusion chromatography (SEC) demonstrated that Org-(129)I was present in both high and low molecular weight fractions of the HA although a slight preference to bond with the lower molecular weight fractions was observed indicating that, after 77 days, the spiked isotope had not fully mixed with the native (127)I pool. Iodine transformations were modelled using first order rate equations and fitted rate coefficients determined. However, extrapolation of the model to 250 days indicated that a pseudo-steady state would be attained after ∼200 days but that the proportion of (129)I incorporated into HA was less than that of (127)I indicating the presence of a recalcitrant pool of (127)I that was unavailable for isotopic mixing.

  20. Bile acid salt binding with colesevelam HCl is not affected by suspension in common beverages.

    PubMed

    Hanus, Martin; Zhorov, Eugene

    2006-12-01

    It has been previously reported that anions in common beverages may bind to bile acid sequestrants (BAS), reducing their capacity for binding bile acid salts. This study examined the ability of the novel BAS colesevelam hydrochloride (HCl), in vitro, to bind bile acid sodium salts following suspension in common beverages. Equilibrium binding was evaluated under conditions of constant time and varying concentrations of bile acid salts in simulated intestinal fluid (SIF). A stock solution of sodium salts of glycochenodeoxycholic acid (GCDC), taurodeoxycholic acid (TDC), and glycocholic acid (GC), was added to each prepared sample of colesevelam HCl. Bile acid salt binding was calculated by high-performance liquid chromatography (HPLC) analysis. Kinetics experiments were conducted using constant initial bile acid salt concentrations and varying binding times. The affinity, capacity, and kinetics of colesevelam HCl binding for GCDC, TDC, and GC were not significantly altered after suspension in water, carbonated water, Coca-Cola, Sprite, grape juice, orange juice, tomato juice, or Gatorade. The amount of bile acid sodium salt bound as a function of time was unchanged by pretreatment with any beverage tested. The in vitro binding characteristics of colesevelam HCl are unchanged by suspension in common beverages.

  1. Calcium-binding capacity of wheat germ protein hydrolysate and characterization of Peptide-calcium complex.

    PubMed

    Liu, Feng-Ru; Wang, Li; Wang, Ren; Chen, Zheng-Xing

    2013-08-07

    This study investigates the ability of various wheat germ protein hydrolysates (WGPHs) to bind calcium and characterizes the peptide-calcium complexes. We demonstrate that the amount of Ca bound depended greatly on the type of enzyme, degree of hydrolysis (DH), amino acid composition, and molecular mass distribution of different hydrolysates. The maximum level of Ca bound (67.5 mg·g(-1)) occurred when Alcalase was used to hydrolyze wheat germ protein at a DH of 21.5%. Peptide fragments exhibiting high calcium-binding capacity had molecular mass <2000 Da. The calcium-binding peptides mainly consisted of Glu, Arg, Asp, and Gly, and the level of Ca bound was related to the hydrophobic amino acid content in WGPHs. UV-visible and Fourier transform infrared spectra demonstrate that amino nitrogen atoms and oxygen atoms on the carboxyl group were involved in complexation. Therefore, wheat germ protein is a promising protein source for the production of calcium-binding peptides and could be utilized as a bioactive ingredient for nutraceutical food production.

  2. Reversible Acid Gas Capture Using CO2-Binding Organic Liquids

    SciTech Connect

    Heldebrant, David J.; Koech, Phillip K.; Yonker, Clement R.; Rainbolt, James E.; Zheng, Feng

    2010-08-31

    Acid gas scrubbing technology is predominantly aqueous alkanolamine based. Of the acid gases, CO2, H2S and SO2 have been shown to be reversible, however there are serious disadvantages with corrosion and high regeneration costs. The primary scrubbing system composed of monoethanolamine is limited to 30% by weight because of the highly corrosive solution. This gravimetric limitation limits the CO2 volumetric (≤108 g/L) and gravimetric capacity (≤7 wt%) of the system. Furthermore the scrubbing system has a large energy penalty from pumping and heating the excess water required to dissolve the MEA bicarbonate salt. Considering the high specific heat of water (4 j/g-1K-1), low capacities and the high corrosion we set out to design a fully organic solvent that can chemically bind all acid gases i.e. CO2 as reversible alkylcarbonate ionic liquids or analogues thereof. Having a liquid acid gas carrier improves process economics because there is no need for excess solvent to pump and to heat. We have demonstrated illustrated in Figure 1, that CO2-binding organic liquids (CO2BOLs) have a high CO2 solubility paired with a much lower specific heat (<1.5 J/g-1K-1) than aqueous systems. CO2BOLs are a subsection of a larger class of materials known as Binding Organic Liquids (BOLs). Our BOLs have been shown to reversibly bind and release COS, CS2, and SO2, which we denote COSBOLS, CS2BOLs and SO2BOLs. Our BOLs are highly tunable and can be designed for post or pre-combustion gas capture. The design and testing of the next generation zwitterionic CO2BOLs and SO2BOLs are presented.

  3. Comparative study of random and oriented antibody immobilization techniques on the binding capacity of immunosensor.

    PubMed

    Kausaite-Minkstimiene, A; Ramanaviciene, A; Kirlyte, J; Ramanavicius, A

    2010-08-01

    A comparative study of four different antibody immobilization techniques that are suitable for modification of surface plasmon resonance (SPR) chip (SPR-chip) is reported. Antibodies against human growth hormone (anti-HGH) were used as the model system. The evaluated SPR-chip modification techniques were (i) random immobilization of intact anti-HGH (intact-anti-HGH) via self-assembled monolayer (SAM) based on 11-mercaptoundecanoic acid (MUA); (ii) random immobilization of intact-anti-HGH within carboxymethyl dextran (CMD) hydrogel by direct covalent amine coupling technique; (iii) oriented coupling of intact-anti-HGH via Fc-fragment to protein-G layer assembled on SAM consisting of MUA (MUA/pG); (iv) oriented immobilization of fragmented anti-HGH antibodies (frag-anti-HGH) via their native thiol-groups directly coupled to the gold. To liberate these thiol groups, the intact-anti-HGH was chemically "divided" into two frag-anti-HGH fragments by chemical reduction with 2-mercaptoethylamine (2-MEA). Optimal concentration of 2-MEA for preparation of anti-HGH was 15 mM. The surface concentration of immobilized antibodies and the antigen binding capacity for all four differently modified SPR-chips was evaluated and compared. The maximum surface concentration of immobilized intact-anti-HGH was obtained by immobilizing the antibody within CMD-hydrogel. The maximal antigen binding capacity was obtained by SPR-chip based on intact-anti-HGH immobilized via MUA/pG. The immobilization based on application of frag-anti-HGH was found to be the most suitable for design of SPR-immunosensor for HGH detection, due to its sufficient antigen binding capacity, simplicity, and low cost in respect to the currently evaluated techniques.

  4. Routes to improve binding capacities of affinity resins demonstrated for Protein A chromatography.

    PubMed

    Müller, Egbert; Vajda, Judith

    2016-05-15

    Protein A chromatography is a well-established platform in downstream purification of monoclonal antibodies. Dynamic binding capacities are continuously increasing with almost every newly launched Protein A resin. Nevertheless, binding capacities of affinity chromatography resins cannot compete with binding capacities obtained with modern ion exchange media. Capacities of affinity resins are roughly 50% lower. High binding capacities of ion exchange media are supported by spacer technologies. In this article, we review existing spacer technologies of affinity chromatography resins. A yet known effective approach to increase the dynamic binding capacity of Protein A resins is oligomerization of the particular Protein A motifs. This resembles the tentacle technology used in ion exchange chromatography. Dynamic binding capacities of a hexameric ligand are roughly twice as high compared to capacities obtained with a tetrameric ligand. Further capacity increases up to 130mg/ml can be realized with the hexamer ligand, if the sodium phosphate buffer concentration is increased from 20 to 100mM. Equilibrium isotherms revealed a BET shape for the hexamer ligand at monoclonal antibody liquid phase concentrations higher than 9mg/ml. The apparent multilayer formation may be due to hydrophobic forces. Other quality attributes such as recovery, aggregate content, and overall purity of the captured monoclonal antibody are not affected.

  5. Pectins from the albedo of immature lemon fruitlets have high water binding capacity.

    PubMed

    Schröder, Roswitha; Clark, Christopher J; Sharrock, Keith; Hallett, Ian C; MacRae, Elspeth A

    2004-04-01

    The white part of citrus peel, the albedo, has a special role in water relations of both fruit and leaves from early on in fruit development. In times of drought, this tissue acts as a water reservoir for juice sacs, seeds and leaves. When water was injected into the albedo, free water was undetectable using magnetic resonance imaging. Microscopy showed tightly packed cells with little intercellular space, and thick cell walls. Cell wall material comprised 21% of the fresh albedo weight, and contained 26.1% galacturonic acid, the main constituent of pectin. From this, we postulated that pectin of the cell wall was responsible for the high water-binding capacity of the immature lemon albedo. Cell wall material was extracted using mild procedures that keep polymers intact, and four pectic fractions were recovered. Of these fractions, the SDS and chelator-soluble fractions showed viscosities ten and twenty times higher than laboratory-grade citrus pectin or the other albedo-derived pectins. The yield of these two pectins represented 28% of the cell walls and 62% of the galacturonic acid content of immature lemon albedo. We concluded that, from viscosity and abundance, these types of pectin account for the high water-binding capacity of this tissue. Compositional analyses showed that the two highly viscous pectic fractions differ in galacturonic acid content, degree of branching and length of side chains from the less viscous albedo-derived pectins. The most striking feature of these highly viscous pectins, however, was their high molecular weight distribution compared to the other pectic fractions.

  6. Effect of chain length on binding of fatty acids to Pluronics in microemulsions.

    PubMed

    James-Smith, Monica A; Shekhawat, Dushyant; Cheung, Sally; Moudgil, Brij M; Shah, Dinesh O

    2008-03-15

    We investigated the effect of fatty acid chain length on the binding capacity of drug and fatty acid to Pluronic F127-based microemulsions. This was accomplished by using turbidity experiments. Pluronic-based oil-in-water microemulsions of various compositions were synthesized and titrated to turbidity with concentrated Amitriptyline, an antidepressant drug. Sodium salts of C(8), C(10), or C(12) fatty acid were used in preparation of the microemulsion and the corresponding binding capacities were observed. It has been previously determined that, for microemulsions prepared with sodium caprylate (C(8) fatty acid soap), a maximum of 11 fatty acid molecules bind to the microemulsion per 1 molecule of Pluronic F127 and a maximum of 12 molecules of Amitriptyline bind per molecule of F127. We have found that with increasing the chain length of the fatty acid salt component of the microemulsion, the binding capacity of both the fatty acid and the Amitriptyline to the microemulsion decreases. For sodium salts of C(8), C(10) and C(12) fatty acids, respectively, a maximum of approximately 11, 8.4 and 8.3 molecules of fatty acid molecules bind to 1 Pluronic F127 molecule. We propose that this is due to the decreasing number of free monomers with increasing chain length. As chain length increases, the critical micelle concentration (cmc) decreases, thus leading to fewer monomers. Pluronics are symmetric tri-block copolymers consisting of propylene oxide (PO) and ethylene oxide (EO). The polypropylene oxide block, PPO is sandwiched between two polyethylene oxide (PEO) blocks. The PEO blocks are hydrophilic while PPO is hydrophobic portion in the Pluronic molecule. Due to this structure, we propose that the fatty acid molecules that are in monomeric form most effectively diffuse between the PEO "tails" and bind to the hydrophobic PPO groups.

  7. Binding of cholesterol and bile acid to hemicelluloses from rice bran.

    PubMed

    Hu, Guohua; Yu, Wenjian

    2013-06-01

    The objective of this study was to investigate the possibility of using hemicellulose from rice bran to scavenge cholesterol and bile acid in vitro study. This paper demonstrates that rice bran hemicellulose A (RBHA), rice bran hemicellulose B (RBHB) and rice bran hemicellulose C (RBHC) have the potential for binding cholesterol and bile acid. The quantity of cholesterol and bile acid bound varies from one rice bran fibre to another. As it can be inferred from the results of the study, RBHB was characterized by the highest capacity for cholesterol binding, followed by RBHC and RBHA. Binding of cholesterol and bile acid to rice bran insoluble dietary fibre (RBDF) and cellulose from rice bran was found to be poor. Lignin from rice bran was the least active fraction for binding cholesterol and bile acid. This confirms that the RBHB preparation from defatted rice bran has great potential in food applications, especially in the development of functional foods.

  8. Suppression of serum iron-binding capacity and bone marrow cellularity in pigs fed aflatoxin

    SciTech Connect

    Harvey, R.B.; Clark, D.E.; Huff, W.E.; Kubena, L.F.; Corrier, D.E.; Phillips, I.D.

    1988-04-01

    Flavus-parasiticus species of the genus Aspergillus are recognized as the primary producers of aflatoxins B/sub 1/, B/sup 2/, G/sup 1/, and G/sup 2/, hereafter referred to as aflatoxin (AF). The effects of feeding AF-contaminated diets to growing and finishing pigs have been described with changes in clinical performance, serum biochemistry, histology, and hematology attributed to aflatoxicosis. However, most of these studies evaluated AF-induced changes for a single AF dosage at a given point in time. The present study was designed to characterize how various AF dosages influence bone marrow histology, hematology, prothrombin and activated thromboplastin times, serum amino acids, and serum iron binding capacity during aflatoxicosis in growing pigs.

  9. Suppression of serum iron-binding capacity and bone marrow cellularity in pigs fed aflatoxin

    SciTech Connect

    Harvey, R.B.; Clark, D.E.; Huff, W.E.; Kubena, L.F.; Corrier, D.E. ); Phillips, T.D. )

    1988-05-01

    Flavus-parasiticus species of the genus Aspergillus are recognized as the primary producers of aflatoxins B{sub 1}, B{sub 2}, G{sub 1}, and G{sub 2}, hereafter referred to as aflatoxin (AF). The effects of feeding AF-contaminated diets to growing and finishing pigs have been described with changes in clinical performance, serum biochemistry, histology, and hematology attributed to aflatoxicosis. However, most of these studies evaluated AF-induced changes for a single AF dosage at a given point in time. The present study was designed to characterize how various AF dosages influence bone marrow histology, hematology, prothrombin and activated thromboplastin times, serum amino acids, and serum iron binding capacity during aflatoxicosis in growing pigs.

  10. Quantitative analysis of the binding strength and adsorption capacity of zinc oxide nanoparticles onto unmodified and modified cotton fiber

    NASA Astrophysics Data System (ADS)

    Printz, Stephen Robert

    Risk of bacterial infection is always a concern in hospitals, so it is important to find ways to minimize this risk. One method for reducing the risk of infection is by using textiles with antimicrobial properties. Zinc oxide nanoparticles have antimicrobial properties, and can be adsorbed onto cotton fibers to pass these properties to the cloth. However, the binding of the zinc oxide nanoparticles to cotton is weak, so the particles desorb from the cloth after repeated washings. The goal of this project was to quantify the binding strength of zinc oxide nanoparticles onto different types of cotton fiber. The cotton was modified by grafting cyclodextrin onto it with citric acid as a crosslinking agent. Adsorption was tested with desized, unbleached cotton print cloth; desized, bleached cotton print cloth; and desized, bleached, mercerized cotton print cloth. As expected, adsorption to unmodified cloth was poor. Unbleached cloth had the highest adsorption capacity (Q 0 = 22 +/- 4 mg ZnO/g cloth), and bleached cloth had the lowest adsorption capacity (Q0 = 17 +/- 4 mg ZnO=g cloth). Mercerized cloth had the lowest strength (b = 0.010 +/- 0.003 ppm-1), and bleached cloth had the highest binding strength (b = 0.04 +/- 0.01 ppm-1). Modification with alpha-cyclodextrin increased adsorption capacity over unmodified cloth by 61, 80, and 70% for mercerized/bleached cloth, bleached cloth, and unbleached cloth, respectively, and increased b by 1601, 126, and 90% respectively. Modification with beta-cyclodextrin increased adsorption capacities by 80, 94, and 112%, respectively, and increased b by 2027, 427, and 46%. As a result, beta-CD modified unbleached cloth had the highest adsorption capacity and one of the lowest binding strengths. However, beta-cyclodextrin modified mercerized cloth has both a high adsorption capacity and a high binding strength, and would likely be the best candidate for use in antimicrobial textiles.

  11. High-capacity calcium-binding chitinase III from pomegranate seeds (Punica granatum Linn.) is located in amyloplasts.

    PubMed

    Lv, Chenyan; Masuda, Taro; Yang, Haixia; Sun, Lei; Zhao, Guanghua

    2011-12-01

    We have recently identified a new class III chitinase from pomegranate seeds (PSC). Interestingly, this new chitinase naturally binds calcium ions with high capacity and low affinity, suggesting that PSC is a Ca-storage protein. Analysis of the amino acid sequence showed that this enzyme is rich in acidic amino acid residues, especially Asp, which are responsible for calcium binding. Different from other known chitinases, PSC is located in the stroma of amyloplasts in pomegranate seeds. Transmission electron microscopy (TEM) analysis indicated that the embryonic cells of pomegranate seeds are rich in calcium ions, most of which are distributed in the stroma and the starch granule of the amyloplasts, consistent with the above idea that PSC is involved in calcium storage, a newly non-defensive function.

  12. Electrostatic contributions to heat capacity changes of DNA-ligand binding.

    PubMed Central

    Gallagher, K; Sharp, K

    1998-01-01

    Significant heat capacity changes (DeltaCp) often accompany protein unfolding, protein binding, and specific DNA-ligand binding reactions. Such changes are widely used to analyze contributions arising from hydrophobic and polar hydration. Current models relate the magnitude of DeltaCp to the solvent accessible surface area (ASA) of the molecule. However, for many binding systems-particularly those involving non-peptide ligands-these models predict a DeltaCp that is significantly different from the experimentally measured value. Electrostatic interactions provide a potential source of heat capacity changes and do not scale with ASA. Using finite-difference Poisson-Boltzmann methods (FDPB), we have determined the contribution of electrostatics to the DeltaCp associated with binding for DNA binding reactions involving the ligands DAPI, netropsin, lexitropsin, and the lambda repressor binding domain. PMID:9675178

  13. Current Understanding of the Binding Sites, Capacity, Affinity, and Biological Significance of Metals in Melanin

    PubMed Central

    Hong, Lian; Simon, John D.

    2008-01-01

    Metal chelation is often invoked as one of the main biological functions of melanin. In order to understand the interaction between metals and melanin, extensive studies have been carried out to determine the nature of the metal binding sites, binding capacity and affinity. These data are central to efforts aimed at elucidating the role metal binding plays in determining the physical, structural, biological, and photochemical properties of melanin. This article examines the current state of understanding of this field. PMID:17580858

  14. Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

    SciTech Connect

    Gangi Setty, Thanuja; Cho, Christine; Govindappa, Sowmya; Apicella, Michael A.; Ramaswamy, S.

    2014-07-01

    Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.

  15. Dynamic Buffer Capacity in Acid-Base Systems.

    PubMed

    Michałowska-Kaczmarczyk, Anna M; Michałowski, Tadeusz

    The generalized concept of 'dynamic' buffer capacity βV is related to electrolytic systems of different complexity where acid-base equilibria are involved. The resulting formulas are presented in a uniform and consistent form. The detailed calculations are related to two Britton-Robinson buffers, taken as examples.

  16. Cellular Retinoic Acid Binding Protein and Breast Cancer

    DTIC Science & Technology

    2006-05-01

    fatty acid probe anilinonaphtalene-8- sulphonic acid (ANS) was measured. ANS readily associates with various FABPs and its fluorescence is highly...DAMD17-03-1-0249 TITLE: Cellular Retinoic Acid Binding Protein and Breast Cancer PRINCIPAL INVESTIGATOR: Leslie J. (Willmert) Donato...DATES COVERED (From - To) 14 Apr 03 – 13 Apr 06 5a. CONTRACT NUMBER Cellular Retinoic Acid Binding Protein and Breast Cancer 5b. GRANT NUMBER

  17. Intracellular binding of the anti-inflammatory drug niflumic acid in the liver.

    PubMed

    Kelmer-Bracht, A M; Ishii-Iwamoto, E L; Bracht, A

    1995-09-01

    Intracellular binding of niflumic acid in the perfused rat liver was analyzed according to the model of Scatchard. The data for the binding isotherm were obtained from previously published indicator dilution experiments. The intracellular bound niflumic acid was calculated as the difference between total concentration and the concentration of the free form. The intracellular concentration of the free form was inferred from the concentration of the free form in the extracellular space under the assumption of equilibrative distribution. A Scatchard model with two classes of binding sites fits very well to the experimental curve. The high affinity class has a dissociation constant of 26.10 +/- 0.69 microM and a maximal binding capacity of 2.21 +/- 0.03 micromol (ml intracellular space)(-1); the low affinity class has a dissociation constant of 721.90 +/- 229.0 microM and a maximal binding capacity of 5.96 +/- 0.67 micromol (ml intracellular space)(-1). Probably, under in vivo conditions, the binding capacity in the cellular space exceeds that of the extracellular space. This phenomenon explains, partly at least, the high intracellular concentrations of niflumic acid found under in vivo conditions.

  18. [Acidity and acid buffering capacity of aerosols during sand-dust storm weather in Beijing].

    PubMed

    Wang, W; Wang, Y; Su, H; Pan, Z; Yue, X; Liu, H; Tang, D

    2001-09-01

    In the spring of 2000, there were 12 sand-dust storms in Beijing. 2 sand-dust storms were experienced in time and mass concentrations, elementary concentrations acidity and acidic buffering capacity of TSP(Total Suspended Particulate, < 100 microns) and PM10(Inhalable particulate, < 10 microns) were sampled and analyzed. Results showed that pollution level of aerosols was extremely high. However, the acidity of aerosols was relatively low and the aerosols had very strong acid buffering capacity for acidification. Therefore, the aerosols brought about by the sand-dust storms could avoid the occurrence of acidic precipitation to some extent.

  19. [Kinetics of ligand binding to nucleic acids at random fillings].

    PubMed

    Arakelian, V B; Babaian, S Iu; Tairian, V I; Arakelian, A V; Parsadanian, M A; Vardevanian, P O

    2006-01-01

    Ligand binding with nucleic acids is described in frames of the theory of random processes. It is shown that the probabilistic description of binding of a ligand to nucleic acid allows one to describe not only the kinetics of changes in the number of bound ligands at arbitrary fillings but also to calculate stationary values of the number of bound ligands and its dispersion. A general analysis of absorption isotherms and the kinetics of ligand binding with nucleic acids allows one to determine the rate constants of formation and decomposition of the ligand-nucleic acid complex. A comparison of the results obtained with the case of low fillings is conducted.

  20. High-capacity composite adsorbents for nucleic acids.

    PubMed

    Tiainen, Peter; Rokebul Anower, M; Larsson, Per-Olof

    2011-08-05

    Cytopore™ is a bead-shaped, macroporous and easily compressible cellulose-based anion-exchange material intended for cultivation of anchor-dependent animal cells. Reticulated vitreous carbon (RVC) is a strong, non-compressible, high voidage (97%) matrix material that can be cut to desired geometrical shapes. Cytopore and RVC were combined to cylindrical composites (25 mm × 10 mm) fitted inside chromatography columns. The composite combined the advantageous properties of both its constituents, making it suitable for column chromatography. The composite could withstand very high flow rates without compaction of the bed (>25 column volumes/min; 4000 cm h(-1)). Chromatography runs with tracers showed a low HETP value (0.3mm), suggesting that pore flow was in operation. The dynamic binding capacities (10% breakthrough) per gram of dry weight Cytopore were determined for several compounds including DNA and RNA and were found to be 240-370 mg/g. The composite was used to isolate pUC 18-type plasmids from a cleared alkaline lysate in a good yield. Confocal microscopy studies showed that plasmids were bound not only to the surface of the Cytopore material but also within the matrix walls, thus offering an explanation to the very high binding capacities observed. The concept of using a composite prepared from a mechanically weak, high-binding material and a strong scaffold material may be applied to other systems as well.

  1. Nucleic acid-binding specificity of human FUS protein

    PubMed Central

    Wang, Xueyin; Schwartz, Jacob C.; Cech, Thomas R.

    2015-01-01

    FUS, a nuclear RNA-binding protein, plays multiple roles in RNA processing. Five specific FUS-binding RNA sequence/structure motifs have been proposed, but their affinities for FUS have not been directly compared. Here we find that human FUS binds all these sequences with Kdapp values spanning a 10-fold range. Furthermore, some RNAs that do not contain any of these motifs bind FUS with similar affinity. FUS binds RNA in a length-dependent manner, consistent with a substantial non-specific component to binding. Finally, investigation of FUS binding to different nucleic acids shows that it binds single-stranded DNA with three-fold lower affinity than ssRNA of the same length and sequence, while binding to double-stranded nucleic acids is weaker. We conclude that FUS has quite general nucleic acid-binding activity, with the various proposed RNA motifs being neither necessary for FUS binding nor sufficient to explain its diverse binding partners. PMID:26150427

  2. Improving the binding capacities of protein A chromatographic materials by means of ligand polymerization.

    PubMed

    Freiherr von Roman, Matthias; Berensmeier, Sonja

    2014-06-20

    Protein A chromatography is one of the most important techniques used in the purification of monoclonal antibodies. Due to the low dynamic binding capacity of protein A chromatographic materials compared to other stationary phases, protein A chromatography is often discussed to be the bottleneck among current purification processes. Several approaches were tested within this study in order to maximize IgG binding capacities of current acrylamido-based based resins. Genetic engineering techniques were used in order to polymerize one of the IgG binding domains (B-domain) of protein A from Staphylococcus aureus (SpA) to achieve ligands with an increased length. The solution-binding ratio and the total size of ligand-antibody complexes were used to characterize the interaction potential of novel ligands, revealing a relatively linear dependency between the number of binding domains upon the amount of bound antibody molecules. This relationship was also valid up to a ligand which was comprised of 8 B-domains after attaching them onto acrylamido-based based stationary phases using epoxy coupling techniques. Equilibrium binding capacities of more than 80mghIgGmL(-1) were achieved using the B8 ligand. Furthermore, static binding capacities, especially for smaller ligands comprised of fewer B-domains, were improved up to 87mghIgGmL(-1) using site-specific coupling chemistry, which is an improvement of more than 20% compared to commercially available materials. In order to evaluate pore exclusion effects due to the use of prolonged affinity ligands, prepared materials were characterized regarding their effective intraparticle porosity and breakthrough capacity.

  3. Serum iron, total iron binding capacity, plasma copper and hemoglobin types in anemic and poikilocytic calves.

    PubMed Central

    McGillivray, S R; Searcy, G P; Hirsch, V M

    1985-01-01

    Ninety-eight calves were studied to determine if anemia and poikilocytosis were related to iron or copper status or hemoglobin type. No significant differences were found in serum iron, total iron binding capacity, marrow iron, plasma copper or hemoglobin type between affected and normal calves. Poikilocytes were strongly inversely correlated (-0.9177) with age. Calves less than six weeks of age had more poikilocytes, lower serum iron, higher total iron binding capacity, less adult hemoglobin and more neonatal and fetal hemoglobin than calves greater than six weeks of age. Images Fig. 1. PMID:2412677

  4. Characterization of the sources of protein-ligand affinity: 1-sulfonato-8-(1')anilinonaphthalene binding to intestinal fatty acid binding protein.

    PubMed Central

    Kirk, W R; Kurian, E; Prendergast, F G

    1996-01-01

    1-Sulfonato-8-(1')anilinonaphthalene (1,8-ANS) was employed as a fluorescent probe of the fatty acid binding site of recombinant rat intestinal fatty acid binding protein (1-FABP). The enhancement of fluorescence upon binding allowed direct determination of binding affinity by fluorescence titration experiments, and measurement of the effects on that affinity of temperature, pH, and ionic strength. Solvent isotope effects were also determined. These data were compared to results from isothermal titration calorimetry. We obtained values for the enthalpy and entropy of this interaction at a variety of temperatures, and hence determined the change in heat capacity of the system consequent upon binding. The ANS-1-FABP is enthalpically driven; above approximately 14 degrees C it is entropically opposed, but below this temperature the entropy makes a positive contribution to the binding. The changes we observe in both enthalpy and entropy of binding with temperature can be derived from the change in heat capacity upon binding by integration, which demonstrates the internal consistency of our results. Bound ANS is displaced by fatty acids and can itself displace fatty acids bound to I-FABP. The binding site for ANS appears to be inside the solvent-containing cavity observed in the x-ray crystal structure, the same cavity occupied by fatty acid. From the fluorescence spectrum and from an inversion of the Debye-Hueckel formula for the activity coefficients as a function of added salt, we inferred that this cavity is fairly polar in character, which is in keeping with inferences drawn from the x-ray structure. The binding affinity of ANS is considered to be a consequence of both electrostatic and conditional hydrophobic effects. We speculate that the observed change in heat capacity is produced mainly by the displacement of strongly hydrogen-bonded waters from the protein cavity. PMID:8770188

  5. Soluble Fiber with High Water-Binding Capacity, Swelling Capacity, and Fermentability Reduces Food Intake by Promoting Satiety Rather Than Satiation in Rats.

    PubMed

    Tan, Chengquan; Wei, Hongkui; Zhao, Xichen; Xu, Chuanhui; Zhou, Yuanfei; Peng, Jian

    2016-10-02

    To understand whether soluble fiber (SF) with high water-binding capacity (WBC), swelling capacity (SC) and fermentability reduces food intake and whether it does so by promoting satiety or satiation or both, we investigated the effects of different SFs with these properties on the food intake in rats. Thirty-two male Sprague-Dawley rats were randomized to four equal groups and fed the control diet or diet containing 2% konjac flour (KF), pregelatinized waxy maize starch (PWMS) plus guar gum (PG), and PWMS starch plus xanthan gum (PX) for three weeks, with the measured values of SF, WBC, and SC in the four diets following the order of PG > KF > PX > control. Food intake, body weight, meal pattern, behavioral satiety sequence, and short-chain fatty acids (SCFAs) in cecal content were evaluated. KF and PG groups reduced the food intake, mainly due to the decreased feeding behavior and increased satiety, as indicated by decreased meal numbers and increased inter-meal intervals. Additionally, KF and PG groups increased concentrations of acetate acid, propionate acid, and SCFAs in the cecal contents. Our results indicate that SF with high WBC, SC, and fermentability reduces food intake-probably by promoting a feeling of satiety in rats to decrease their feeding behavior.

  6. Soluble Fiber with High Water-Binding Capacity, Swelling Capacity, and Fermentability Reduces Food Intake by Promoting Satiety Rather Than Satiation in Rats

    PubMed Central

    Tan, Chengquan; Wei, Hongkui; Zhao, Xichen; Xu, Chuanhui; Zhou, Yuanfei; Peng, Jian

    2016-01-01

    To understand whether soluble fiber (SF) with high water-binding capacity (WBC), swelling capacity (SC) and fermentability reduces food intake and whether it does so by promoting satiety or satiation or both, we investigated the effects of different SFs with these properties on the food intake in rats. Thirty-two male Sprague-Dawley rats were randomized to four equal groups and fed the control diet or diet containing 2% konjac flour (KF), pregelatinized waxy maize starch (PWMS) plus guar gum (PG), and PWMS starch plus xanthan gum (PX) for three weeks, with the measured values of SF, WBC, and SC in the four diets following the order of PG > KF > PX > control. Food intake, body weight, meal pattern, behavioral satiety sequence, and short-chain fatty acids (SCFAs) in cecal content were evaluated. KF and PG groups reduced the food intake, mainly due to the decreased feeding behavior and increased satiety, as indicated by decreased meal numbers and increased inter-meal intervals. Additionally, KF and PG groups increased concentrations of acetate acid, propionate acid, and SCFAs in the cecal contents. Our results indicate that SF with high WBC, SC, and fermentability reduces food intake—probably by promoting a feeling of satiety in rats to decrease their feeding behavior. PMID:27706095

  7. Tropical tannin-rich fodder intake modifies saliva-binding capacity in growing sheep.

    PubMed

    Vargas-Magaña, J J; Aguilar-Caballero, A J; Torres-Acosta, J F J; Sandoval-Castro, C A; Hoste, H; Capetillo-Leal, C M

    2013-12-01

    We evaluated the effect of feeding dietary tannins from Lysiloma latisiliquum fresh forage on the saliva tannin-binding capacity of hair sheep lambs without previous exposure to tannin-rich (TR) fodder. Twenty-four hair sheep lambs (13.6±3.04 kg LW) were fed a tannin-free diet at the beginning of the experimental period (from day 10 to 13). On day 14, lambs were distributed into three groups (n=8): control group (CG), fed with the tannin-free diet (from D10 to D112); tannin short-term group (TST), fed the basal diet and 650 g of L. latisiliquum forage (from D14 to D55); tannin long-term group (TLT), fed the basal diet and 650 g of L. latisiliquum forage (from D14 to D112). Saliva samples were collected from the mouth of each lamb in the morning before feeding time on D10 and D14 (baseline period), on D49 and D56 (period 1) and on D97 and D112 (period 2). The tannin binding response of salivary protein (∆% turbidity) was determined with the haze development test (HDT) using either tannic acid or L. latisiliquum forage acetone extract. A turbidity protein index (TPI) was calculated as (∆% turbidity/[salivary protein (mg)]). Differences in HDT and TPI in the different groups were compared by repeated measures ANOVA using Proc Mixed. All groups had similar ∆% turbidity throughout the experiment (P>0.05). At baseline and period 1, the TPI of the different groups was similar (P>0.05). On period 2 the TLT group showed higher TPI compared with CG (P<0.05). Meanwhile, CG and TST showed similar salivary TPI. The saliva of hair sheep lambs consuming TR L. latisiliquum fresh fodder (TLT group) increased their TPI compared with control lambs not exposed to tannins.

  8. Capture and release of acid-gasses with acid-gas binding organic compounds

    DOEpatents

    Heldebrant, David J; Yonker, Clement R; Koech, Phillip K

    2015-03-17

    A system and method for acid-gas capture wherein organic acid-gas capture materials form hetero-atom analogs of alkyl-carbonate when contacted with an acid gas. These organic-acid gas capture materials include combinations of a weak acid and a base, or zwitterionic liquids. This invention allows for reversible acid-gas binding to these organic binding materials thus allowing for the capture and release of one or more acid gases. These acid-gas binding organic compounds can be regenerated to release the captured acid gasses and enable these organic acid-gas binding materials to be reused. This enables transport of the liquid capture compounds and the release of the acid gases from the organic liquid with significant energy savings compared to current aqueous systems.

  9. Oil binding capacities of triacylglycerol crystalline nanoplatelets: nanoscale models of tristearin solids in liquid triolein.

    PubMed

    Razul, M Shajahan G; MacDougall, Colin J; Hanna, Charles B; Marangoni, Alejandro G; Peyronel, Fernanda; Papp-Szabo, Erzsebet; Pink, David A

    2014-10-01

    Polycrystalline particles composed of triacylglycerol (TAG) molecules, and their networks, in anhydrous TAG oils find extensive use as edible oils in the food industry. Although modelling studies of TAG systems, have been carried out, none have attempted to address a problem of central concern to food science and technology: the "oil binding capacity" of a system of such edible oils. Crystalline nanoparticles (CNPs) have recently been identified as the fundamental components of solid fats in oils. Oil binding capacity is an important concept regarding the ability of fats particles to retain oil, and the ability of these CNPs to bind oil is important in designing healthy foods. We have carried out atomic scale molecular dynamics computer simulations to understand the behavior of a triacylglycerol oil (triolein) in nanoscale confinements between tristearin CNPs. We define a nanoscale oil binding capacity function by utilizing the average oil number density, 〈Φ(d)〉, between two CNPs as a function of their separation, d. We modelled pure tristearin CNPs as well as tristearin CNPs in which the surfaces are covered with an interface comprising soft permanent coatings. Their surfaces are "hard" and "soft" respectively. We found that for a pair of hard-surface tristearin CNPs a distance d apart, (i) triolein exhibits number density, and therefore density, oscillations as a function of d, and (ii) the average number density between two such CNPs decreases as d decreases, viz. the oil binding capacity is lowered. When a soft layer of oil covers the CNP surfaces, we found that the oscillations are smeared out and that the average number density between the two CNPs remained approximately constant as d decreased indicating a high oil binding capacity. Our results might have identified important nanoscale aspects to aid in healthy food design.

  10. Decreased angiotensin II binding affinity and binding capacity in the anterior pituitary gland of adult spontaneously hypertensive rats

    SciTech Connect

    Gutkind, J.S.; Castren, E.; Saavedra, J.M.

    1988-01-01

    Angiotensin II (ANG) binding sites were quantified in single pituitary glands from 4-week-old and 14-week-old male spontaneously hypertensive rats (SHR) and age-matched male normotensive Wistar-Kyoto (WKY) control rats after incubation with /sup 125/I-(Sar/sup 1/)-ANG, autoradiography with computerized densitometry, and comparison to /sup 125/I-standards. The maximum binding capacity (B/sub max/) decreased while the dissociation constant (K/sub d/) for ANG increased in 14-week-old SHR when compared to age-matched WKY control rats. Conversely, no difference between rat strains was found in 4-week-old animals. Our results suggest that pituitary ANG binding sites may play a role in the pathophysiology of established genetic hypertension.

  11. Antioxidant capacity and phenolic acids of virgin coconut oil.

    PubMed

    Marina, A M; Man, Y B Che; Nazimah, S A H; Amin, I

    2009-01-01

    The antioxidant properties of virgin coconut oil produced through chilling and fermentation were investigated and compared with refined, bleached and deodorized coconut oil. Virgin coconut oil showed better antioxidant capacity than refined, bleached and deodorized coconut oil. The virgin coconut oil produced through the fermentation method had the strongest scavenging effect on 1,1-diphenyl-2-picrylhydrazyl and the highest antioxidant activity based on the beta-carotene-linoleate bleaching method. However, virgin coconut oil obtained through the chilling method had the highest reducing power. The major phenolic acids detected were ferulic acid and p-coumaric acid. Very high correlations were found between the total phenolic content and scavenging activity (r=0.91), and between the total phenolic content and reducing power (r=0.96). There was also a high correlation between total phenolic acids and beta-carotene bleaching activity. The study indicated that the contribution of antioxidant capacity in virgin coconut oil could be due to phenolic compounds.

  12. Premature capacity-loss mechanisms in lead/acid batteries

    NASA Astrophysics Data System (ADS)

    Hollenkamp, A. F.; Constanti, K. K.; Huey, A. M.; Koop, M. J.; Aputeanu, L.

    The phenomenon known as 'premature capacity loss' (PCL) causes the early demise of lead/acid batteries based on a variety of grid alloys. It is also known to be a problem specific to the positive plate and is usually invoked by duties that involve repetitive deep-discharge cycling. In order to determine the cause(s) of the problem, an extensive study of the behaviour of cells based on a range of positive grid alloys is being conducted. Examples of PCL have been generated by subjecting three-plate cells to 100% depth-of-discharge, at I = C8/8, with 110% overcharge. Cells based on antimony-free grids exhibit capacity loss at a rate of up to 5% of the initial capacity per cycle, with both constant-current and constant-voltage charging. With the latter charging method, most of the cells also develop extremely poor charge acceptance within 10 to 15 cycles. The performance of cells with high-antimony positive grids is significantly better, although substantial capacity loss is still observed. The latter cannot be explained by any of the classic failure modes for lead/acid batteries. Poor charge acceptance is not displayed by these cells. Plates show signs of physical degradation, but these represent a minor contribution to capacity loss. Phase composition of positive material does not vary with grid alloy and is typical of healthy plates. Investigations of corrosion-layer morphology have shown that Pb-Ca grids give rise to weak corrosion products that are prone to fracture and separation, while the corrosion layers on Pb-Sb plates are apparently more coherent and more strongly bonded to the underlying grid.

  13. Quantitative evaluation of E. coli F4 and Salmonella Typhimurium binding capacity of yeast derivatives.

    PubMed

    Ganner, Anja; Stoiber, Christian; Uhlik, Jakob Tizian; Dohnal, Ilse; Schatzmayr, Gerd

    2013-10-22

    The target of the present study was to quantify the capacity of different commercially available yeast derivatives to bind E. coli F4 and Salmonella Typhimurium. In addition, a correlation analysis was performed for the obtained binding numbers and the mannan-, glucan- and protein contents of the products, respectively. In a subsequent experiment, different yeast strains were fermented and treated by autolysis or French press to obtain a concentrated yeast cell wall. The capacity of yeast cell wall products to bind E. coli F4 and Salmonella Typhimurium was assessed with a quantitative microbiological microplate-based assay by measuring the optical density (OD) as the growth parameter of adhering bacteria. Total mannan and glucan were determined by HPLC using an isocratic method and a Refractive Index (RI) Detector. Total protein was determined by Total Kjeldahl Nitrogen (TKN). Statistical analyses were performed with IBM SPSS V19 using Spearman correlation and Mann Whitney U Test.Different yeast derivatives show different binding numbers, which indicate differences in product quality.Interestingly, the binding numbers for Salmonella Typhimurium are consistently higher (between one and two orders of magnitude) than for E. coli F4.We could demonstrate some statistical significant correlations between the mannan- and glucan content of different yeast derivatives and pathogen binding numbers; however, for the different yeast strains fermented under standardized laboratory conditions, no statistically significant correlations between the mannan- and glucan content and the binding numbers for E. coli and Salmonella Typhimurium were found.Interestingly, we could demonstrate that the yeast autolysis had a statistically significant difference on E. coli binding in contrast to the French press treatment. Salmonella binding was independent of these two treatments.As such, we could not give a clear statement about the binding factors involved. We propose that many more factors

  14. Binding of actin to thioglycolic acid modified superparamagnetic nanoparticles for antibody conjugation.

    PubMed

    Maltas, Esra; Ertekin, Betul

    2015-01-01

    Thioglycolic acid modified superparamagnetic iron oxide nanoparticles (TG-APTS-SPION) were synthesized by using (3-aminopropyl) triethoxysilane (APTS) and thioglycolic acid (TG). Actin was immobilized on the nanoparticle surfaces. Binding amount of the actin (Act) on TG-APTS-SPIONs was determined by using a calibration curve equation that was drawn using fluorescence spectra at 280 and 342 nm of excitation and emission wavelengths. Anti-Actin (anti-Act) was interacted with the actin immobilized TG-APTS-SPIONs as primary antibody. Horse radish peroxidase (HRP) was also interacted with antibody conjugated nanoparticles as secondary antibody. The binding capacity of primary and secondary antibodies was also estimated by fluorescence spectroscopy. Scanning electron microscopy (SEM), Infrared spectroscopy (FTIR) and energy dispersive X-ray (EDX) analysis were also clarified binding of the protein and antibodies to the nanoparticles' surfaces. Western blot analysis was also done for actin conjunction with anti Act antibody to confirm binding of the antibody to the protein.

  15. Heat Capacity of Poly(L-lactic acid)

    NASA Astrophysics Data System (ADS)

    Pyda, Marek; Bopp, C. Bopp Richard C.; Richard, C.; Wunderlich, Bernhard

    2002-03-01

    The heat capacity of poly(L-lactic acid) (PLA) is reported from 5-520 K from standard differential scanning calorimetry (DSC), temperature modulated DSC (TMDSC), and adiabatic calorimetry. The semicrystalline PLA has a melting endotherm between 418 and 432 K, with variable heats of fusion, depending on thermal history. The thermodynamic heat of fusion is 6.15 kJ/mol. The heat capacity is linked to its group vibrational spectrum and the skeletal vibrations described by the Tarasov equation (theta parameters 574 and 52 K, N = 9). Calculated and experimental heat capacities agree to ±3compares within ±0.5contributions of other polymers with the same constituent groups. The glass transition temperature of liquid PLA is at 333 K with a change in heat capacity of about 41 J/(K mol). With these results, the enthalpy, entropy, and Gibbs function were obtained. For semicrystalline samples one can then discuss the crystallinity changes with temperature, the question of a rigid-amorphous fraction, and the reversible melting. --- Supported by NSF, Polymers Program, DMR-9703692, and the Div. of Mat. Sci., BES, DOE at ORNL, managed by UT-Batelle, LLC, for the U.S. Department of Energy, under contract number DOE-AC05-00OR22725.

  16. Characterization of a domoic acid binding site from Pacific razor clam.

    PubMed

    Trainer, Vera L; Bill, Brian D

    2004-08-10

    The Pacific razor clam, Siliqua patula, is known to retain domoic acid, a water-soluble glutamate receptor agonist produced by diatoms of the genus Pseudo-nitzschia. The mechanism by which razor clams tolerate high levels of the toxin, domoic acid, in their tissues while still retaining normal nerve function is unknown. In our study, a domoic acid binding site was solubilized from razor clam siphon using a combination of Triton X-100 and digitonin. In a Scatchard analysis using [3H]kainic acid, the partially-purified membrane showed two distinct receptor sites, a high affinity, low capacity site with a KD (mean +/- S.E.) of 28 +/- 9.4 nM and a maximal binding capacity of 12 +/- 3.8 pmol/mg protein and a low affinity, high capacity site with a mM affinity for radiolabeled kainic acid, the latter site which was lost upon solubilization. Competition experiments showed that the rank order potency for competitive ligands in displacing [3H]kainate binding from the membrane-bound receptors was quisqualate > ibotenate > iodowillardiine = AMPA = fluorowillardiine > domoate > kainate > L-glutamate. At high micromolar concentrations, NBQX, NMDA and ATPA showed little or no ability to displace [3H]kainate. In contrast, Scatchard analysis using [3H]glutamate showed linearity, indicating the presence of a single binding site with a KD and Bmax of 500 +/- 50 nM and 14 +/- 0.8 pmol/mg protein, respectively. These results suggest that razor clam siphon contains both a high and low affinity receptor site for kainic acid and may contain more than one subtype of glutamate receptor, thereby allowing the clam to function normally in a marine environment that often contains high concentrations of domoic acid.

  17. Specific binding of /sup 125/I-labeled human chorionic gonadotropin to gonadal tissue: comparison of limited-point saturation analyses to Scatchard analyses for determining binding capacities and factors affecting estimates of binding capacity

    SciTech Connect

    Spicer, L.J.; Ireland, J.J.

    1986-07-01

    Experiments were conducted to compare gonadotropin binding capacity calculated from limited-point saturation analyses to those obtained from Scatchard analyses, and to test the effects of membrane purity and source of gonadotropin receptors on determining the maximum percentage of radioiodinated hormone bound to receptors (maximum bindability). One- to four-point saturation analyses gave results comparable to results by Scatchard analyses when examining relative binding capacities of receptors. Crude testicular homogenates had lower estimates of maximum bindability of /sup 125/I-labeled human chorionic gonadotropin than more purified gonadotropin receptor preparations. Under similar preparation techniques, some gonadotropin receptor sources exhibited low maximum bindability.

  18. Apo B100 similarities to viral proteins suggest basis for LDL-DNA binding and transfection capacity.

    PubMed

    Guevara, Juan; Prashad, Nagindra; Ermolinsky, Boris; Gaubatz, John W; Kang, Dongcheul; Schwarzbach, Andrea E; Loose, David S; Guevara, Natalia Valentinova

    2010-07-01

    LDL mediates transfection with plasmid DNA in a variety of cell types in vitro and in several tissues in vivo in the rat. The transfection capacity of LDL is based on apo B100, as arginine/lysine clusters, suggestive of nucleic acid-binding domains and nuclear localization signal sequences, are present throughout the molecule. Apo E may also contribute to this capacity because of its similarity to the Dengue virus capsid proteins and its ability to bind DNA. Synthetic peptides representing two apo B100 regions with prominent Arg/Lys clusters were shown to bind DNA. Region 1 (0014Lys-Ser0160) shares sequence motifs present in DNA binding domains of Interferon Regulatory Factors and Flaviviridae capsid/core proteins. It also contains a close analog of the B/E receptor ligand of apo E. Region 1 peptides, B1-1 (0014Lys-Glu0054) and B1-2 (0055Leu-Ala0096), mediate transfection of HeLa cells but are cytotoxic. Region 2 (3313Asp-Thr3431), containing the known B/E receptor ligand, shares analog motifs with the human herpesvirus 5 immediate-early transcriptional regulator (UL122) and Flaviviridae NS3 helicases. Region 2 peptides, B2-1 (3313Asp-Glu3355), and B2-2 (3356Gly-Thr3431) are ineffective in cell transfection and are noncytotoxic. These results confirm the role of LDL as a natural transfection vector in vivo, a capacity imparted by the apo B100, and suggest a basis for Flaviviridae cell entry.

  19. Nucleic acids encoding a cellulose binding domain

    DOEpatents

    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.

  20. Nucleic acids encoding a cellulose binding domain

    DOEpatents

    Shoseyov, O.; Shpiegl, I.; Goldstein, M.A.; Doi, R.H.

    1996-03-05

    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. 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. 15 figs.

  1. Sedimentation properties in density gradients correspond with levels of sperm DNA fragmentation, chromatin compaction and binding affinity to hyaluronic acid.

    PubMed

    Torabi, Forough; Binduraihem, Adel; Miller, David

    2017-03-01

    Mature spermatozoa bind hyaluronic acid in the extracellular matrix via hyaladherins. Immature spermatozoa may be unable to interact because they do not express the appropriate hyaladherins on their surface. Fresh human semen samples were fractionated using differential density gradient centrifugation (DDGC) and the ability of these fractions to bind hyaluronic acid was evaluated. The presence of sperm hyaladherins was also assessed. CD44 was located mainly on the acrosome and equatorial segment and became more restricted to the equatorial segment in capacitated spermatozoa. Hyaluronic acid-TRITC (hyaluronic acid conjugated with tetramethylrhodamine isothiocyanante), a generic hyaluronic-acid-binding reagent, labelled the membrane and the neck region, particularly after capacitation. Sperm populations obtained after DDGC or after interaction with hyaluronic acid were assessed for DNA fragmentation and chromatin maturity. Strong relationships between both measures and sperm sedimentation and hyaluronic-acid-binding profiles were revealed. Capacitation enhanced hyaluronic acid binding of both DDGC-pelleted sperm and sperm washed free of seminal fluid. In conclusion, hyaladherins were detected on human sperm and a higher capacity for sperm hyaluronic-acid-binding was shown to correspond with their DDGC sedimentation profiles and with lower levels of DNA fragmentation and better chromatin maturity. Capacitation induced changes in the distribution and presence of hyaladherins may enhance hyaluronic-acid-binding.

  2. Specific erythrocyte binding capacity and biological activity of Plasmodium falciparum erythrocyte binding ligand 1 (EBL-1)-derived peptides

    PubMed Central

    Curtidor, Hernando; Rodríguez, Luis E.; Ocampo, Marisol; López, Ramses; García, Javier E.; Valbuena, John; Vera, Ricardo; Puentes, Álvaro; Vanegas, Magnolia; Patarroyo, Manuel E.

    2005-01-01

    Erythrocyte binding ligand 1 (EBL-1) is a member of the ebl multigene family involved in Plasmodium falciparum invasion of erythrocytes. We found that five EBL-1 high-activity binding peptides (HABPs) bound specifically to erythrocytes: 29895 (41HKKKSGELNNNKSGILRSTY60), 29903 (201LYECGK-KIKEMKWICTDNQF220), 29923 (601CNAILGSYADIGDIVRGLDV620), 29924(621WRDINTNKLSEK-FQKIFMGGY640), and 30018 (2481LEDIINLSKKKKKSINDTSFY2500). We also show that binding was saturable, not sialic acid-dependent, and that all peptides specifically bound to a 36-kDa protein on the erythrocyte membrane. The five HABPs inhibited in vitro merozoite invasion depending on the peptide concentration used, suggesting their possible role in the invasion process. PMID:15659376

  3. Novel oral phosphate binder with nanocrystalline maghemite-phosphate binding capacity and pH effect.

    PubMed

    Nguyen, T M-H; Müller, R H; Taupitz, M; Schnorr, J; Hamm, B; Wagner, S

    2015-03-30

    Hyperphosphatemia is one of the main risk factors contributing to morbidity and mortality in patients with end stage renal disease. The demand for a new phosphate binder is continuously increasing since the number of patients suffering under hyperphosphatemia is growing. However, side effects and high pill burden of currently available phosphate binders are the main reasons for low compliance and uncontrolled serum phosphate levels. Therefore, the aim of this study was to develop a novel phosphate binder with a high phosphate binding capacity over the entire gastrointestinal (GI) pH range. This novel phosphate binder C-PAM-10 is based on d-mannose coated nanocrystalline maghemite and belongs to the new class of phosphate binders, called the "iron based agents". It was possible to obtain a phosphate binding product that showed very high phosphate binding capacities with the characteristic of being pH independent at relevant pH ranges. The simulation of a GI passage ranging from pH 1.2 to pH 7.5 showed a 2.5 times higher phosphate binding capacity compared to the commonly used phosphate binder sevelamer carbonate. The simulation of a pH sensitive coating that releases the iron based phosphate binder at pH values ≥4.5 still showed a very high phosphate binding capacity combined with very low iron release which might decrease iron related side effects in vivo. Therefore, C-PAM-10 and its variations may be very promising candidates as a superior phosphate binder.

  4. Steam Cooking Significantly Improves In Vitro Bile Acid Binding of Collard Greens, Kale, Mustard Greens, Broccoli, Green Bell Pepper and Cabbage

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bile acid binding capacity has been related to the cholesterol-lowering potential of foods and food fractions. Lowering recirculating bile acids results in utilization of cholesterol to synthesize bile acid and reduced fat absorption. Secondary bile acids have been associated with increasing the r...

  5. Heat Capacity Changes for Transition-State Analogue Binding and Catalysis with Human 5'-Methylthioadenosine Phosphorylase.

    PubMed

    Firestone, Ross S; Cameron, Scott A; Karp, Jerome M; Arcus, Vickery L; Schramm, Vern L

    2017-02-17

    Human 5'-methylthioadenosine phosphorylase (MTAP) catalyzes the phosphorolysis of 5'-methylthioadenosine (MTA). Its action regulates cellular MTA and links polyamine synthesis to S-adenosylmethionine (AdoMet) salvage. Transition state analogues with picomolar dissociation constants bind to MTAP in an entropically driven process at physiological temperatures, suggesting increased hydrophobic character or dynamic structure for the complexes. Inhibitor binding exhibits a negative heat capacity change (-ΔCp), and thus the changes in enthalpy and entropy upon binding are strongly temperature-dependent. The ΔCp of inhibitor binding by isothermal titration calorimetry does not follow conventional trends and is contrary to that expected from the hydrophobic effect. Thus, ligands of increasing hydrophobicity bind with increasing values of ΔCp. Crystal structures of MTAP complexed to transition-state analogues MT-DADMe-ImmA, BT-DADMe-ImmA, PrT-ImmA, and a substrate analogue, MT-tubercidin, reveal similar active site contacts and overall protein structural parameters, despite large differences in ΔCp for binding. In addition, ΔCp values are not correlated with Kd values. Temperature dependence of presteady state kinetics revealed the chemical step for the MTAP reaction to have a negative heat capacity for transition state formation (-ΔCp(‡)). A comparison of the ΔCp(‡) for MTAP presteady state chemistry and ΔCp for inhibitor binding revealed those transition-state analogues most structurally and thermodynamically similar to the transition state. Molecular dynamics simulations of MTAP apoenzyme and complexes with MT-DADMe-ImmA and MT-tubercidin show small, but increased dynamic motion in the inhibited complexes. Variable temperature CD spectroscopy studies for MTAP-inhibitor complexes indicate remarkable protein thermal stability (to Tm = 99 °C) in complexes with transition-state analogues.

  6. Newborn jaundice technologies: unbound bilirubin and bilirubin binding capacity in neonates.

    PubMed

    Amin, Sanjiv B; Lamola, Angelo A

    2011-06-01

    Neonatal jaundice (hyperbilirubinemia), which is extremely common in neonates, can be associated with neurotoxicity. A safe level of bilirubin has not been defined in either premature or term infants. Emerging evidence suggest that the level of unbound (or "free") bilirubin has a better sensitivity and specificity than total serum bilirubin for bilirubin-induced neurotoxicity. Although recent studies suggest the usefulness of free bilirubin measurements in managing high-risk neonates, including premature infants, no widely available method exists to assay the serum free bilirubin concentration. To keep pace with the growing demand, in addition to reevaluation of old methods, several promising new methods are being developed for sensitive, accurate, and rapid measurement of free bilirubin and bilirubin binding capacity. These innovative methods need to be validated before adopting for clinical use. We provide an overview of some promising methods for free bilirubin and binding capacity measurements with the goal to enhance research in this area of active interest and apparent need.

  7. Serum ferritin and total iron-binding capacity to estimate iron storage in pigs.

    PubMed

    Smith, J E; Moore, K; Boyington, D; Pollmann, D S; Schoneweis, D

    1984-11-01

    The inability to accurately determine storage iron in baby pigs limits the development of new treatment programs. In pigs treated neonatally with iron dextran, serum ferritin had increased dramatically at ten days of age and then returned to near preinjection levels by 50 days of age. In contrast, serum ferritin in untreated pigs declined until they were offered creep feed at 21 days of age. When serum ferritin, serum iron, serum total iron-binding capacity, erythrocyte number, packed cell volume, and blood hemoglobin were measured in three-week-old pigs, serum ferritin combined with serum total iron-binding capacity correlated significantly with the total nonheme iron in the liver and spleen. The nonheme iron (in mg) could be predicted (r2 = 0.71) by the following expression: 8.7 + 0.6 (ferritin in ng/ml).

  8. Improved rapid methods for the determination of iron content and binding capacity of serum.

    PubMed

    BEALE, R N; BOSTROM, J O; TAYLOR, R F

    1962-03-01

    Improved methods are described for the determination of serum iron (SeFe) and latent iron-binding capacity under controlled conditions of pH. As before, no precipitation or heating of the serum is required, and controls are not used. Unbuffered sulphonated bathophenanthroline is employed for colour development. Statistical data for 40 normals are presented and comparisons with other data are given; the effects of ageing of serum are studied.

  9. Comparison and trend study on acidity and acidic buffering capacity of particulate matter in China

    NASA Astrophysics Data System (ADS)

    Ren, Lihong; Wang, Wei; Wang, Qingyue; Yang, XiaoYang; Tang, Dagang

    2011-12-01

    The acidity of about 2000 particulate matter samples from aircraft and ground-based monitoring is analyzed by the method similar to soil acidity determination. The ground-based samples were collected at about 50 urban or background sites in northern and southern China. Moreover, the acidic buffering capacity of those samples is also analyzed by the method of micro acid-base titration. Results indicate that the acidity level is lower in most northern areas than those in the south, and the acidic buffering capacity showed inverse tendency, correspondingly. This is the most important reason why the pollution of acidic-precipitation is much more serious in Southern China than that in Northern China. The acidity increases and the acidic buffering capacity drops with the decreasing of the particle sizes, indicating that fine particle is the main influencing factor of the acidification. The ionic results show that Ca salt is the main alkaline substance in particulate matter, whereas the acidification of particulate matter is due to the SO 2 and NO x emitted from the fossil fuel burning. And among of them, coal burning is the main contributor of SO 2, however the contribution of NO x that emitted from fuel burning of motor vehicles has increased in recent years. By comparison of the experimental results during the past 20 years, it can be concluded that the acid precipitation of particulate matter has not been well controlled, and it even shows an increasing tendency in China lately. The acid precipitation of particulate matter has begun to frequently attack in part of the northern areas. Multiple regression analysis indicates that coefficient value of the ions is the lowest at the urban sites and the highest at the regional sites, whereas the aircraft measurement results are intermediate between those two kinds of sites.

  10. Heavy metal binding capacity (HMBC) of municipal solid waste landfill leachates.

    PubMed

    Ward, Marnie L; Bitton, Gabriel; Townsend, Timothy

    2005-07-01

    This research describes the use of a toxicity assay for the identification of metal toxicity, bioavailability and heavy metal binding capacity (HMBC) of municipal solid waste (MSW) landfill leachates. MetPLATE, an assay specific for heavy metal toxicity, was used to determine the HMBC of MSW leachates collected from 14 sites in Florida, with a wide range of chemical and physical characteristics. The leachates displayed a low toxicity which was attributed to the site-specific parameters, including, high concentrations of both organic and inorganic ligands. The HMBC test was undertaken to measure the effect of these site-specific parameters on metal toxicity. The potential for MSW leachate to bind and, thus, detoxify heavy metals was investigated with copper, zinc, and mercury. The HMBC values obtained ranged from 3 to 115, 5 to 93 and 4 to 101 for HMBC-Cu+2, HMBC-Zn+2, and HMBC-Hg+2, respectively. Additionally, the high strength leachates displayed the highest binding capacities, although the landfills sampled represented a wide range of characteristics. For comparison, the HMBC values reported with local lake water, Lake Alice and Lake Beverly, and a wastewater treatment plant effluent were all below 3. A partial fractionation of MSW leachate samples from sites 1, 5 and 8, was conducted to further investigate the influence of selected site-specific physico-chemical parameters on metal binding. The fractionation revealed that the HMBC of the leachate samples was heavily influenced by the concentration of solids, organics and hardness.

  11. Binding sites of retinol and retinoic acid with serum albumins.

    PubMed

    Belatik, A; Hotchandani, S; Bariyanga, J; Tajmir-Riahi, H A

    2012-02-01

    Retinoids are effectively transported in the bloodstream via serum albumins. We report the complexation of bovine serum albumin (BSA) with retinol and retinoic acid at physiological conditions, using constant protein concentration and various retinoid contents. FTIR, CD and fluorescence spectroscopic methods and molecular modeling were used to analyze retinoid binding site, the binding constant and the effects of complexation on BSA stability and secondary structure. Structural analysis showed that retinoids bind BSA via hydrophilic and hydrophobic interactions with overall binding constants of K(Ret)(-BSA) = 5.3 (±0.8) × 10(6) M(-1) and K(Retac-BSA) = 2.3 (±0.4) × 10(6) M(-1). The number of bound retinoid molecules (n) was 1.20 (±0.2) for retinol and 1.8 (±0.3) for retinoic acid. Molecular modeling showed the participation of several amino acids in retinoid-BSA complexes stabilized by H-bonding network. The retinoid binding altered BSA conformation with a major reduction of α-helix from 61% (free BSA) to 36% (retinol-BSA) and 26% (retinoic acid-BSA) with an increase in turn and random coil structures indicating a partial protein unfolding. The results indicate that serum albumins are capable of transporting retinoids in vitro and in vivo.

  12. Novel Peptide with Specific Calcium-Binding Capacity from Schizochytrium sp. Protein Hydrolysates and Calcium Bioavailability in Caco-2 Cells

    PubMed Central

    Cai, Xixi; Lin, Jiaping; Wang, Shaoyun

    2016-01-01

    Peptide-calcium can probably be a suitable supplement to improve calcium absorption in the human body. In this study, a specific peptide Phe-Tyr (FY) with calcium-binding capacity was purified from Schizochytrium sp. protein hydrolysates through gel filtration chromatography and reversed phase HPLC. The calcium-binding capacity of FY reached 128.77 ± 2.57 μg/mg. Results of ultraviolet spectroscopy, fluorescence spectroscopy, and infrared spectroscopy showed that carboxyl groups, amino groups, and amido groups were the major chelating sites. FY-Ca exhibited excellent thermal stability and solubility, which were beneficial to be absorbed and transported in the basic intestinal tract of the human body. Moreover, the calcium bioavailability in Caco-2 cells showed that FY-Ca could enhance calcium uptake efficiency by more than three times when compared with CaCl2, and protect calcium ions against dietary inhibitors, such as tannic acid, oxalate, phytate, and Zn2+. Our findings further the progress of algae-based peptide-calcium, suggesting that FY-Ca has the potential to be developed as functionally nutraceutical additives. PMID:28036002

  13. A Specific Peptide with Calcium-Binding Capacity from Defatted Schizochytrium sp. Protein Hydrolysates and the Molecular Properties.

    PubMed

    Cai, Xixi; Yang, Qian; Lin, Jiaping; Fu, Nanyan; Wang, Shaoyun

    2017-03-29

    Marine microorganisms have been proposed as a new kind of protein source. Efforts are needed in order to transform the protein-rich biological wastes left after lipid extraction into value-added bio-products. Thus, the utilization of protein recovered from defatted Schizochytrium sp. by-products presents an opportunity. A specific peptide Tyr-Leu (YL) with calcium-binding capacity was purified from defatted Schizochytrium sp. protein hydrolysates through gel filtration chromatography and RP-HPLC. The calcium-binding activity of YL reached 126.34 ± 3.40 μg/mg. The calcium-binding mechanism was investigated through ultraviolet, fluorescence and infrared spectroscopy. The results showed that calcium ions could form dative bonds with carboxyl oxygen atoms and amino nitrogen atoms as well as the nitrogen and oxygen atoms of amide bonds. YL-Ca exhibited excellent thermal stability and solubility, which was beneficial for its absorption and transport in the basic intestinal tract of the human body. Moreover, the cellular uptake of calcium in Caco-2 cells showed that YL-Ca could enhance calcium uptake efficiency and protect calcium ions against precipitation caused by dietary inhibitors such as tannic acid, oxalate, phytate and metal ions. The findings indicate that the by-product of Schizochytrium sp. is a promising source for making peptide-calcium bio-products as algae-based functional supplements for human beings.

  14. Hydration properties and binding capacities of dietary fibers from bamboo shoot shell and its hypolipidemic effects in mice.

    PubMed

    Luo, Xianliang; Wang, Qi; Zheng, Baodong; Lin, Liangmei; Chen, Bingyan; Zheng, Yafeng; Xiao, Jianbo

    2017-02-22

    In the bamboo shoot processing industries, bamboo shoot shells are discarded without any utilization. As a cheap potential dietary fibers resource, bamboo shoot (Leleba oldhami Nakal) shell was decomposed to dietary fibers by multiple enzymes. The extraction yields of insoluble dietary fiber and soluble dietary fiber were 56.21% and 8.67%, respectively. The resulting fibers showed significant swelling capacity, water holding capacity and exhibit in vitro binding capacities to fat, cholesterol, bile acids and nitrites. The administration of bamboo shoot shell fibers improved the lipid metabolism disorderly situation of hyperlipidemia mice. Compared with normal group, total dietary fiber supplement could exhibit the lowest body weight gain (2.84%) in mice, and decrease total cholesterol, triglyceride and low density lipoprotein-cholesterol by 31.53%, 21.35% and 31.53%, respectively; while it can increase high density lipoprotein-cholesterol by 37.6%. The bamboo shoot shell fibers could be a potentially available dietary ingredient in functional food industries.

  15. A diclofenac suppository-nabumetone combination therapy for arthritic pain relief and a monitoring method for the diclofenac binding capacity of HSA site II in rheumatoid arthritis.

    PubMed

    Setoguchi, Nao; Takamura, Norito; Fujita, Ken-ichi; Ogata, Kenji; Tokunaga, Jin; Nishio, Toyotaka; Chosa, Etsuo; Arimori, Kazuhiko; Kawai, Keiichi; Yamamoto, Ryuichi

    2013-03-01

    Diclofenac suppository, a non-steroidal anti-inflammatory drug (NSAID), is used widely in rheumatoid arthritis (RA) patients with severe arthritic pain. As the binding percentage of diclofenac to serum proteins is high, its free (unbound) concentration after rectal administration is low. To increase temporarily the free concentration of diclofenac and to enhance its analgesic effect by inhibiting the protein binding of diclofenac, the analgesic effect of diclofenac was examined before and after the start of an inhibitor administration to RA patients with insufficient control of arthritic pain, and the protein binding capacity of diclofenac was evaluated. Binding experiments were performed by ultrafiltration, and arthritic pain was recorded by the face scale. Free fractions of diazepam and diclofenac were augmented by increasing 6-methoxy-2-naphthylacetic acid (6-MNA; the active metabolite of the NSAID nabumetone) concentrations. The free fraction of diazepam increased after the start of nabumetone administration to RA patients, and arthritic pain relief was observed. These results suggest that 6-MNA has an inhibitory effect on the protein binding of diclofenac and the free fraction of diazepam can be used to evaluate the binding capacity of diclofenac. It is considered that diclofenac suppository-nabumetone combination therapy and the method for protein binding monitoring by diazepam can positively benefit RA patients with insufficient control of arthritic pain.

  16. Nucleic acid binding and other biomedical properties of artificial oligolysines

    PubMed Central

    Roviello, Giovanni N; Vicidomini, Caterina; Costanzo, Vincenzo; Roviello, Valentina

    2016-01-01

    In the present study, we report the interaction of an artificial oligolysine (referred to as AOL) realized in our laboratory with targets of biomedical importance. These included polyinosinic acid (poly rI) and its complex with polycytidylic acid (poly I:C), RNAs with well-known interferon-inducing ability, and double-stranded (ds) DNA. The ability of the peptide to bind both single-stranded poly rI and ds poly I:C RNAs emerged from our circular dichroism (CD) and ultraviolet (UV) studies. In addition, we found that AOL forms complexes with dsDNA, as shown by spectroscopic binding assays and UV thermal denaturation experiments. These findings are encouraging for the possible use of AOL in biomedicine for nucleic acid targeting and oligonucleotide condensation, with the latter being a key step preceding their clinical application. Moreover, we tested the ability of AOL to bind to proteins, using serum albumin as a model protein. We demonstrated the oligolysine–protein binding by CD experiments which suggested that AOL, positively charged under physiological conditions, binds to the protein regions rich in anionic residues. Finally, the morphology characterization of the solid oligolysine, performed by scanning electron microscopy, showed different crystal forms including cubic-shaped crystals confirming the high purity of AOL. PMID:28115843

  17. CD36 binds oxidized low density lipoprotein (LDL) in a mechanism dependent upon fatty acid binding.

    PubMed

    Jay, Anthony G; Chen, Alexander N; Paz, Miguel A; Hung, Justin P; Hamilton, James A

    2015-02-20

    The association of unesterified fatty acid (FA) with the scavenger receptor CD36 has been actively researched, with focuses on FA and oxidized low density lipoprotein (oxLDL) uptake. CD36 has been shown to bind FA, but this interaction has been poorly characterized to date. To gain new insights into the physiological relevance of binding of FA to CD36, we characterized FA binding to the ectodomain of CD36 by the biophysical method surface plasmon resonance. Five structurally distinct FAs (saturated, monounsaturated (cis and trans), polyunsaturated, and oxidized) were pulsed across surface plasmon resonance channels, generating association and dissociation binding curves. Except for the oxidized FA HODE, all FAs bound to CD36, with rapid association and dissociation kinetics similar to HSA. Next, to elucidate the role that each FA might play in CD36-mediated oxLDL uptake, we used a fluorescent oxLDL (Dii-oxLDL) live cell assay with confocal microscopy imaging. CD36-mediated uptake in serum-free medium was very low but greatly increased when serum was present. The addition of exogenous FA in serum-free medium increased oxLDL binding and uptake to levels found with serum and affected CD36 plasma membrane distribution. Binding/uptake of oxLDL was dependent upon the FA dose, except for docosahexaenoic acid, which exhibited binding to CD36 but did not activate the uptake of oxLDL. HODE also did not affect oxLDL uptake. High affinity FA binding to CD36 and the effects of each FA on oxLDL uptake have important implications for protein conformation, binding of other ligands, functional properties of CD36, and high plasma FA levels in obesity and type 2 diabetes.

  18. Urinary intestinal fatty acid binding protein predicts necrotizing enterocolitis.

    PubMed

    Gregory, Katherine E; Winston, Abigail B; Yamamoto, Hidemi S; Dawood, Hassan Y; Fashemi, Titilayo; Fichorova, Raina N; Van Marter, Linda J

    2014-06-01

    Necrotizing enterocolitis, characterized by sudden onset and rapid progression, remains the most significant gastrointestinal disorder among premature infants. In seeking a predictive biomarker, we found intestinal fatty acid binding protein, an indicator of enterocyte damage, was substantially increased within three and seven days before the diagnosis of necrotizing enterocolitis.

  19. Ion exchange chromatography of monoclonal antibodies: effect of resin ligand density on dynamic binding capacity.

    PubMed

    Hardin, Ann Marie; Harinarayan, Chithkala; Malmquist, Gunnar; Axén, Andreas; van Reis, Robert

    2009-05-15

    Dynamic binding capacity (DBC) of a monoclonal antibody on agarose based strong cation exchange resins is determined as a function of resin ligand density, apparent pore size of the base matrix, and protein charge. The maximum DBC is found to be unaffected by resin ligand density, apparent pore size, or protein charge within the tested range. The critical conductivity (conductivity at maximum DBC) is seen to vary with ligand density. It is hypothesized that the maximum DBC is determined by the effective size of the proteins and the proximity to which they can approach one another. Once a certain minimum resin ligand density is supplied, additional ligand is not beneficial in terms of resin capacity. Additional ligand can provide flexibility in designing ion exchange resins for a particular application as the critical conductivity could be matched to the feedstock conductivity and it may also affect the selectivity.

  20. Protein immobilization capacity and covalent binding coverage of pulsed plasma polymer surfaces

    NASA Astrophysics Data System (ADS)

    Yin, Yongbai; Bax, Daniel; McKenzie, David R.; Bilek, Marcela M. M.

    2010-06-01

    Three carbon surfaces were deposited using pulsed plasma enhanced chemical vapour deposition method: a low and a high nitrogen-containing plasma polymer surfaces and a diamond-like carbon surface. The surfaces were analysed using both X-ray photoelectron spectroscopy (XPS) technique and the enzyme-linked immunosorbent assay (ELISA) method combining with sodium dodecyl sulphate (SDS) cleaning to investigate the capacity and covalent binding of the immobilized proteins. A good correlation was found on quantification of remaining protein after SDS cleaning using the ELISA method and the XPS technique. All surfaces had similar initial capacity of protein attachment but with large different resistance to SDS cleaning. The analysis showed that the high nitrogen-containing plasma polymer was the best biocompatible material due to its highest resistance to SDS cleaning, i.e. with the highest quantity (˜80%) of proteins bound covalently.

  1. Modulation of linoleic acid-binding properties of human serum albumin by divalent metal cations.

    PubMed

    Nemashkalova, Ekaterina L; Permyakov, Eugene A; Permyakov, Sergei E; Litus, Ekaterina A

    2017-03-16

    Human serum albumin (HSA) is an abundant multiligand carrier protein, linked to progression of Alzheimer's disease (AD). Blood HSA serves as a depot of amyloid β (Aβ) peptide. Aβ peptide-buffering properties of HSA depend on interaction with its ligands. Some of the ligands, namely, linoleic acid (LA), zinc and copper ions are involved into AD progression. To clarify the interplay between LA and metal ion binding to HSA, the dependence of LA binding to HSA on Zn(2+), Cu(2+), Mg(2+) and Ca(2+) levels and structural consequences of these interactions have been explored. Seven LA molecules are bound per HSA molecule in the absence of the metal ions. Zn(2+) binding to HSA causes a loss of one bound LA molecule, while the other metals studied exert an opposite effect (1-2 extra LA molecules are bound). In most cases, the observed effects are not related to the metal-induced changes in HSA quaternary structure. However, the Zn(2+)-induced decline in LA capacity of HSA could be due to accumulation of multimeric HSA forms. Opposite to Ca(2+)/Mg(2+)-binding, Zn(2+) or Cu(2+) association with HSA induces marked changes in its hydrophobic surface. Overall, the divalent metal ions modulate LA capacity and affinity of HSA to a different extent. LA- and Ca(2+)-binding to HSA synergistically support each other. Zn(2+) and Cu(2+) induce more pronounced changes in hydrophobic surface and quaternary structure of HSA and its LA capacity. A misbalanced metabolism of these ions in AD could modify interactions of HSA with LA, other fatty acids and hydrophobic substances, associated with AD.

  2. Characterisation of a fatty acid and retinol binding protein orthologue from the hookworm Ancylostoma ceylanicum.

    PubMed

    Fairfax, Keke C; Vermeire, Jon J; Harrison, Lisa M; Bungiro, Richard D; Grant, Wayne; Husain, Sohail Z; Cappello, Michael

    2009-12-01

    Hookworms, bloodfeeding intestinal nematodes, infect nearly one billion people in resource limited countries and are a leading cause of anaemia and malnutrition. Like other nematodes, hookworms lack the capacity to synthesise essential fatty acids de novo and therefore must acquire those from exogenous sources. The cDNA corresponding to a putative Ancylostoma ceylanicum fatty acid and retinol binding protein-1 (AceFAR-1) was amplified from adult hookworm mRNA. Studies using quantitative reverse transcriptase real-time PCR demonstrate that AceFAR-1 transcripts are most abundant in the earliest developmental stages of the parasite, and greater in females than males. Using in vitro assays, the recombinant AceFAR-1 (rAceFAR-1) was shown to bind individual fatty acids with equilibrium dissociation constants in the low micromolar range. The pattern of fatty acid uptake by live adult worms cultured ex vivo was similar to the in vitro binding profile of rAceFAR-1, raising the possibility that the native protein may be involved in acquisition of fatty acids by A. ceylanicum. Animals vaccinated orally with rAceFAR-1 and the mucosal adjuvant cholera toxin exhibited a statistically significant (40-47%) reduction in intestinal worm burden compared with controls immunized with antigen or adjuvant alone. Together, these data suggest a potential role for AceFAR-1 in hookworm biology, making it a potentially valuable target for drug and vaccine development.

  3. Characterization of a fatty acid and retinol binding protein orthologue from the hookworm Ancylostoma ceylanicum✯

    PubMed Central

    Fairfax, Keke C.; Vermeire, Jon J.; Harrison, Lisa M.; Bungiro, Richard D.; Grant, Wayne; Husain, Sohail Z.; Cappello, Michael

    2009-01-01

    Hookworms, bloodfeeding intestinal nematodes, infect nearly one billion people in resource limited countries and are a leading cause of anemia and malnutrition. Like other nematodes, hookworms lack the capacity to synthesize essential fatty acids de novo and therefore must acquire those from exogenous sources. The cDNA corresponding to a putative Ancylostoma ceylanicum fatty acid and retinol binding protein-1 (AceFAR-1) was amplified from adult hookworm mRNA. Studies using quantitative reverse transcriptase real time-PCR demonstrate that AceFAR-1 transcripts are most abundant in the earliest developmental stages of the parasite, and greater in females than males. Using in vitro assays, the recombinant AceFAR-1 (rAceFAR-1) was shown to bind individual fatty acids with equilibrium dissociation constants in the low micromolar range. The pattern of fatty acid uptake by live adult worms cultured ex vivo was similar to the in vitro binding profile of rAceFAR-1, raising the possibility that the native protein may be involved in acquisition of fatty acids by A. ceylanicum. Animals vaccinated orally with rAceFAR-1 and the mucosal adjuvant cholera toxin exhibited a statistically significant (40–47%) reduction in intestinal worm burden compared with controls immunized with antigen or adjuvant alone. Together, these data suggest a potential role for AceFAR-1 in hookworm biology, making it a potentially valuable target for drug and vaccine development. PMID:19591834

  4. Disulfide bridge regulates ligand-binding site selectivity in liver bile acid-binding proteins.

    PubMed

    Cogliati, Clelia; Tomaselli, Simona; Assfalg, Michael; Pedò, Massimo; Ferranti, Pasquale; Zetta, Lucia; Molinari, Henriette; Ragona, Laura

    2009-10-01

    Bile acid-binding proteins (BABPs) are cytosolic lipid chaperones that play central roles in driving bile flow, as well as in the adaptation to various pathological conditions, contributing to the maintenance of bile acid homeostasis and functional distribution within the cell. Understanding the mode of binding of bile acids with their cytoplasmic transporters is a key issue in providing a model for the mechanism of their transfer from the cytoplasm to the nucleus, for delivery to nuclear receptors. A number of factors have been shown to modulate bile salt selectivity, stoichiometry, and affinity of binding to BABPs, e.g. chemistry of the ligand, protein plasticity and, possibly, the formation of disulfide bridges. Here, the effects of the presence of a naturally occurring disulfide bridge on liver BABP ligand-binding properties and backbone dynamics have been investigated by NMR. Interestingly, the disulfide bridge does not modify the protein-binding stoichiometry, but has a key role in modulating recognition at both sites, inducing site selectivity for glycocholic and glycochenodeoxycholic acid. Protein conformational changes following the introduction of a disulfide bridge are small and located around the inner binding site, whereas significant changes in backbone motions are observed for several residues distributed over the entire protein, both in the apo form and in the holo form. Site selectivity appears, therefore, to be dependent on protein mobility rather than being governed by steric factors. The detected properties further establish a parallelism with the behaviour of human ileal BABP, substantiating the proposal that BABPs have parallel functions in hepatocytes and enterocytes.

  5. Modeling lanthanide series binding sites on humic acid.

    PubMed

    Pourret, Olivier; Martinez, Raul E

    2009-02-01

    Lanthanide (Ln) binding to humic acid (HA) has been investigated by combining ultrafiltration and ICP-MS techniques. A Langmuir-sorption-isotherm metal-complexation model was used in conjunction with a linear programming method (LPM) to fit experimental data representing various experimental conditions both in HA/Ln ratio (varying between 5 and 20) and in pH range (from 2 to 10) with an ionic strength of 10(-3) mol L(-1). The LPM approach, not requiring prior knowledge of surface complexation parameters, was used to solve the existing discrepancies in LnHA binding constants and site densities. The application of the LPM to experimental data revealed the presence of two discrete metal binding sites at low humic acid concentrations (5 mg L(-1)), with log metal complexation constants (logK(S,j)) of 2.65+/-0.05 and 7.00 (depending on Ln). The corresponding site densities were 2.71+/-0.57x10(-8) and 0.58+/-0.32x10(-8) mol of Ln(3+)/mg of HA (depending on Ln). Total site densities of 3.28+/-0.28x10(-8), 4.99+/-0.02x10(-8), and 5.01+/-0.01x10(-8) mol mg(-1) were obtained by LPM for humic acid, for humic acid concentrations of 5, 10, and 20 mg L(-1), respectively. These results confirm that lanthanide binding occurs mainly at weak sites (i.e., ca. 80%) and second at strong sites (i.e., ca. 20%). The first group of discrete metal binding sites may be attributed to carboxylic groups (known to be the main binding sites of Ln in HA), and the second metal binding group to phenolic moieties. Moreover, this study evidences heterogeneity in the distribution of the binding sites among Ln. Eventually, the LPM approach produced feasible and reasonable results, but it was less sensitive to error and did not require an a priori assumption of the number and concentration of binding sites.

  6. Structural analysis of ibuprofen binding to human adipocyte fatty-acid binding protein (FABP4).

    PubMed

    González, Javier M; Fisher, S Zoë

    2015-02-01

    Inhibition of human adipocyte fatty-acid binding protein (FABP4) has been proposed as a treatment for type 2 diabetes, fatty liver disease and atherosclerosis. However, FABP4 displays a naturally low selectivity towards hydrophobic ligands, leading to the possibility of side effects arising from cross-inhibition of other FABP isoforms. In a search for structural determinants of ligand-binding selectivity, the binding of FABP4 towards a group of small molecules structurally related to the nonsteroidal anti-inflammatory drug ibuprofen was analyzed through X-ray crystallography. Several specific hydrophobic interactions are shown to enhance the binding affinities of these compounds, whereas an aromatic edge-to-face interaction is proposed to determine the conformation of bound ligands, highlighting the importance of aromatic interactions in hydrophobic environments.

  7. An experimental and modeling study of humic acid concentration effect on H(+) binding: Application of the NICA-Donnan model.

    PubMed

    Vidali, Roza; Remoundaki, Emmanouela; Tsezos, Marios

    2009-11-15

    Humic substances are the most abundant components of the colloidal and the dissolved fraction of natural organic matter (NOM) and they are characterized by a strong binding capacity for both metals and organic pollutants, affecting their mobility and bioavailability. The understanding of the humic acidic character is the first necessary step for the study of the mechanisms of binding of other positively charged soluble metal species by humic molecules. The present work, which constitutes part of the Ph.D. thesis of Roza Vidali, reports results on the influence of the concentration of humic acids on the binding of protons obtained through both an experimental and a modeling approach. A reference purified peat humic acid (PPHA) isolated by the International Humic Substances Society (IHSS) and a humic acid from a Greek soil (GHA) were experimentally studied at various humic acid concentrations, ranging from 20 to 200mgL(-1). The proton binding isotherms obtained at different humic acid concentrations have shown that proton binding is dependent on the concentration of both humic acids. Proton binding experimental data were fitted to the NICA-Donnan model and the model parameter values were calculated for humic acid concentrations of 20 and >or=100mgL(-1). The results obtained for the NICA-Donnan parameters at humic acid concentrations >or=100mgL(-1) are in excellent agreement with those reported in the literature. However, these model parameter values cannot be used for modeling and predicting cation binding in natural aquatic systems, where humic acid concentrations are much lower. Two sets of the NICA-Donnan parameters are reported: one for humic acid concentrations of >or=100mgL(-1) and one for humic acid concentration of 20mgL(-1). The significance of the parameters values for each concentration level is also discussed.

  8. Increased flexibility and liposome-binding capacity of CD1e at endosomal pH.

    PubMed

    Bushmarina, Natalia; Tourne, Sylvie; Giacometti, Gaëlle; Signorino-Gelo, François; Garcia-Alles, Luis F; Cazenave, Jean-Pierre; Hanau, Daniel; de la Salle, Henri

    2011-06-01

    The plasma membrane proteins CD1a, CD1b and CD1c are expressed by human dendritic cells, the professional antigen-presenting cells of the immune system, and present lipid antigens to T lymphocytes. CD1e belongs to the same family of molecules, but accumulates as a membrane-associated form in the Golgi compartments of immature dendritic cells and as a soluble cleaved form in the lysosomes of mature dendritic cells. In lysosomes, the N-terminal propeptide of CD1e is also cleaved, but the functional consequences of this step are unknown. Here, we investigated how the pH changes encountered during transport to lysosomes affect the structure of CD1e and its ligand-binding properties. Circular dichroism studies demonstrated that the secondary and tertiary structures of recombinant CD1e were barely altered by pH changes. Nevertheless, at acidic pH, guanidium chloride-induced unfolding of CD1e molecules required lower concentrations of denaturing agent. The nonfunctional L194P allelic variant was found to be structurally less stable at acidic pH than the functional forms, providing an explanation for the lack of its detection in lysosomes. The number of water-exposed hydrophobic patches that bind 8-anilinonaphthalene-1-sulfonate was higher in acidic conditions, especially for the L194P variant. CD1e molecules interacted with lipid surfaces enriched in anionic lipids, such as bis(monoacylglycero)phosphate, a late endosomal/lysosomal lipid, especially at acidic pH, or when the propeptide was present. Altogether, these data indicate that, in the late endosomes/lysosomes of DCs, the acid pH promotes the binding of lipid antigens to CD1e through increased hydrophobic and ionic interactions.

  9. Binding of bile acids by pastry products containing bioactive substances during in vitro digestion.

    PubMed

    Dziedzic, Krzysztof; Górecka, Danuta; Szwengiel, Artur; Smoczyńska, Paulina; Czaczyk, Katarzyna; Komolka, Patrycja

    2015-03-01

    The modern day consumer tends to choose products with health enhancing properties, enriched in bioactive substances. One such bioactive food component is dietary fibre, which shows a number of physiological properties including the binding of bile acids. Dietary fibre should be contained in everyday, easily accessible food products. Therefore, the aim of this study was to determine sorption capacities of primary bile acid (cholic acid - CA) and secondary bile acids (deoxycholic - DCA and lithocholic acids - LCA) by muffins (BM) and cookies (BC) with bioactive substances and control muffins (CM) and cookies (CC) in two sections of the in vitro gastrointestinal tract. Variations in gut flora were also analysed in the process of in vitro digestion of pastry products in a bioreactor. Enzymes: pepsin, pancreatin and bile salts: cholic acid, deoxycholic acid and lithocholic acid were added to the culture. Faecal bacteria, isolated from human large intestine, were added in the section of large intestine. The influence of dietary fibre content in cookies and concentration of bile acids in two stages of digestion were analysed. Generally, pastry goods with bioactive substances were characterized by a higher content of total fibre compared with the control samples. These products also differ in the profile of dietary fibre fractions. Principal Component Analysis (PCA) showed that the bile acid profile after two stages of digestion depends on the quality and quantity of fibre. The bile acid profile after digestion of BM and BC forms one cluster, and with the CM and CC forms a separate cluster. High concentration of H (hemicellulose) is positively correlated with LCA (low binding effect) and negatively correlated with CA and DCA contents. The relative content of bile acids in the second stage of digestion was in some cases above the content in the control sample, particularly LCA. This means that the bacteria introduced in the 2nd stage of digestion synthesize the LCA.

  10. Relationship between laminin binding capacity and laminin expression on tumor cells sensitive or resistant to natural cell-mediated cytotoxicity

    SciTech Connect

    Laybourn, K.A.; Varani, J.; Fligiel, S.E.G.; Hiserodt, J.C.

    1986-03-01

    Previous studies have identified the presence of laminin binding sites on murine NK and NC sensitive tumor cells by /sup 125/I-laminin binding and laminin induced cell-cell aggregation. The finding that the addition of exogenous laminin inhibits NK/NC binding to sensitive tumor cells suggests laminin binding sites may serve as target antigens for NK cells. The present study extends earlier reports by analyzing a large panel of tumor cells for laminin binding capacity, laminin expression and sensitivity to NK/NC killing. The data indicate that all tumor cells which bind to NK/NC cells (8 lines tested) express laminin binding sites. All of these tumor cells were capable of competing for NK lysis of YAC-1 cells in cold target competition assays, and all bound enriched NK cells in direct single cell binding assays. In contrast, tumor cells expressing high levels of surface laminin (B16 melanomas, C57B1/6 fibrosarcomas, and RAS transfected 3T3 fibroblasts) but low levels of laminin binding capacity did not bind NK/NC cells and were resistant to lysis. These data support the hypothesis that expression of laminin/laminin binding sites may contribute to tumor cell sensitivity to NK/NC binding and/or killing.

  11. Acid Gas Capture Using CO2-Binding Organic Liquids

    SciTech Connect

    Heldebrant, David J.; Koech, Phillip K.; Rainbolt, James E.; Zheng, Feng

    2010-11-10

    Current chemical CO2 scrubbing technology is primarily aqueous alkanolamine based. These systems rapidly bind CO2 (forming water-soluble carbamate and bicarbonate salts) however, the process has serious disadvantages. The concentration of monoethanolamine rarely exceeds 30 wt % due to the corrosive nature of the solution, and this reduces the maximum CO2 volumetric (≤108 g/L) and gravimetric capacity (≤7 wt%) of the CO2 scrubber. The ≤30 wt % loading of ethanolamine also means that a large excess of water must be pumped and heated during CO2 capture and release, and this greatly increases the energy requirements especially considering the high specific heat of water (4 j/g-1K-1). Our approach is to switch to organic systems that chemically bind CO2 as liquid alkylcarbonate salts. Our CO2-binding organic liquids have higher CO2 solubility, lower specific heats, potential for less corrosion and lower binding energies for CO2 than aqueous systems. CO2BOLs also reversibly bind and release mixed sulfur oxides. Furthermore the CO2BOL system can be direct solvent replacements for any solvent based CO2 capture systems because they are commercially available reagents and because they are fluids they would not require extensive process re-engineering.

  12. Effects of phorbol 12-myristate 13-acetate and cortisol interaction on steroid-binding capacity in the rat.

    PubMed Central

    Janssens, J P; de Loecker, W

    1979-01-01

    The specificity of the cortisol-receptor protein is examined in plasma and liver cytosol of rats. Phorbol 12-myristate 13-acetate does not inhibit the binding of cortisol to transcortin, nor does it affect the binding capacity of dexamethasone to the intracellular glucocorticoid receptor, but, by interacting with the cortisol molecule, it interferes with hormone-mediated processes in the cell. PMID:534535

  13. Acetic acid enhances endurance capacity of exercise-trained mice by increasing skeletal muscle oxidative properties.

    PubMed

    Pan, Jeong Hoon; Kim, Jun Ho; Kim, Hyung Min; Lee, Eui Seop; Shin, Dong-Hoon; Kim, Seongpil; Shin, Minkyeong; Kim, Sang Ho; Lee, Jin Hyup; Kim, Young Jun

    2015-01-01

    Acetic acid has been shown to promote glycogen replenishment in skeletal muscle during exercise training. In this study, we investigated the effects of acetic acid on endurance capacity and muscle oxidative metabolism in the exercise training using in vivo mice model. In exercised mice, acetic acid induced a significant increase in endurance capacity accompanying a reduction in visceral adipose depots. Serum levels of non-esterified fatty acid and urea nitrogen were significantly lower in acetic acid-fed mice in the exercised mice. Importantly, in the mice, acetic acid significantly increased the muscle expression of key enzymes involved in fatty acid oxidation and glycolytic-to-oxidative fiber-type transformation. Taken together, these findings suggest that acetic acid improves endurance exercise capacity by promoting muscle oxidative properties, in part through the AMPK-mediated fatty acid oxidation and provide an important basis for the application of acetic acid as a major component of novel ergogenic aids.

  14. Fatty acid-binding site environments of serum vitamin D-binding protein and albumin are different

    PubMed Central

    Swamy, Narasimha; Ray, Rahul

    2008-01-01

    Vitamin D-binding protein (DBP) and albumin (ALB) are abundant serum proteins and both possess high-affinity binding for saturated and unsaturated fatty acids. However, certain differences exist. We surmised that in cases where serum albumin level is low, DBP presumably can act as a transporter of fatty acids. To explore this possibility we synthesized several alkylating derivatives of 14C-palmitic acid to probe the fatty acid binding pockets of DBP and ALB. We observed that N-ethyl-5-phenylisooxazolium-3′-sulfonate-ester (WRK ester) of 14C-palmitic acid specifically labeled DBP; but p-nitrophenyl- and N-hydroxysuccinimidyl-esters failed to do so. However, p-nitrophenyl ester of 14C-palmitic acid specifically labeled bovine ALB, indicating that the micro-environment of the fatty acid-binding domains of DBP and ALB may be different; and DBP may not replace ALB as a transporter of fatty acids. PMID:18374965

  15. MARCKS actin-binding capacity mediates actin filament assembly during mitosis in human hepatic stellate cells.

    PubMed

    Rombouts, Krista; Mello, Tommaso; Liotta, Francesco; Galli, Andrea; Caligiuri, Alessandra; Annunziato, Francesco; Pinzani, Massimo

    2012-08-15

    Cross-linking between the actin cytoskeleton and plasma membrane actin-binding proteins is a key interaction responsible for the mechanical properties of the mitotic cell. Little is known about the identity, the localization, and the function of actin filament-binding proteins during mitosis in human hepatic stellate cells (hHSC). The aim of the present study was to identify and analyze the cross talk between actin and myristoylated alanine-rich kinase C substrate (MARCKS), an important PKC substrate and actin filament-binding protein, during mitosis in primary hHSC. Confocal analysis and chromosomal fraction analysis of mitotic hHSC demonstrated that phosphorylated (P)-MARCKS displays distinct phase-dependent localizations, accumulates at the perichromosomal layer, and is a centrosomal protein belonging to the chromosomal cytosolic fraction. Aurora B kinase (AUBK), an important mitotic regulator, β-actin, and P-MARCKS concentrate at the cytokinetic midbody during cleavage furrow formation. This localization is critical since MARCKS-depletion in hHSC is characterized by a significant loss in cytosolic actin filaments and cortical β-actin that induces cell cycle inhibition and dislocation of AUBK. A depletion of AUBK in hHSC affects cell cycle, resulting in multinucleation. Quantitative live cell imaging demonstrates that the actin filament-binding capacity of MARCKS is key to regulate mitosis since the cell cycle inhibitory effect in MARCKS-depleted cells caused abnormal cell morphology and an aberrant cytokinesis, resulting in a significant increase in cell cycle time. These findings implicate that MARCKS, an important PKC substrate, is essential for proper cytokinesis and that MARCKS and its partner actin are key mitotic regulators during cell cycle in hHSC.

  16. Quantification of oil binding capacity of structuring fats: A novel method and its application.

    PubMed

    Omonov, Tolibjon S; Bouzidi, Laziz; Narine, Suresh S

    2010-09-01

    A robust, well-defined and reproducible method to accurately measure the oil binding capacity (OBC) of structuring fats was developed. The method was validated using two oil/fat model systems, i.e., fully hydrogenated canola oil (FHCO) in canola oil (CO) (FHCO/CO) and fully hydrogenated soybean oil (FHSO) in CO (FHSO/CO). The mixtures were crystallized from the melt down to three different temperatures (15, 25 and 35 degrees C) at constant rates of cooling and the OBC was measured after different periods of storage time. The critical concentration of hard fat at which the solid fat network is stable and effectively binds oil has been also measured for mixtures crystallized at temperatures close to room temperature, i.e., 25 degrees C. Crystal structure, melting behavior, microstructure, and solid fat content of these binary systems have been investigated in relation to the OBC of the solid fat network using X-ray diffraction (XRD), differential scanning calorimetry (DSC), polarized light microscopy (PLM), and wide-line pulsed nuclear magnetic resonance (pNMR) techniques. The two model systems exhibited similar trends in OBC over time, a behavior attributed to their similar TAG composition and polymorphism. However, relatively smaller OBC values were achieved by the CO/FHSO compared to CO/FHCO samples, largely due to differences in their solid network structure. Four successive decreasing linear segments, identifying successive mechanisms of oil migration/binding, were observed in the experimental OBC versus fat weight fraction curves. The critical concentration of hard fat, at which the solid fat network is effective in binding oil, was also determined and found to be approximately 6wt% for both systems.

  17. DNA Mutagenic Activity and Capacity for HIV-1 Restriction of the Cytidine Deaminase APOBEC3G Depends on Whether DNA or RNA Binds to Tyrosine 315.

    PubMed

    Polevoda, Bogdan; Joseph, Rebecca; Friedman, Alan E; Bennett, Ryan P; Greiner, Rebecca; De Zoysa, Thareendra; Stewart, Ryan A; Smith, Harold C

    2017-04-05

    APOBEC3G (A3G) belongs to the AID/APOBEC protein family of cytidine deaminases (CDA) that bind to nucleic acids. A3G mutates the HIV genome by deamination of dC to dU, leading to accumulation of virus-inactivating mutations. Binding to cellular RNAs inhibits A3G binding to substrate single-stranded (ss) DNA and CDA activity. RNA and ssDNA bind to the same three A3G tryptic peptides (amino acids 181-194, 314-320, and 345-374) that form parts of a continuously exposed protein surface extending from the catalytic domain in the C-terminus of A3G to its N-terminus. We show here that the A3G tyrosines 181 and 315 directly cross-link ssDNA. Binding experiments showed that a Y315A mutation alone significantly reduced A3G binding to both ssDNA and RNA, whereas Y181A and Y182A mutations only moderately affected A3G nucleic acid binding. Consistent with these findings, the Y315A mutant exhibited little to no deaminase activity in an E. coli DNA mutator reporter, while Y181A and Y182A mutants retained ~50% of wild-type A3G activity. The Y315A mutant also showed a markedly reduced ability to assemble into viral particles and had reduced antiviral activity. In uninfected cells, the impaired RNA-binding capacity of Y315A was evident by a shift of A3G from high-molecular-mass ribonucleoprotein complexes to low-molecular-mass complexes. We conclude that Y315 is essential for coordinating ssDNA interaction with or entry to the deaminase domain and hypothesize that RNA bound to Y315 may be sufficient to competitively inhibit ssDNA deaminase-dependent antiviral activity.

  18. Retinoic acid binding protein in normal and neopolastic rat prostate.

    PubMed

    Gesell, M S; Brandes, M J; Arnold, E A; Isaacs, J T; Ueda, H; Millan, J C; Brandes, D

    1982-01-01

    Sucrose density gradient analysis of cytosol from normal and neoplastic rat prostatic tissues exhibited a peak of (3H) retinoic acid binding in the 2S region, corresponding to the cytoplasmic retinoic acid binding protein (cRABP). In the Fisher-Copenhagen F1 rat, cRABP was present in the lateral lobe, but could not be detected in the ventral nor in the dorsal prostatic lobes. Four sublines of the R-3327 rat prostatic tumor contained similar levels of this binding protein. The absence of cRABP in the normal tissue of origin of the R-3327 tumor, the rat dorsal prostate, and reappearance in the neoplastic tissues follows a pattern described in other human and animal tumors. The occurrence of cRABP in the well-differentiated as well as in the anaplastic R-3327 tumors in which markers which reflect a state of differentiation and hormonal regulation, such as androgen receptor, 5 alpha reductase, and secretory acid phosphatase are either markedly reduced or absent, points to cRABP as a marker of malignant transformation.

  19. Effects of microgravity on the binding of acetylsalicylic acid by Rhizobium leguminosarum bv. trifolii

    NASA Astrophysics Data System (ADS)

    Urban, James E.; Gerren, Richard; Zoelle, Jeffery

    1995-07-01

    Bacteroids can be induced in vitro by treating growing Rhizobium leguminosarum bv. trifolii with succinic acid or succinic acid structural analogs like acetylsalicylic acid. Quantitating bacteroid induction by measuring acetylsalicylic binding under normal (1 g) conditions showed two forms of binding to occur. In one form of binding cells immediately bound comparatively high levels of acetylsalicylic acid, but the binding was quickly reversed. The second form of binding increased with time by first-order kinetics, and reached saturation in 40 s. Similar experiments performed in the microgravity environment aboard the NASA 930 aircraft showed only one form of binding and total acetylsalicylic acid bound was 32% higher than at 1 g.

  20. [Is cobalt-binding capacity of serum a new perspective diagnostic marker?].

    PubMed

    Litus, E A; Zaĭtsev, V G; Verovskiĭ, V E; Goncharova, L V; Dudchenko, G P; Ostrovskiĭ, O V

    2014-01-01

    The aim of this study was to determine the reference interval of serum cobalt-binding capacity (CoBC) and to estimate the effect of factors unrelated to oxidative modification of serum albumin on this diagnostic marker. A group of healthy volunteers (n=194), a group of patients with autoimmune diseases (n=44) and a group of patients with diabetes type 2 (n=50) participated in this study. The regional reference interval was found to be 0,462-0,744 mmol Co 2+ /l of serum. The study of serum CoBC in two groups of patients showed that the CoBC level strongly depends on the serum protein profile. Therefore, this diagnostic test may be used for diagnosing ischemia, but other pathologies associated with changes in a ratio of blood protein fractions can also influence the serum CoBC level.

  1. Stimulation and binding of myocardial phospholipase C by phosphatidic acid.

    PubMed

    Henry, R A; Boyce, S Y; Kurz, T; Wolf, R A

    1995-08-01

    Exposure of adult ventricular myocytes to exogenous natural phosphatidic acid results in the production of inositol phosphates by unknown mechanism(s). We characterized stimulation of myocytic phosphoinositide-specific phospholipase C (PLC) by synthetic dioleoyl phosphatidic acid (PA) as a potential mechanism for modulation of inositol phosphate production. Our data demonstrate that exogenous PA, at 10(-8)-10(-5) M, caused a concentration-dependent increase in inositol 1,4,5-trisphosphate in adult rabbit ventricular myocytes. PA also caused a concentration-dependent increase in in vitro activity of myocytic PLC in the presence or absence of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). PLC-delta 1, the predominant isozyme of PLC expressed in adult rabbit ventricular myocytes, bound to liposomes of PA with high affinity in the presence of EGTA. The phosphomonoester group of PA was critical to in vitro stimulation of myocytic PLC activity and high-affinity binding of PLC-delta 1. We propose that binding of PLC-delta 1 to phosphatidic acid may be a novel mechanism for dynamic membrane association and modulation of PLC in adult ventricular myocytes.

  2. Identify the key amino acid of BAFF binding with TACI.

    PubMed

    Wang, Renxi; Wang, Ru; Ma, Ning; Guo, Yueling; Xiao, He; Chen, Guojiang; Han, Gencheng; Hou, Chunmei; Shen, Beifen; Feng, Jiannan; Li, Yan

    2013-01-01

    B-cell activating factor (BAFF) has been used as a therapeutic target. To develop BAFF-specific small molecular inhibitors, it is necessary to know the key amino acid in the BAFF binding with its receptor. The key binding amino acid of BAFF interacting with its receptor TACI (trans-membrane activator and calcium modulator and cyclophilin ligand interactor) was analyzed based on the computer-guided molecular modeling method. According to theoretical prediction, a series of key amino acid mutants of BAFF, including M204 (Lys(204) to Ala), M208 (Met(208) to Ala), M209 (Gly(209) to Ala), M210 (His(210) to Ala), M234 (Gln(234) to Ala), M236 (Met(236) to Ala), and M237 (Pro(237) to Ala) were designed and evaluated with biological experiments. The results show that M208, M209, M236, and M237 of BAFF were the key amino acids and in accord with the theoretical results. The results highlight clues for the further development of BAFF-specific small molecular inhibitors.

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

    SciTech Connect

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

    1991-08-01

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

  4. Prediction of nucleic acid binding probability in proteins: a neighboring residue network based score.

    PubMed

    Miao, Zhichao; Westhof, Eric

    2015-06-23

    We describe a general binding score for predicting the nucleic acid binding probability in proteins. The score is directly derived from physicochemical and evolutionary features and integrates a residue neighboring network approach. Our process achieves stable and high accuracies on both DNA- and RNA-binding proteins and illustrates how the main driving forces for nucleic acid binding are common. Because of the effective integration of the synergetic effects of the network of neighboring residues and the fact that the prediction yields a hierarchical scoring on the protein surface, energy funnels for nucleic acid binding appear on protein surfaces, pointing to the dynamic process occurring in the binding of nucleic acids to proteins.

  5. Increases thermal stability and cellulose-binding capacity of Cryptococcus sp. S-2 lipase by fusion of cellulose binding domain derived from Trichoderma reesei

    SciTech Connect

    Thongekkaew, Jantaporn; Ikeda, Hiroko; Iefuji, Haruyuki

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer The CSLP and fusion enzyme were successfully expressed in the Pichia pastoris. Black-Right-Pointing-Pointer The fusion enzyme was stable at 80 Degree-Sign C for 120-min. Black-Right-Pointing-Pointer The fusion enzyme was responsible for cellulose-binding capacity. Black-Right-Pointing-Pointer The fusion enzyme has an attractive applicant for enzyme immobilization. -- Abstract: To improve the thermal stability and cellulose-binding capacity of Cryptococcus sp. S-2 lipase (CSLP), the cellulose-binding domain originates from Trichoderma reesei cellobiohydrolase I was engineered into C-terminal region of the CSLP (CSLP-CBD). The CSLP and CSLP-CBD were successfully expressed in the Pichia pastoris using the strong methanol inducible alcohol oxidase 1 (AOX1) promoter and the secretion signal sequence from Saccharomyces cerevisiae ({alpha} factor). The recombinant CSLP and CSLP-CBD were secreted into culture medium and estimated by SDS-PAGE to be 22 and 27 kDa, respectively. The fusion enzyme was stable at 80 Degree-Sign C and retained more than 80% of its activity after 120-min incubation at this temperature. Our results also found that the fusion of fungal exoglucanase cellulose-binding domain to CSLP is responsible for cellulose-binding capacity. This attribute should make it an attractive applicant for enzyme immobilization.

  6. Characterization of the binding sites for dicarboxylic acids on bovine serum albumin.

    PubMed Central

    Tonsgard, J H; Meredith, S C

    1991-01-01

    Dicarboxylic acids are prominent features of several diseases, including Reye's syndrome and inborn errors of mitochondrial and peroxisomal fatty acid oxidation. Moreover, dicarboxylic acids are potentially toxic to cellular processes. Previous studies [Tonsgard, Mendelson & Meredith (1988) J. Clin. Invest. 82, 1567-1573] demonstrated that long-chain dicarboxylic acids have a single high-affinity binding site and between one and three lower-affinity sites on albumin. Medium-chain-length dicarboxylic acids have a single low-affinity site. We further characterized dicarboxylic acid binding to albumin in order to understand the potential effects of drugs and other ligands on dicarboxylic acid binding and toxicity. Progesterone and oleate competitively inhibit octadecanedioic acid binding to the single high-affinity site. Octanoate inhibits binding to the low-affinity sites. Dansylated probes for subdomain 2AB inhibit dodecanedioic acid binding whereas probes for subdomain 3AB do not. In contrast, low concentrations of octadecanedioic acid inhibit the binding of dansylated probes to subdomain 3AB and 2AB. L-Tryptophan, which binds in subdomain 3AB, inhibits hexadecanedioic acid binding but has no effect on dodecanedioic acid. Bilirubin and acetylsalicylic acid, which bind in subdomain 2AB, inhibit the binding of medium-chain and long-chain dicarboxylic acids. Our results suggest that long-chain dicarboxylic acids bind in subdomains 2C, 3AB and 2AB. The single low-affinity binding site for medium-chain dicarboxylic acids is in subdomain 2AB. These studies suggest that dicarboxylic acids are likely to be unbound in disease states and may be potentially toxic. PMID:2064600

  7. Steam cooking significantly improves in vitro bile acid binding of collard greens, kale, mustard greens, broccoli, green bell pepper, and cabbage.

    PubMed

    Kahlon, Talwinder Singh; Chiu, Mei-Chen M; Chapman, Mary H

    2008-06-01

    Bile acid binding capacity has been related to the cholesterol-lowering potential of foods and food fractions. Lowered recirculation of bile acids results in utilization of cholesterol to synthesize bile acid and reduced fat absorption. Secondary bile acids have been associated with increased risk of cancer. Bile acid binding potential has been related to lowering the risk of heart disease and that of cancer. Previously, we have reported bile acid binding by several uncooked vegetables. However, most vegetables are consumed after cooking. How cooking would influence in vitro bile acid binding of various vegetables was investigated using a mixture of bile acids secreted in human bile under physiological conditions. Eight replicate incubations were conducted for each treatment simulating gastric and intestinal digestion, which included a substrate only, a bile acid mixture only, and 6 with substrate and bile acid mixture. Cholestyramine (a cholesterol-lowering, bile acid binding drug) was the positive control treatment and cellulose was the negative control. Relative to cholestyramine, in vitro bile acid binding on dry matter basis was for the collard greens, kale, and mustard greens, 13%; broccoli, 10%; Brussels sprouts and spinach, 8%; green bell pepper, 7%; and cabbage, 5%. These results point to the significantly different (P < or = .05) health-promoting potential of collard greens = kale = mustard greens > broccoli > Brussels sprouts = spinach = green bell pepper > cabbage as indicated by their bile acid binding on dry matter basis. Steam cooking significantly improved the in vitro bile acid binding of collard greens, kale, mustard greens, broccoli, green bell pepper, and cabbage compared with previously observed bile acid binding values for these vegetables raw (uncooked). Inclusion of steam-cooked collard greens, kale, mustard greens, broccoli, green bell pepper, and cabbage in our daily diet as health-promoting vegetables should be emphasized. These green

  8. Expression of the high capacity calcium-binding domain of calreticulin increases bioavailable calcium stores in plants

    NASA Technical Reports Server (NTRS)

    Wyatt, Sarah E.; Tsou, Pei-Lan; Robertson, Dominique; Brown, C. S. (Principal Investigator)

    2002-01-01

    Modulation of cytosolic calcium levels in both plants and animals is achieved by a system of Ca2+-transport and storage pathways that include Ca2+ buffering proteins in the lumen of intracellular compartments. To date, most research has focused on the role of transporters in regulating cytosolic calcium. We used a reverse genetics approach to modulate calcium stores in the lumen of the endoplasmic reticulum. Our goals were two-fold: to use the low affinity, high capacity Ca2+ binding characteristics of the C-domain of calreticulin to selectively increase Ca2+ storage in the endoplasmic reticulum, and to determine if those alterations affected plant physiological responses to stress. The C-domain of calreticulin is a highly acidic region that binds 20-50 moles of Ca2+ per mole of protein and has been shown to be the major site of Ca2+ storage within the endoplasmic reticulum of plant cells. A 377-bp fragment encoding the C-domain and ER retention signal from the maize calreticulin gene was fused to a gene for the green fluorescent protein and expressed in Arabidopsis under the control of a heat shock promoter. Following induction on normal medium, the C-domain transformants showed delayed loss of chlorophyll after transfer to calcium depleted medium when compared to seedlings transformed with green fluorescent protein alone. Total calcium measurements showed a 9-35% increase for induced C-domain transformants compared to controls. The data suggest that ectopic expression of the calreticulin C-domain increases Ca2+ stores, and that this Ca2+ reserve can be used by the plant in times of stress.

  9. Effects of linoleic and gamma-linolenic acids (efamol evening primrose oil) on fatty acid-binding proteins of rat liver.

    PubMed

    Dutta-Roy, A K; Demarco, A C; Raha, S K; Shay, J; Garvey, M; Horrobin, D F

    We have studied the effects of Efamol evening primrose oil (EPO) on fatty acid-binding proteins (L-FABP) of rat liver. EPO contains 72% cis-linoleic acid and 9% cis-gamma linolenic acid. EPO has been clinically used for treatment of a number of diseases in humans and animals. EPO is also known to lower cholesterol level in humans and animals. Feeding of an EPO supplemented diet to rats (n = 9) for 2 months decreases the oleate binding capacity of purified L-FABP of rat liver whereas the palmitate binding activity was increased by 38%. However, EPO feeding did not alter the L-FABP concentrations significantly as measured by using the fluorescence fatty acid probe, dansylamino undecanoic acid. Endogenous fatty acid analysis of L-FABPs revealed significant qualitative and quantitative changes in fatty acid pattern after EPO feeding. EPO feeding decreased the endogenous palmitate level by 53% and oleate level by 64% in L-FABPs and also EPO feeding decreased the total endogenous fatty acid content from 62 nanomole per mg of protein to 42 nanomole per mg of L-FABP (n = 3).

  10. In vitro bile acid binding of mustard greens, kale, broccoli, cabbage and green bell pepper improves with sautéing compared with raw or other methods of preparation.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bile acid binding capacity has been related to cholesterol-lowering potential of foods and food fractions. Lowered recirculating bile acids results in utilization of cholesterol to synthesize bile acid and reduced fat absorption. Secondary bile acids have been associated with increased risk of can...

  11. Liver fatty acid binding protein is required for high rates of hepatic fatty acid oxidation but not for the action of PPARalpha in fasting mice.

    PubMed

    Erol, Erdal; Kumar, Leena S; Cline, Gary W; Shulman, Gerald I; Kelly, Daniel P; Binas, Bert

    2004-02-01

    Liver fatty acid binding protein (L-FABP) has been proposed to limit the availability of long-chain fatty acids (LCFA) for oxidation and for peroxisome proliferator-activated receptor alpha (PPAR-alpha), a fatty acid binding transcription factor that determines the capacity of hepatic fatty acid oxidation. Here, we used L-FABP null mice to test this hypothesis. Under fasting conditions, this mutation reduced beta-hydroxybutyrate (BHB) plasma levels as well as BHB release and palmitic acid oxidation by isolated hepatocytes. However, the capacity for ketogenesis was not reduced: BHB plasma levels were restored by octanoate injection; BHB production and palmitic acid oxidation were normal in liver homogenates; and hepatic expression of key PPAR-alpha target (MCAD, mitochondrial HMG CoA synthase, ACO, CYP4A3) and other (CPT1, LCAD) genes of mitochondrial and extramitochondrial LCFA oxidation and ketogenesis remained at wild-type levels. During standard diet, mitochondrial HMG CoA synthase mRNA was selectively reduced in L-FABP null liver. These results suggest that under fasting conditions, hepatic L-FABP contributes to hepatic LCFA oxidation and ketogenesis by a nontranscriptional mechanism, whereas L-FABP can activate ketogenic gene expression in fed mice. Thus, the mechanisms whereby L-FABP affects fatty acid oxidation may vary with physiological condition.

  12. Structure and oxidation capacity of amino acid chloramine derivatives and their effects on platelet aggregation.

    PubMed

    Murina, M A; Chudina, N A; Roshchupkin, D I; Belakina, N S; Sergienko, V I

    2004-12-01

    Comparison of antiaggregation capacity of N-chloramine acids with different position of the chloramine group in the molecule showed that in the most efficient compounds the distance between the chloramine and carboxyl groups was 3-5 carbon atoms. This feature of antiaggregation activity was not related to the difference in oxidation capacity of N-chloramine acids. It was hypothesized that the revealed structural dependence of antiaggregation activity of N-chloramine acids is determined by the structure of platelet membrane, in particular, the presence of a negatively charged group near the site of interaction between N-chloramine acids and platelet membrane.

  13. The p.R1819_C1948delinsS mutation makes von Willebrand factor ADAMTS13-resistant and reduces its collagen-binding capacity.

    PubMed

    Daidone, Viviana; Saga, Giorgia; Barbon, Giovanni; Pontara, Elena; Cattini, Maria G; Morpurgo, Margherita; Zanotti, Giuseppe; Casonato, Alessandra

    2015-08-01

    This report concerns abnormal ADAMTS13 (a disintegrin and metalloprotease with a thrombospondin type 1 motif, member 13) and collagen interactions coinciding with the p.R1819_C1948delinsS von Willebrand factor (VWF) mutation associated with the deletion of the C-terminus of the A3 domain (amino acids 1819-1947) in a patient with a history of bleeding. The von Willebrand disease (VWD) phenotype of the patient featured low plasma and platelet VWF, multimers with smears extending over the highest normal oligomers in plasma, but not platelets, and an impaired collagen-binding capacity. In vitro full-length p.R1819_C1948delinsS VWF expression showed impaired VWF release, increased cellular content with normally-multimerized VWF and impaired collagen binding. The recombinant p.R1819_C1948delinsS VWF fragment, extending from domains A2 to B3 (p.R1819_C1948delinsS A2-B3 VWF), was completely resistant to proteolysis by ADAMTS13 in the presence of 1·5 mol/l urea, unlike its normal counterpart. The defect stems from impaired ADAMTS13 binding to p.R1819_C1948delinsS A2-B3, analysed under static conditions. Partial deletion of the C-terminus of the A3 domain thus makes VWF resistant to ADAMTS13, interfering with ADAMTS13 binding to VWF, and impairing the collagen-binding capacity of VWF. The p.R1819_C1948delinsS mutation has both haemorrhagic features (defective collagen binding, reduced VWF levels) and prothrombotic (ADAMTS13 resistance) features, and the latter probably mitigate the patient's bleeding symptoms.

  14. Proton-binding study of standard and reference fulvic acids, humic acids, and natural organic matter

    NASA Astrophysics Data System (ADS)

    Ritchie, Jason D.; Perdue, E. Michael

    2003-01-01

    The acid-base properties of 14 standard and reference materials from the International Humic Substances Society (IHSS) were investigated by potentiometric titration. Titrations were conducted in 0.1 M NaCl under a nitrogen atmosphere, averaging 30 min from start to finish. Concentrations of carboxyl groups and phenolic groups were estimated directly from titration curves. Titration data were also fit to a modified Henderson-Hasselbalch model for two classes of proton-binding sites to obtain "best fit" parameters that describe proton-binding curves for the samples. The model was chosen for its simplicity, its ease of implementation in computer spreadsheets, and its excellent ability to describe the shapes of the titration curves. The carboxyl contents of the IHSS samples are in the general order: terrestrial fulvic acids > aquatic fulvic acids > Suwannee River natural organic matter (NOM) > aquatic humic acids > terrestrial humic acids. Overall, fulvic acids and humic acids have similar phenolic contents; however, all of the aquatically derived samples have higher phenolic contents than the terrestrially derived samples. The acid-base properties of reference Suwannee River NOM are surprisingly similar to those of standard Suwannee River humic acid. Results from titrations in this study were compared with other published results from both direct and indirect titrations. Typically, carboxyl contents for the IHSS samples were in agreement with the results from both methods of titration. Phenolic contents for the IHSS samples were comparable to those determined by direct titrations, but were significantly less than estimates of phenolic content that were based on indirect titrations with Ba(OH) 2 and Ca(OAc) 2. The average phenolic-to-carboxylic ratio of the IHSS samples is approximately 1:4. Models that assume a 1:2 ratio of phenolic-to-carboxylic groups may overestimate the relative contribution of phenolic groups to the acid-base chemistry of humic substances.

  15. NMR studies reveal the role of biomembranes in modulating ligand binding and release by intracellular bile acid binding proteins.

    PubMed

    Pedò, Massimo; Löhr, Frank; D'Onofrio, Mariapina; Assfalg, Michael; Dötsch, Volker; Molinari, Henriette

    2009-12-18

    Bile acid molecules are transferred vectorially between basolateral and apical membranes of hepatocytes and enterocytes in the context of the enterohepatic circulation, a process regulating whole body lipid homeostasis. This work addresses the role of the cytosolic lipid binding proteins in the intracellular transfer of bile acids between different membrane compartments. We present nuclear magnetic resonance (NMR) data describing the ternary system composed of the bile acid binding protein, bile acids, and membrane mimetic systems, such as anionic liposomes. This work provides evidence that the investigated liver bile acid binding protein undergoes association with the anionic membrane and binding-induced partial unfolding. The addition of the physiological ligand to the protein-liposome mixture is capable of modulating this interaction, shifting the equilibrium towards the free folded holo protein. An ensemble of NMR titration experiments, based on nitrogen-15 protein and ligand observation, confirm that the membrane and the ligand establish competing binding equilibria, modulating the cytoplasmic permeability of bile acids. These results support a mechanism of ligand binding and release controlled by the onset of a bile salt concentration gradient within the polarized cell. The location of a specific protein region interacting with liposomes is highlighted.

  16. Biothermodynamic characterization of monocarboxylic and dicarboxylic aliphatic acids binding to human serum albumin: a flow microcalorimetric study.

    PubMed

    Aki, H; Yamamoto, M

    1993-02-01

    Thermodynamic parameters have been evaluated for the binding of unbranched monocarboyxlic aliphatic acids (MCAs) of 4 to 16 carbons (MC4 to MC16) and dicarboxylic aliphatic acids (DCAs) of 4 to 16 carbons (DC4 to DC16) to human serum albumin (HSA) on the basis of microcalorimetric measurement at pH 7.4 and 37 degrees C by computer-fitting to single- and two-class binding models. Long-chain MCAs (MC10 to MC16) and DCAs (DC14 and DC16) had the first class of binding sites with high affinity (large binding constant) of 10(5) to 10(6) M-1 and the second class with lower affinity and high capacity (large numbers of binding sites). Short- or medium-chain MCAs and DCAs bound to HSA at some low affinity binding sites. The binding constants of MCAs were ten times larger than those of DCAs. All the relationships between the thermodynamic parameters and alkyl-chain length of the acids showed clear-cut inflections in their plots around eight or nine methylene units. The free energy change of the first class of binding sites (- delta G1) became more negative with an increment of -1.0 kJ mol-1 CH2(-1) as the alkyl-chain length increased, but there were steep rises between MC9 and MC11 with -2.90 kJ mol-1 CH2(-1) and between DC9 and DC12 with -2.02 kJ mol-1 CH2(-1). The enthalpy change (- delta H) increased at the rate of -7.4 kJ mol-1 CH2(-1) to the maximum at MC9 and DC10, then decreased due to hydrophobicity of the alkyl-chains. From compensation analyses (delta H vs. delta S and delta G), HSA binding sites were characterized into three groups.

  17. A Sialic Acid Binding Site in a Human Picornavirus

    PubMed Central

    Frank, Martin; Hähnlein-Schick, Irmgard; Ekström, Jens-Ola; Arnberg, Niklas; Stehle, Thilo

    2014-01-01

    The picornaviruses coxsackievirus A24 variant (CVA24v) and enterovirus 70 (EV70) cause continued outbreaks and pandemics of acute hemorrhagic conjunctivitis (AHC), a highly contagious eye disease against which neither vaccines nor antiviral drugs are currently available. Moreover, these viruses can cause symptoms in the cornea, upper respiratory tract, and neurological impairments such as acute flaccid paralysis. EV70 and CVA24v are both known to use 5-N-acetylneuraminic acid (Neu5Ac) for cell attachment, thus providing a putative link between the glycan receptor specificity and cell tropism and disease. We report the structures of an intact human picornavirus in complex with a range of glycans terminating in Neu5Ac. We determined the structure of the CVA24v to 1.40 Å resolution, screened different glycans bearing Neu5Ac for CVA24v binding, and structurally characterized interactions with candidate glycan receptors. Biochemical studies verified the relevance of the binding site and demonstrated a preference of CVA24v for α2,6-linked glycans. This preference can be rationalized by molecular dynamics simulations that show that α2,6-linked glycans can establish more contacts with the viral capsid. Our results form an excellent platform for the design of antiviral compounds to prevent AHC. PMID:25329320

  18. LRRK2 kinase activity is dependent on LRRK2 GTP binding capacity but independent of LRRK2 GTP binding.

    PubMed

    Taymans, Jean-Marc; Vancraenenbroeck, Renée; Ollikainen, Petri; Beilina, Alexandra; Lobbestael, Evy; De Maeyer, Marc; Baekelandt, Veerle; Cookson, Mark R

    2011-01-01

    Leucine rich repeat kinase 2 (LRRK2) is a Parkinson's disease (PD) gene that encodes a large multidomain protein including both a GTPase and a kinase domain. GTPases often regulate kinases within signal transduction cascades, where GTPases act as molecular switches cycling between a GTP bound "on" state and a GDP bound "off" state. It has been proposed that LRRK2 kinase activity may be increased upon GTP binding at the LRRK2 Ras of complex proteins (ROC) GTPase domain. Here we extensively test this hypothesis by measuring LRRK2 phosphorylation activity under influence of GDP, GTP or non-hydrolyzable GTP analogues GTPγS or GMPPCP. We show that autophosphorylation and lrrktide phosphorylation activity of recombinant LRRK2 protein is unaltered by guanine nucleotides, when co-incubated with LRRK2 during phosphorylation reactions. Also phosphorylation activity of LRRK2 is unchanged when the LRRK2 guanine nucleotide binding pocket is previously saturated with various nucleotides, in contrast to the greatly reduced activity measured for the guanine nucleotide binding site mutant T1348N. Interestingly, when nucleotides were incubated with cell lysates prior to purification of LRRK2, kinase activity was slightly enhanced by GTPγS or GMPPCP compared to GDP, pointing to an upstream guanine nucleotide binding protein that may activate LRRK2 in a GTP-dependent manner. Using metabolic labeling, we also found that cellular phosphorylation of LRRK2 was not significantly modulated by nucleotides, although labeling is significantly reduced by guanine nucleotide binding site mutants. We conclude that while kinase activity of LRRK2 requires an intact ROC-GTPase domain, it is independent of GDP or GTP binding to ROC.

  19. Tested Demonstrations: Buffer Capacity of Various Acetic Acid-Sodium Acetate Systems: A Lecture Experiment.

    ERIC Educational Resources Information Center

    Donahue, Craig J.; Panek, Mary G.

    1985-01-01

    Background information and procedures are provided for a lecture experiment which uses indicators to illustrate the concept of differing buffer capacities by titrating acetic acid/sodium acetate buffers with 1.0 molar hydrochloric acid and 1.0 molar sodium hydroxide. A table with data used to plot the titration curve is included. (JN)

  20. Adrenocortical nuclear progesterone-binding protein: Identification by photoaffinity labeling and evidence for deoxyribonucleic acid binding and stimulation by adrenocorticotropin

    SciTech Connect

    Demura, T.; Driscoll, W.J.; Lee, Y.C.; Strott, C.A. )

    1991-01-01

    Nuclei of the guinea pig adrenal cortex contain a protein that specifically binds progesterone and that, biochemically, is clearly distinct from the classical progesterone receptor. The adrenocortical nuclear progesterone-binding protein has now been purified more than 2000-fold by steroid-affinity chromatography with a 75% yield. The purified protein preparation demonstrated three major bands on sodium dodecyl sulfate-polyacrylamide gel of 79K, 74K, and 50K. To determine which of the three might represent the progesterone-binding protein, steroid photoaffinity labeling was performed which resulted in the specific and exclusive labeling of a 50K band. Thus, the adrenocortical nuclear progesterone-binding protein appears to be distinct from the classical progesterone receptor not only biochemically, but also on the basis of molecular size. To test whether the adrenocortical nuclear progesterone-binding protein can be hormonally stimulated, guinea pigs were treated with ACTH. The chronic administration of ACTH caused a 4- to 6-fold increase in the specific progesterone binding capacity without a change in the binding affinity. There appeared to be no significant difference in nuclear progesterone binding between the zona fasciculata and zona reticularis. This finding suggests a mediating role for the progesterone-binding protein in ACTH action. In addition, the nuclear progesterone-binding protein bound to nonspecific DNA sequences, further suggesting a possible transcriptional regulatory role.

  1. Uranium Binding Mechanisms of the Acid-Tolerant Fungus Coniochaeta fodinicola.

    PubMed

    Vázquez-Campos, Xabier; Kinsela, Andrew S; Collins, Richard N; Neilan, Brett A; Aoyagi, Noboru; Waite, T David

    2015-07-21

    The uptake and binding of uranium [as (UO2)(2+)] by a moderately acidophilic fungus, Coniochaeta fodinicola, recently isolated from a uranium mine site, is examined in this work in order to better understand the potential impact of organisms such as this on uranium sequestration in hydrometallurgical systems. Our results show that the viability of the fungal biomass is critical to their capacity to remove uranium from solution. Indeed, live biomass (viable cells based on vital staining) were capable of removing ∼16 mg U/g dry weight in contrast with dead biomass (autoclaved) which removed ∼45 mg U/g dry weight after 2 h. Furthermore, the uranium binds with different strength, with a fraction ranging from ∼20-50% being easily leached from the exposed biomass by a 10 min acid wash. Results from X-ray absorption spectroscopy measurements show that the strength of uranium binding is strongly influenced by cell viability, with live cells showing a more well-ordered uranium bonding environment, while the distance to carbon or phosphorus second neighbors is similar in all samples. When coupled with time-resolved laser fluorescence and Fourier transformed infrared measurements, the importance of organic acids, phosphates, and polysaccharides, likely released with fungal cell death, appear to be the primary determinants of uranium binding in this system. These results provide an important progression to our understanding with regard to uranium sequestration in hydrometallurgical applications with implications to the unwanted retention of uranium in biofilms and/or its mobility in a remediation context.

  2. Reversible binding of ethacrynic acid to human serum albumin: difference circular dichroism study.

    PubMed

    Bertucci, C; Nanni, B; Salvadori, P

    1999-01-01

    The reversible binding of ethacrynic acid was characterized by a difference circular dichroism method. A 2/1 stoichiometry was determined for the [drug]/[HSA] (human serum albumin) complex. The reversible binding of ethacrynic acid to HSA determines direct competition with ligands that selectivity bind to site II and to the fatty acid site. Furthermore, indirect competition was shown for ligands for site I (anti-cooperative) and to site III (cooperative).

  3. Identification of novel PTEN-binding partners: PTEN interaction with fatty acid binding protein FABP4.

    PubMed

    Gorbenko, O; Panayotou, G; Zhyvoloup, A; Volkova, D; Gout, I; Filonenko, V

    2010-04-01

    PTEN is a tumor suppressor with dual protein and lipid-phosphatase activity, which is frequently deleted or mutated in many human advanced cancers. Recent studies have also demonstrated that PTEN is a promising target in type II diabetes and obesity treatment. Using C-terminal PTEN sequence in pEG202-NLS as bait, yeast two-hybrid screening on Mouse Embryo, Colon Cancer, and HeLa cDNA libraries was carried out. Isolated positive clones were validated by mating assay and identified through automated DNA sequencing and BLAST database searches. Sequence analysis revealed a number of PTEN-binding proteins linking this phosphatase to a number of different signaling cascades, suggesting that PTEN may perform other functions besides tumor-suppressing activity in different cell types. In particular, the interplay between PTEN function and adipocyte-specific fatty-acid-binding protein FABP4 is of notable interest. The demonstrable tautology of PTEN to FABP4 suggested a role for this phosphatase in the regulation of lipid metabolism and adipocyte differentiation. This interaction was further studied using coimmunoprecipitation and gel-filtration assays. Finally, based on Biacore assay, we have calculated the K(D) of PTEN-FABP4 complex, which is around 2.8 microM.

  4. Alteration of the Copper-Binding Capacity of Iron-Rich Humic Colloids during Transport from Peatland to Marine Waters.

    PubMed

    Muller, François L L; Cuscov, Marco

    2017-02-28

    Blanket bogs contain vast amounts of Sphagnum-derived organic substances which can act as powerful chelators for dissolved iron and thus enhance its export to the coastal ocean. To investigate the variations in quantity and quality of these exports, adsorptive cathodic stripping voltammetry (CSV) was used to characterize the metal binding properties of molecular weight-fractionated dissolved organic matter (MW-fractionated DOM) in the catchment and coastal plume of a small peat-draining river over a seasonal cycle. Within the plume, both iron- and copper-binding organic ligands showed a linear, conservative distribution with increasing salinity, illustrating the high stability of peatland-derived humic substances (HS). Within the catchment, humic colloids lost up to 50% of their copper-binding capacity, expressed as a molar ratio to organic carbon, after residing for 1 week or more in the main reservoir of the catchment. Immediately downstream of the reservoir, the molar ratio [L2]/[Corg], where L2 was the second strongest copper-binding ligand, was 0.75 × 10(-4) when the reservoir residence time was 5 h but 0.34 × 10(-4) when it was 25 days. Residence time did not affect the carbon specific iron-binding capacity of the humic substances which was [L]/[Corg] = (0.80 ± 0.20) × 10(-2). Our results suggest that the loss of copper-binding capacity with increasing residence time is caused by intracolloidal interactions between iron and HS during transit from peat soil to river mouth.

  5. Phenolic acid induced growth of gold nanoshells precursor composites and their application in antioxidant capacity assay.

    PubMed

    Ma, Xiaoyuan; Qian, Weiping

    2010-11-15

    In the present work, the gold nanoshells (GNSs) precursor composites were preadsorbed onto the surface of ITO substrates. With the treatment of modified electrodes immersed in the gold nanoparticles (GNPs) growth solution containing different phenolic acids, the GNSs precursor composites were enlarged to varying degrees. Phenolic acids with one or more phenolic hydroxyl groups served as reductants for the growth of GNPs. The enlargement conditions varied with the different reducing capacity of phenolic acids, exhibiting specific morphologies differ from the complete GNSs. Consequently, the UV-vis-NIR spectra and cyclic voltammetry curves for the phenolic acid-treated ITO electrode were gradually changed. Results showed that the higher reducing capacity for phenolic acid to reduce AuCl(4)(-) to Au(0) resulted in the intensified localized surface plasmon resonance features and reduced cathodic currents. The spectral wavelength peaks red shifted hundreds of nanometers across the visible region. Moreover, the antioxidant capacity of phenolic acids correlates well with their reducing activity, both of which reflect their tendency to donate electrons. Thus, the optical and electrochemical results could be used to evaluate the antioxidant capacity of phenolic acids by utilizing GNSs precursor composites as nanoprobes. The method is simple, rapid and could be used in visual analysis to a certain extent.

  6. CD44 Binding to Hyaluronic Acid Is Redox Regulated by a Labile Disulfide Bond in the Hyaluronic Acid Binding Site

    PubMed Central

    Kellett-Clarke, Helena; Stegmann, Monika; Barclay, A. Neil; Metcalfe, Clive

    2015-01-01

    CD44 is the primary leukocyte cell surface receptor for hyaluronic acid (HA), a component of the extracellular matrix. Enzymatic post translational cleavage of labile disulfide bonds is a mechanism by which proteins are structurally regulated by imparting an allosteric change and altering activity. We have identified one such disulfide bond in CD44 formed by Cys77 and Cys97 that stabilises the HA binding groove. This bond is labile on the surface of leukocytes treated with chemical and enzymatic reducing agents. Analysis of CD44 crystal structures reveal the disulfide bond to be solvent accessible and in the–LH hook configuration characteristic of labile disulfide bonds. Kinetic trapping and binding experiments on CD44-Fc chimeric proteins show the bond is preferentially reduced over the other disulfide bonds in CD44 and reduction inhibits the CD44-HA interaction. Furthermore cells transfected with CD44 no longer adhere to HA coated surfaces after pre-treatment with reducing agents. The implications of CD44 redox regulation are discussed in the context of immune function, disease and therapeutic strategies. PMID:26379032

  7. Ascorbic acid enables reversible dopamine receptor /sup 3/H-agonist binding

    SciTech Connect

    Leff, S.; Sibley, D.R.; Hamblin, M.; Creese, I.

    1981-11-16

    The effects of ascorbic acid on dopaminergic /sup 3/H-agonist receptor binding were studied in membrane homogenates of bovine anterior pituitary and caudate, and rat striatum. In all tissues virtually no stereospecific binding (defined using 1uM (+)butaclamol) of the /sup 3/H-agonists N-propylnorapomorphine (NPA), apomorphine, or dopamine could be demonstrated in the absence of ascorbic acid. Although levels of total /sup 3/H-agonist binding were three to five times greater in the absence than in the presence of 0.1% ascorbic acid, the increased binding was entirely non-stereospecific. Greater amounts of dopamine-inhibitable /sup 3/H-NPA binding could be demonstrated in the absence of 0.1% ascorbic acid, but this measure of ''specific binding'' was demonstrated not to represent dopamine receptor binding since several other catecholamines and catechol were equipotent with dopamine and more potent than the dopamine agonist (+/-)amino-6,7-dihydroxy-1,2,3,4-tetrahydronapthalene (ADTN) in inhibiting this binding. High levels of dopamine-displaceable /sup 3/H-agonist binding were detected in fresh and boiled homogenates of cerebellum, an area of brain which receives no dopaminergic innervation, further demonstrating the non-specific nature of /sup 3/H-agonist binding in the absence of ascorbic acid. These studies emphasize that under typical assay conditions ascorbic acid is required in order to demonstrate reversible and specific /sup 3/H-agonist binding to dopamine receptors.

  8. Models of metal binding structures in fulvic acid from the Suwannee River, Georgia

    USGS Publications Warehouse

    Leenheer, J.A.; Brown, G.K.; MacCarthy, P.; Cabaniss, S.E.

    1998-01-01

    Fulvic acid, isolated from the Suwannee River, Georgia, was assessed for its ability to bind Ca2+, Cd2+, Cu2+, Ni2+, and Zn2+ ions at pH 6 before and after extensive fractionation that was designed to reveal the nature of metal binding functional groups. The binding constant for Ca2+ ion had the greatest increase of all the ions in a metal binding fraction that was selected for intensive characterization for the purpose of building quantitative average model structures. The 'metal binding' fraction was characterized by quantitative 13C NMR, 1H NMR, and FT-1R spectrometry and elemental, titrimetric, and molecular weight determinations. The characterization data revealed that carboxyl groups were clustered in short- chain aliphatic dibasic acid structures. The Ca2+ binding data suggested that ether-substituted oxysuccinic acid structures are good models for the metal binding sites at pH 6. Structural models were derived based upon oxidation and photolytic rearrangements of cutin, lignin, and tannin precursors. These structural models rich in substituted dibasic acid structures revealed polydentate binding sites with the potential for both inner-sphere and outer-sphere type binding. The majority of the fulvic acid molecule was involved with metal binding rather than a small substructural unit.Fulvic acid, isolated from the Suwannee River, Georgia, was assessed for its ability to bind Ca2+, Cd2+, Cu2+, Ni2+, and Zn2+ ions at pH 6 before and after extensive fractionation that was designed to reveal the nature of metal binding functional groups. The binding constant for Ca2+ ion had the greatest increase of all the ions in a metal binding fraction that was selected for intensive characterization for the purpose of building quantitative average model structures. The `metal binding' fraction was characterized by quantitative 13C NMR, 1H NMR, and FT-IR spectrometry and elemental, titrimetric, and molecular weight determinations. The characterization data revealed that

  9. Ab initio Design of Ca-Decorated Organic Frameworks for High Capacity Molecular Hydrogen Storage with Enhanced Binding

    SciTech Connect

    Sun, Y. Y.; Lee, K.; Kim, Y. H.; Zhang, S. B.

    2009-01-01

    Ab initio calculations show that Ca can decorate organic linkers of metal-organic framework, MOF-5, with a binding energy of 1.25 eV. The Ca-decorated MOF-5 can store molecular hydrogen (H{sub 2}) in both high gravimetric (4.6 wt %) and high volumetric (36 g/l) capacities. Even higher capacities (5.7 wt % and 45 g/l) can be obtained in a rationally designed covalent organic framework system, COF-{alpha}, with decorated Ca. Both density functional theory and second-order Moller-Plesset perturbation calculations show that the H{sub 2} binding in these systems is significantly stronger than the van der Waals interactions, which is required for H{sub 2} storage at near ambient conditions.

  10. Nucleic acid binding property of the gene products of rice stripe virus.

    PubMed

    Liang, Delin; Ma, Xiangqiang; Qu, Zhicai; Hull, Roger

    2005-10-01

    GST fusion proteins of the six gene products from RNAs 2,3 and 4 of the tenuivirus, Rice stripe virus (RSV), were used to study the nucleic acid binding activities in vitro. Three of the proteins, p3, pc3 and pc4, bound both single- and double-stranded cDNA of RSV RNA4 and also RNA3 transcribed from its cDNA clone, while p2, pc2-N (the N-terminal part of pc2) nor p4 bound the cDNA or RNA transcript. The binding activity of p3 is located in the carboxyl-terminus amino acid 154-194, which contains basic amino acid rich beta-sheets. The acidic amino acid-rich amino-terminus (amino acids 1-100) of p3 did not have nucleic acid binding activity. The related analogous gene product of the tenuivirus, Rice hoja blanca virus, is a suppressor of gene silencing and the possibility of the nucleic acid binding ability of RSV p3 being associated with this property is discussed. The C-terminal part of the RSV nucleocapsid protein, which also contains a basic region, binds nucleic acids, which is consistent with its function. The central and C-terminal regions of pc4 bind nucleic acid. It has been suggested that this protein is a cell-to-cell movement protein and nucleic acid binding would be in accord with this function.

  11. Elucidating the influence of gold nanoparticles on the binding of salvianolic acid B and rosmarinic acid to bovine serum albumin.

    PubMed

    Peng, Xin; Qi, Wei; Huang, Renliang; Su, Rongxin; He, Zhimin

    2015-01-01

    Salvianolic acid B and rosmarinic acid are two main water-soluble active ingredients from Salvia miltiorrhiza with important pharmacological activities and clinical applications. The interactions between salvianolic acid B (or rosmarinic acid) and bovine serum albumin (BSA) in the presence and absence of gold nanoparticles (Au NPs) with three different sizes were investigated by using biophysical methods for the first time. Experimental results proved that two components quenched the fluorescence of BSA mainly through a static mechanism irrespective of the absence or presence of Au NPs. The presence of Au NPs decreased the binding constants of salvianolic acid B with BSA from 27.82% to 10.08%, while Au NPs increased the affinities of rosmarinic acid for BSA from 0.4% to 14.32%. The conformational change of BSA in the presence of Au NPs (caused by a noncompetitive binding between Au NPs and drugs at different albumin sites) induced changeable affinity and binding distance between drugs and BSA compared with no Au NPs. The competitive experiments revealed that the site I (subdomain IIA) of BSA was the primary binding site for salvianolic acid B and rosmarinic acid. Additionally, two compounds may induce conformational and micro-environmental changes of BSA. The results would provide valuable binding information between salvianolic acid B (or rosmarinic acid) and BSA, and also indicated that the Au NPs could alter the interaction mechanism and binding capability of drugs to BSA, which might be beneficial to understanding the pharmacokinetics and biological activities of the two drugs.

  12. Effects of soil salinity on the content, composition, and ion binding capacity of glomalin-related soil protein (GRSP).

    PubMed

    Zhang, Zhonghua; Wang, Qiong; Wang, Hua; Nie, Siming; Liang, Zhengwei

    2017-03-01

    Soil aggregation, an ecosystem function correlated with the concentration of glomalin-related soil protein (GRSP), is highly disturbed in saline soil. However, few studies have focused on differences in amount, composition, and ion binding capacity of GRSP in typical sodic-saline soils. In this study, a field study was performed in Songnen Plain. Combined indicators of soil salinity (Q value) were significant negatively correlated with GRSP concentration by Principal Component Analysis. Multiple linear regression models showed that soil salinity might account for 46%, 25% and 44% variation in total GRSP (T-GRSP), easily-extractable GRSP (EE-GRSP) and difficultly-extractable GRSP (DE-GRSP), respectively. Soil bulk density had most important impact on GRSP concentration, followed by the pH, soil EC had the weak influence. Comparative analysis was carried out between low-salinity and high-salinity soil. Purified T-GRSP of high-saline soil contained higher N content (13.13%), lower C content (43.41%) and lower functional groups relative content (e.g. CO and SiOSi). Purified T-GRSP of high-salinity soil had a greater binding capacity with calcium and phosphorus, the binding capacity could compensate the GRSP loss about 29.8% and 14.1%, respectively. Our findings suggested that sodic salinization of the soil led to a decrease in GRSP concentration and a change in the component percentages. This change in composition might be related to adaptation of fungi-plant systems to varied environments. The calcium and phosphorus binding capacity had a positive dependent of soil salinization, which was possible to develop ecological management or recovery technology in the future.

  13. Ligand specificity and conformational stability of human fatty acid-binding proteins.

    PubMed

    Zimmerman, A W; van Moerkerk, H T; Veerkamp, J H

    2001-09-01

    Fatty acid binding proteins (FABPs) are small cytosolic proteins with virtually identical backbone structures that facilitate the solubility and intracellular transport of fatty acids. At least eight different types of FABP occur, each with a specific tissue distribution and possibly with a distinct function. To define the functional characteristics of all eight human FABPs, viz. heart (H), brain (B), myelin (M), adipocyte (A), epidermal (E), intestinal (I), liver (L) and ileal lipid-binding protein (I-LBP), we studied their ligand specificity, their conformational stability and their immunological crossreactivity. Additionally, binding of bile acids to I-LBP was studied. The FABP types showed differences in fatty acid binding affinity. Generally, the affinity for palmitic acid was lower than for oleic and arachidonic acid. All FABP types, except E-FABP, I-FABP and I-LBP interacted with 1-anilinonaphtalene-8-sulphonic acid (ANS). Only L-FABP, I-FABP and M-FABP showed binding of 11-((5-dimethylaminonaphtalene-1-sulfonyl)amino)undecanoic acid (DAUDA). I-LBP showed increasing binding of bile acids in the order taurine-conjugated>glycine-conjugated>unconjugated bile acids. A hydroxylgroup of bile acids at position 7 decreased and at position 12 increased the binding affinity to I-LBP. The fatty acid-binding affinity and the conformation of FABP types were differentially affected in the presence of urea. Our results demonstrate significant differences in ligand binding, conformational stability and surface properties between different FABP types which may point to a specific function in certain cells and tissues. The preference of I-LBP (but not L-FABP) for conjugated bile acids is in accordance with a specific role in bile acid reabsorption in the ileum.

  14. Drug-binding properties of rat alpha 1-foetoprotein. Binding of warfarin, phenylbutazone, azapropazone, diazepam, digitoxin and cholic acid.

    PubMed Central

    Hervé, F; Rajkowski, K; Martin, M T; Dessen, P; Cittanova, N

    1984-01-01

    As part of an investigation into whether alpha 1-foetoprotein (alpha 1-FP) plays the same transport role in foetal serum as albumin does in the adult, the binding properties of both proteins were compared with respect to the binding of a series of compounds known to be bound by albumin's specific drug-binding sites. The binding of warfarin, phenylbutazone, azapropazone, diazepam, digitoxin and cholic acid by rat alpha 1-FP and serum albumin was studied by equilibrium dialysis at 4 degrees C. Rat alpha 1-FP was shown to have neither albumin's high-affinity site II (diazepam as marker) nor its site III (digitoxin and cholic acid as markers). High-affinity binding by alpha 1-FP was found for the specific markers (warfarin, phenylbutazone, azapropazone) of albumin's drug-binding site I. However, instead of albumin's one high-affinity site/molecule, a mean value of 0.5 site/molecule was obtained with rat alpha 1-FP. Charcoal treatment at neutral pH of rat serum albumin did not affect its measured binding properties, but treatment of the alpha 1-FP led to an increased affinity for warfarin, phenylbutazone and azapropazone without a change in the measured number of sites, indicating competition for binding at this site by (an) endogenous ligand(s). These results are discussed in terms of the structures of the two proteins and with respect to the physiological implications of the differences found. PMID:6206846

  15. Protein imprinted ionic liquid polymer on the surface of multiwall carbon nanotubes with high binding capacity for lysozyme.

    PubMed

    Yuan, Shifang; Deng, Qiliang; Fang, Guozhen; Wu, Jianhua; Li, Wangwang; Wang, Shuo

    2014-06-01

    In this research, ionic liquid as functional monomer to prepare molecularly imprinted polymers for protein recognition was for the first time demonstrated, in which, 1-vinyl-3-butylimidazolium chloride was selected as functional monomer, acrylamide as co-functional monomer and lysozyme (Lyz) as template protein to synthesize imprinted polymers on the surface of multiwall carbon nanotubes in aqueous medium. The results indicated that ionic liquid was helpful to improve binding capacity of imprinted polymers, which had a maximum binding capacity of 763.36 mg/g in the optimum adsorption conditions. The prepared imprinted polymers had a fast adsorption rate and a much higher adsorption capacity than the corresponding non-imprinted polymers, with the difference in adsorption capacity up to 258.31 mg/g. The obtained polymer was evaluated by Lyz, bovine serum albumin (BSA), bovine hemoglobin (BHb), equine myoglobin (MB) and cytochrome c (Cyt c). The selectivity factor (β) for Lyz/BSA, Lyz/Mb, Lyz/BHb, and Lyz/Cyt c were 7.17, 2.12, 1.76 and 1.57, respectively, indicating the imprinted polymers had a good selectivity. Easy preparation of the imprinted polymers as well as high ability and selectivity to adsorb template proteins makes this polymer attractive and broadly applicable in biomacromolecular separation, biotechnology and sensors.

  16. Locating the binding sites of folic acid with milk α- and β-caseins.

    PubMed

    Bourassa, P; Tajmir-Riahi, H A

    2012-01-12

    We located the binding sites of folic acid with milk α- and β-caseins at physiological conditions, using constant protein concentration and various folic acid contents. FTIR, UV-visible, and fluorescence spectroscopic methods as well as molecular modeling were used to analyze folic acid binding sites, the binding constant, and the effect of folic acid interaction on the stability and conformation of caseins. Structural analysis showed that folic acid binds caseins via both hydrophilic and hydrophobic contacts with overall binding constants of K(folic acid-α-caseins) = 4.8 (±0.6) × 10(4) M(-1) and K(folic acid-β-caseins) = 7.0 (±0.9) × 10(4) M(-1). The number of bound acid molecules per protein was 1.5 (±0.4) for α-casein and 1.4 (±0.3) for β-casein complexes. Molecular modeling showed different binding sites for folic acid on α- and β-caseins. The participation of several amino acids in folic acid-protein complexes was observed, which was stabilized by hydrogen bonding network and the free binding energy of -7.7 kcal/mol (acid-α-casein) and -8.1 kcal/mol (acid-β-casein). Folic acid complexation altered protein secondary structure by the reduction of α-helix from 35% (free α-casein) to 33% (acid-complex) and 32% (free β-casein) to 26% (acid-complex) indicating a partial protein destabilization. Caseins might act as carriers for transportation of folic acid to target molecules.

  17. Progesterone binding to the tryptophan residues of human alpha1-acid glycoprotein.

    PubMed

    Albani, J R

    2006-11-06

    Binding studies between progesterone and alpha1-acid glycoprotein allowed us to demonstrate that the binding site of progesterone contains one hydrophobic tryptophan residue and that the structure of the protein is not altered upon binding. The data obtained at saturated concentrations of progesterone clearly reveal the type of interaction at physiological levels.

  18. Dihedral angle preferences of DNA and RNA binding amino acid residues in proteins.

    PubMed

    Ponnuraj, Karthe; Saravanan, Konda Mani

    2017-04-01

    A protein can interact with DNA or RNA molecules to perform various cellular processes. Identifying or analyzing DNA/RNA binding site amino acid residues is important to understand molecular recognition process. It is quite possible to accurately model DNA/RNA binding amino acid residues in experimental protein-DNA/RNA complex by using the electron density map whereas, locating/modeling the binding site amino acid residues in the predicted three dimensional structures of DNA/RNA binding proteins is still a difficult task. Considering the above facts, in the present work, we have carried out a comprehensive analysis of dihedral angle preferences of DNA and RNA binding site amino acid residues by using a classical Ramachandran map. We have computed backbone dihedral angles of non-DNA/RNA binding residues and used as control dataset to make a comparative study. The dihedral angle preference of DNA and RNA binding site residues of twenty amino acid type is presented. Our analysis clearly revealed that the dihedral angles (φ, ψ) of DNA/RNA binding amino acid residues prefer to occupy (-89° to -60°, -59° to -30°) bins. The results presented in this paper will help to model/locate DNA/RNA binding amino acid residues with better accuracy.

  19. Pyrazinamide, but not pyrazinoic acid, is a competitive inhibitor of NADPH binding to Mycobacterium tuberculosis fatty acid synthase I.

    PubMed

    Sayahi, Halimah; Zimhony, Oren; Jacobs, William R; Shekhtman, Alexander; Welch, John T

    2011-08-15

    Pyrazinamide (PZA), an essential component of short-course anti-tuberculosis chemotherapy, was shown by Saturation Transfer Difference (STD) NMR methods to act as a competitive inhibitor of NADPH binding to purified Mycobacterium tuberculosis fatty acid synthase I (FAS I). Both PZA and pyrazinoic acid (POA) reversibly bind to FAS I but at different binding sites. The competitive binding of PZA and NADPH suggests potential FAS I binding sites. POA was not previously known to have any specific binding interactions. The STD NMR of NADPH bound to the mycobacterial FAS I was consistent with the orientation reported in published single crystal X-ray diffraction studies of fungal FAS I. Overall the differences in binding between PZA and POA are consistent with previous recognition of the importance of intracellular accumulation of POA for anti-mycobacterial activity.

  20. Trends for isolated amino acids and dipeptides: Conformation, divalent ion binding, and remarkable similarity of binding to calcium and lead

    NASA Astrophysics Data System (ADS)

    Ropo, M.; Blum, V.; Baldauf, C.

    2016-11-01

    We derive structural and binding energy trends for twenty amino acids, their dipeptides, and their interactions with the divalent cations Ca2+, Ba2+, Sr2+, Cd2+, Pb2+, and Hg2+. The underlying data set consists of more than 45,000 first-principles predicted conformers with relative energies up to ~4 eV (~400 kJ/mol). We show that only very few distinct backbone structures of isolated amino acids and their dipeptides emerge as lowest-energy conformers. The isolated amino acids predominantly adopt structures that involve an acidic proton shared between the carboxy and amino function. Dipeptides adopt one of two intramolecular-hydrogen bonded conformations C5 or . Upon complexation with a divalent cation, the accessible conformational space shrinks and intramolecular hydrogen bonding is prevented due to strong electrostatic interaction of backbone and side chain functional groups with cations. Clear correlations emerge from the binding energies of the six divalent ions with amino acids and dipeptides. Cd2+ and Hg2+ show the largest binding energies–a potential correlation with their known high acute toxicities. Ca2+ and Pb2+ reveal almost identical binding energies across the entire series of amino acids and dipeptides. This observation validates past indications that ion-mimicry of calcium and lead should play an important role in a toxicological context.

  1. Trends for isolated amino acids and dipeptides: Conformation, divalent ion binding, and remarkable similarity of binding to calcium and lead

    PubMed Central

    Ropo, M.; Blum, V.; Baldauf, C.

    2016-01-01

    We derive structural and binding energy trends for twenty amino acids, their dipeptides, and their interactions with the divalent cations Ca2+, Ba2+, Sr2+, Cd2+, Pb2+, and Hg2+. The underlying data set consists of more than 45,000 first-principles predicted conformers with relative energies up to ~4 eV (~400 kJ/mol). We show that only very few distinct backbone structures of isolated amino acids and their dipeptides emerge as lowest-energy conformers. The isolated amino acids predominantly adopt structures that involve an acidic proton shared between the carboxy and amino function. Dipeptides adopt one of two intramolecular-hydrogen bonded conformations C5 or . Upon complexation with a divalent cation, the accessible conformational space shrinks and intramolecular hydrogen bonding is prevented due to strong electrostatic interaction of backbone and side chain functional groups with cations. Clear correlations emerge from the binding energies of the six divalent ions with amino acids and dipeptides. Cd2+ and Hg2+ show the largest binding energies–a potential correlation with their known high acute toxicities. Ca2+ and Pb2+ reveal almost identical binding energies across the entire series of amino acids and dipeptides. This observation validates past indications that ion-mimicry of calcium and lead should play an important role in a toxicological context. PMID:27808109

  2. Stability of the Octameric Structure Affects Plasminogen-Binding Capacity of Streptococcal Enolase

    PubMed Central

    Law, Ruby H. P.; Casey, Lachlan W.; Valkov, Eugene; Bertozzi, Carlo; Stamp, Anna; Jovcevski, Blagojce; Aquilina, J. Andrew; Whisstock, James C.; Walker, Mark J.; Kobe, Bostjan

    2015-01-01

    Group A Streptococcus (GAS) is a human pathogen that has the potential to cause invasive disease by binding and activating human plasmin(ogen). Streptococcal surface enolase (SEN) is an octameric α-enolase that is localized at the GAS cell surface. In addition to its glycolytic role inside the cell, SEN functions as a receptor for plasmin(ogen) on the bacterial surface, but the understanding of the molecular basis of plasmin(ogen) binding is limited. In this study, we determined the crystal and solution structures of GAS SEN and characterized the increased plasminogen binding by two SEN mutants. The plasminogen binding ability of SENK312A and SENK362A is ~2- and ~3.4-fold greater than for the wild-type protein. A combination of thermal stability assays, native mass spectrometry and X-ray crystallography approaches shows that increased plasminogen binding ability correlates with decreased stability of the octamer. We propose that decreased stability of the octameric structure facilitates the access of plasmin(ogen) to its binding sites, leading to more efficient plasmin(ogen) binding and activation. PMID:25807546

  3. Influence of phosphate ions on buffer capacity of soil humic acids

    NASA Astrophysics Data System (ADS)

    Boguta, P.; Sokołowska, Z.

    2012-02-01

    The object of this study was to determine change of natural buffer capacity of humic acids by strong buffering agents, which were phosphate ions. Studies were carried out on the humic acids extracted from peat soils. Additional information was obtained by determination of water holding capacity, density, ash and pH for peats and optical parameter Q4/6 for humic acids. Humic acid suspensions exhibited the highest buffer properties at low pH and reached maximum at pH ~ 4. Phosphates possessed buffer properties in the pH range from 4.5 to 8.0. The maximum of buffering was at pH~6.8 and increased proportionally with an increase in the concentration of phosphate ions. The study indicated that the presence of phosphate ions may strongly change natural buffer capacity of humic acids by shifting buffering maximum toward higher pH values. Significant correlations were found for the degree of the secondary transformation with both the buffer capacity and the titrant volume used during titration.

  4. The 'helix clamp' in HIV-1 reverse transcriptase: a new nucleic acid binding motif common in nucleic acid polymerases.

    PubMed Central

    Hermann, T; Meier, T; Götte, M; Heumann, H

    1994-01-01

    Amino acid sequences homologous to 259KLVGKL (X)16KLLR284 of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) are conserved in several nucleotide polymerizing enzymes. This amino acid motif has been identified in the crystal structure model as an element of the enzyme's nucleic acid binding apparatus. It is part of the helix-turn-helix structure, alpha H-turn-alpha I, within the 'thumb' region of HIV-1 RT. The motif grasps the complexed nucleic acid at one side. Molecular modeling studies on HIV-1 RT in complex with a nucleic acid fragment suggest that the motif has binding function in the p66 subunit as well as in the p51 subunit, acting as a kind of 'helix clamp'. Given its wide distribution within the nucleic acid polymerases, the helix clamp motif is assumed to be a structure of general significance for nucleic acid binding. Images PMID:7527138

  5. Analysis of (/sup 3/H) Kainic acid binding with rat and Frog brain membranes

    SciTech Connect

    Zharkovskii, A.M.; Zharkovskaya, T.A.

    1985-10-01

    This paper analyzes the binding of (H 3)-KA with membrances in vitro and the effect of various neuroactive amino acids, suggested as endogenous ligands for binding sites of (H 3)-KA, on binding. Experiments were carried out on male albino rats and on winter frogs. Choice of the frog's brain was determined by the high density of high-affinity binding sites of (H 3)-KA. The concentrations of substances inhibiting binding (H 3)-KA by 50% were calculated by logit-probit analysis, and inhibition constants were also calculated. It is shown that although L-glutamate and folic acid inhibit binding of (H 3)-KA, they do not satisfy the criteria to be met by endogenous ligands, and this inhibition of binding is noncompetitive in character. This suggests that KA binding sites and glutamate receptors are not identical, although they may perhaps be subunits of a single complex.

  6. Non-intercalative, deoxyribose binding of boric acid to calf thymus DNA.

    PubMed

    Ozdemir, Ayse; Gursaclı, Refiye Tekiner; Tekinay, Turgay

    2014-05-01

    The present study characterizes the effects of the boric acid binding on calf thymus DNA (ct-DNA) by spectroscopic and calorimetric methods. UV-Vis absorbance spectroscopy, circular dichroism (CD) spectroscopy, transmission electron microscopy (TEM), isothermal titration calorimetry (ITC), and Fourier transform infrared (FT-IR) spectroscopy were employed to characterize binding properties. Changes in the secondary structure of ct-DNA were determined by CD spectroscopy. Sizes and morphologies of boric acid-DNA complexes were determined by transmission electron microscopy (TEM). The kinetics of boric acid binding to calf thymus DNA (ct-DNA) was investigated by isothermal titration calorimetry (ITC). ITC results revealed that boric acid exhibits a moderate affinity to ct-DNA with a binding constant (K a) of 9.54 × 10(4) M(-1). FT-IR results revealed that boric acid binds to the deoxyribose sugar of DNA without disrupting the B-conformation at tested concentrations.

  7. Modulatory effects of unsaturated fatty acids on the binding of glucocorticoids to rat liver glucocorticoid receptors.

    PubMed

    Vallette, G; Vanet, A; Sumida, C; Nunez, E A

    1991-09-01

    Binding of the synthetic glucocorticoid dexamethasone to the rat liver cytosol glucocorticoid receptor was inhibited by physiological concentrations of nonesterified fatty acids as a function of increasing dose, degree of unsaturation, and chain length of the fatty acid. Polyunsaturated fatty acids were the most potent inhibitors. Scatchard analysis and Line-weaver-Burk plots of the binding data revealed that both the association constants and number of binding sites decreased and that polyunsaturated fatty acids inhibition was of a mixed non-competitive type. The dissociation rate constant of [3H]dexamethasone from glucocorticoid receptors was increased by up to 10 times in the presence of docosahexaenoic acid, whereas a competitive inhibitor like the glucocorticoid antagonist RU 38486 had no effect. Moreover, sucrose density gradient analysis showed that docosahexaenoic acid inhibited the binding of [3H] dexamethasone to both the 8.8S and 4S forms. The results strongly suggest that unsaturated fatty acids are interacting at a site on the receptor different from the hormone binding site and the heat shock protein and that by binding to a second site unsaturated fatty acids greatly change the conformation of the hormone binding site to reduce its affinity for the hormone, either partially or completely depending on the concentration and the class of the fatty acid.

  8. Identification of a fatty acid binding protein4-UCP2 axis regulating microglial mediated neuroinflammation.

    PubMed

    Duffy, Cayla M; Xu, Hongliang; Nixon, Joshua P; Bernlohr, David A; Butterick, Tammy A

    2017-02-16

    Hypothalamic inflammation contributes to metabolic dysregulation and the onset of obesity. Dietary saturated fats activate microglia via a nuclear factor-kappa B (NFκB) mediated pathway to release pro-inflammatory cytokines resulting in dysfunction or death of surrounding neurons. Fatty acid binding proteins (FABPs) are lipid chaperones regulating metabolic and inflammatory pathways in response to fatty acids. Loss of FABP4 in peripheral macrophages via either molecular or pharmacologic mechanisms results in reduced obesity-induced inflammation via a UCP2-redox based mechanism. Despite the widespread appreciation for the role of FABP4 in mediating peripheral inflammation, the expression of FABP4 and a potential FABP4-UCP2 axis regulating microglial inflammatory capacity is largely uncharacterized. To that end, we hypothesized that microglial cells express FABP4 and that inhibition would upregulate UCP2 and attenuate palmitic acid (PA)-induced pro-inflammatory response. Gene expression confirmed expression of FABP4 in brain tissue lysate from C57Bl/6J mice and BV2 microglia. Treatment of microglial cells with an FABP inhibitor (HTS01037) increased expression of Ucp2 and arginase in the presence or absence of PA. Moreover, cells exposed to HTS01037 exhibited attenuated expression of inducible nitric oxide synthase (iNOS) compared to PA alone indicating reduced NFκB signaling. Hypothalamic tissue from mice lacking FABP4 exhibit increased UCP2 expression and reduced iNOS, tumor necrosis factor-alpha (TNF-α), and ionized calcium-binding adapter molecule 1 (Iba1; microglial activation marker) expression compared to wild type mice. Further, this effect is negated in microglia lacking UCP2, indicating the FABP4-UCP2 axis is pivotal in obesity induced neuroinflammation. To our knowledge, this is the first report demonstrating a FABP4-UCP2 axis with the potential to modulate the microglial inflammatory response.

  9. Mechanism of ligand binding to alpha 1-acid glycoprotein (orosomucoid): correlated thermodynamic factors and molecular parameters of polarity.

    PubMed Central

    Urien, S; Giroud, Y; Tsai, R S; Carrupt, P A; Brée, F; Testa, B; Tillement, J P

    1995-01-01

    Eight ligands were used in this study, four basic, three neutral and one acidic. Their binding to serum alpha 1-acid glycoprotein (orosomucoid) was measured at several temperatures, and the data were analysed together by a general model with three unknowns, number of binding sites, delta H0 and delta S0. The partition coefficients of the ligands were measured in octanol/water and heptane/water systems (log Poct. and log Phep.), and their molecular volumes were calculated by molecular modelling techniques. These structural properties allow determination of polarity parameters (delta log Poct.-hep., lambda oct. and lambda hep.) which encode in different proportions the various polar interactions between the solute and the aqueous and organic phases, i.e. hydrogen-bonding capacity and dipolarity/polarizability. This study shows that good correlations exist between delta H0 or delta S0 and polarity parameters, such that the enthalpic contribution to binding increases with increasing polarity of the ligands, mainly hydrogen-bond-donor acidity, whereas their entropic contribution to binding decreases. PMID:7887909

  10. Models of metal binding structures in fulvic acid from the Suwannee River, Georgia

    SciTech Connect

    Leenheer, J.A.; Brown, G.K.; Cabaniss, S.E.; MacCarthy, P.

    1998-08-15

    Fulvic acid, isolated from the Suwannee River, Georgia, was assessed for its ability to bind Ca{sup 2+}, Cd{sup 2+}, Cu{sup 2+}, Ni{sup 2+}, and Zn{sup 2+} ions at pH 6 before and after extensive fractionation that was designed to reveal the nature of metal binding functional groups. The binding constant for Ca{sup 2+} ion had the greatest increase of all the ions in a metal binding fraction that was selected for intensive characterization for the purpose of building quantitative average model structures. The metal binding fraction was characterized by quantitative {sup 13}C NMR, {sup 1}H NMR, and FT-IR spectrometry and elemental, titrimetric, and molecular weight determinations. The characterization data revealed that carboxyl groups were clustered in short-chain aliphatic dibasic acid structures. The Ca{sup 2+} binding data suggested that ether-substituted oxysuccinic acid structures are good models for the metal binding sites at pH 6. Structural models were derived based upon oxidation and photolytic rearrangements of cutin, lignin, and tannin precursors. These structural models rich in substituted dibasic acid structures revealed polydentate binding sites with the potential for both inner-sphere and outer-sphere type binding. The majority of the fulvic acid molecule was involved with metal binding rather than a small substructural unit.

  11. A study of metal concentrations and metallothionein binding capacity in liver, kidney and brain tissues of three Arctic seal species.

    PubMed

    Sonne, Christian; Aspholm, Ole; Dietz, Rune; Andersen, Steen; Berntssen, Marc H G; Hylland, Ketil

    2009-12-01

    Arctic seals are known to accumulate relatively high concentrations of potential toxic heavy metals in their vital organs, such as livers and kidneys, as well as in their central nervous system. We therefore decided to determine whether mercury, copper, cadmium and zinc levels in liver, kidney and brain tissues of three Arctic seal species were associated with the intracellular metal-binding protein metallothionein (MT) as a sign of toxic exposure. Samples from four ringed (Phoca hispida), five harp (P.groenlandica) and five hooded (Cystophora cristata) seals taken during field trips to Central West Greenland (Godhavn) and the Barents Sea in the spring of 1999 were used for the present study. In all three seal species concentrations of mercury, zinc and copper were highest in the liver, except for cadmium which was highest in the kidneys. Metal concentrations increased significantly in the order: ringed sealbinding capacity was highest in the kidneys for all three species and increased in the same order: ringed seals (2-10%)binding capacity is sufficient to protect tissues (i.e. kidney) from metal toxicosis. MT with its binding capacity could be a useful marker for environmental exposure to metals and their potential toxicity in the Arctic.

  12. Application of a continuous distribution model for proton binding by humic acids extracted from acidic lake sediments

    SciTech Connect

    Rhea, J.R.; Young, T.C. )

    1987-01-01

    The proton binding characteristics of humic acids extracted from the sediments of Cranberry Pond, an acidic water body located in the Adirondack Mountain region of New York State, were explored by the application of a nultiligand distribution model. The model characterizes a class of proton binding sites by mean log K values and the standard deviations of log K values and the mean. Mean log K values and their relative abundances were determined directly from experimental titration data. The model accurately predicts the binding of protons by the humic acids for pH values in the range 3.5 to 10.0.

  13. Application of a continuous distribution model for proton binding by humic acids extracted from acidic lake sediments

    NASA Astrophysics Data System (ADS)

    Rhea, James R.; Young, Thomas C.

    1987-10-01

    The proton binding characteristics of humic acids extracted from the sediments of Cranberry Pond, an acidic water body located in the Adirondack Mountain region of New York State, were explored by the application of a multiligand distribution model. The model characterizes a class of proton binding sites by mean log K values and the standard deviations of log K values about the mean. Mean log K values and their relative abundances were determined directly from experimental titration data. The model accurately predicts the binding of protons by the humic acids for pH values in the range 3.5 to 10.0.

  14. Binding characteristics of Cu(2+) to natural humic acid fractions sequentially extracted from the lake sediments.

    PubMed

    He, En; Lü, Changwei; He, Jiang; Zhao, Boyi; Wang, Jinghua; Zhang, Ruiqing; Ding, Tao

    2016-11-01

    Humic acids (HAs) determine the distribution, toxicity, bioavailability, and ultimate fate of heavy metals in the environment. In this work, ten HA fractions (F1-F10) were used as adsorbent, which were sequentially extracted from natural sediments of Lake Wuliangsuhai, to investigate the binding characteristics of Cu(2+) to HA. On the basis of the characterization results, differences were found between the ten extracted HA fractions responding to their elemental compositions and acidic functional groups. The characterization results reveal that the responses of ten extracted HA fractions to their elemental compositions and acidic functional groups were different. The O/C and (O + N)/C ratio of F1-F8 approximately ranged from 0.66 to 0.53 and from 0.72 to 0.61, respectively; the measured results showed that the contents of phenolic groups and carboxyl groups decreased from 4.46 to 2.60 mmol/g and 1.60 to 0.58 mmol/g, respectively. The binding characteristics of Cu(2+) to the ten HA fractions were well modeled by the bi-Langmuir model; the binding behavior of Cu(2+) to all the ten HA fractions were strongly impacted by pH and ionic strength. The FTIR and SEM-EDX image of HA fractions (pre- and post-adsorption) revealed that carboxyl and phenolic groups were responsible for the Cu(2+) sorption on the ten sequentially extracted HA fractions process, which is the same with the analysis of the ligand binding and bi-Langmuir models Accordingly, the adsorption capacity of the former HA fractions on Cu(2+) were higher than the latter ones, which may be attributed to the difference of carboxyl and phenolic group contents between the former and latter extracted HA fractions. Additionally, the functional groups with N and S should not be neglected. This work is hopeful to understand the environmental effect of humic substances, environmental geochemical behavior, and bioavailability of heavy metals in lakes.

  15. Crystal structure of axolotl (Ambystoma mexicanum) liver bile acid-binding protein bound to cholic and oleic acid.

    PubMed

    Capaldi, Stefano; Guariento, Mara; Perduca, Massimiliano; Di Pietro, Santiago M; Santomé, José A; Monaco, Hugo L

    2006-07-01

    The family of the liver bile acid-binding proteins (L-BABPs), formerly called liver basic fatty acid-binding proteins (Lb-FABPs) shares fold and sequence similarity with the paralogous liver fatty acid-binding proteins (L-FABPs) but has a different stoichiometry and specificity of ligand binding. This article describes the first X-ray structure of a member of the L-BABP family, axolotl (Ambystoma mexicanum) L-BABP, bound to two different ligands: cholic and oleic acid. The protein binds one molecule of oleic acid in a position that is significantly different from that of either of the two molecules that bind to rat liver FABP. The stoichiometry of binding of cholate is of two ligands per protein molecule, as observed in chicken L-BABP. The cholate molecule that binds buried most deeply into the internal cavity overlaps well with the analogous bound to chicken L-BABP, whereas the second molecule, which interacts with the first only through hydrophobic contacts, is more external and exposed to the solvent.

  16. [Method for determining the acid-reducing capacity of antacid preparations].

    PubMed

    Chakŭrski, I; Dobrev, Kh; Dobreva, P

    1986-01-01

    A method is proposed for the determination of acid-binding ability of antacids, enabling the automatic reading of pH changes. Changes are provided for work with original gastric juice. The method allows the determination of individual dosage of antacid for each patient.

  17. Regulation of amantadine hydrochloride binding with IIA subdomain of human serum albumin by fatty acid chains.

    PubMed

    Yang, Feng; Lee, Philbert; Ma, Zhiyuan; Ma, Li; Yang, Guoping; Wu, Xiaoyang; Liang, Hong

    2013-01-01

    Human serum albumin (HSA) is a major protein component of blood plasma that has been exploited to bind and transport a wide variety of endogenous and exogenous organic compounds. Although anionic drugs readily associate with the IIA subdomain of HSA, most cationic drugs poorly associate with HSA at this subdomain. In this study, we propose to improve the association between cationic drugs and HSA by modifying HSA with fatty acid chains. For our experiments, we tested amantadine hydrochloride, a cationic drug with antiviral and antiparkinsonian effects. Our results suggest that extensive myristoylation of HSA can help stabilize the interaction between amantadine and HSA in vitro. Our X-ray crystallography data further elucidate the structural basis of this regulation. Additionally, our crystallography data suggest that anionic drugs, with a functional carboxylate group, may enhance the association between amantadine and HSA by a mechanism similar to myristoylation. Ultimately, our results provide critical structural insight into this novel association between cationic drugs and the HSA IIA subdomain, raising the tempting possibility to fully exploit the unique binding capacity of HSA's IIA subdomain to achieve simultaneous delivery of anionic and cationic drugs.

  18. Phenolic acids and antioxidant capacity of distillers dried grains with solubles (DDGS) as compared with corn

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Three sets of ground corn and the corresponding distillers dried grains with solubles (DDGS) were collected from three commercial plants and analyzed for individual phenolic acids by high performance liquid chromatography coupled with diode array and/or mass spectrometry and for antioxidant capacity...

  19. Binding Preferences of Amino Acids for Gold Nanoparticles: A Molecular Simulation Study.

    PubMed

    Shao, Qing; Hall, Carol K

    2016-08-09

    A better understanding of the binding preference of amino acids for gold nanoparticles of different diameters could aid in the design of peptides that bind specifically to nanoparticles of a given diameter. Here we identify the binding preference of 19 natural amino acids for three gold nanoparticles with diameters of 1.0, 2.0, and 4.0 nm, and investigate the mechanisms that govern these preferences. We calculate potentials of mean force between 36 entities (19 amino acids and 17 side chains) and the three gold nanoparticles in explicit water using well-tempered metadynamics simulations. Comparing these potentials of mean force determines the amino acids' nanoparticle binding preferences and if these preferences are controlled by the backbone, the side chain, or both. Twelve amino acids prefer to bind to the 4.0 nm gold nanoparticle, and seven prefer to bind to the 2.0 nm one. We also use atomistic molecular dynamics simulations to investigate how water molecules near the nanoparticle influence the binding of the amino acids. The solvation shells of the larger nanoparticles have higher water densities than those of the smaller nanoparticles while the orientation distributions of the water molecules in the shells of all three nanoparticles are similar. The nanoparticle preferences of the amino acids depend on whether their binding free energy is determined mainly by their ability to replace or to reorient water molecules in the nanoparticle solvation shell. The amino acids whose binding free energy depends mainly on the replacement of water molecules are likely to prefer to bind to the largest nanoparticle and tend to have relatively simple side chain structures. Those whose binding free energy depends mainly on their ability to reorient water molecules prefer a smaller nanoparticle and tend to have more complex side chain structures.

  20. Stream chemistry in the eastern United States. 2. Current sources of acidity in acidic and low acid-neutralizing-capacity streams

    SciTech Connect

    Herlihy, A.T.; Kaufmann, P.R.; Mitch, M.E.

    1991-01-01

    The authors examined anion composition in National Stream Survey (NSS) data in order to evaluate the most probable sources of current acidity in acidic and low acid neutralizing capacity (ANC) streams in the eastern United States. Acidic streams that had almost no organic influence (less than 10% of total anions) and sulfate and nitrate concentrations indicative of evaporative concentration of atmospheric deposition were classified as acidic due to acidic deposition. These acidic streams were located in small forested watersheds in the Mid-Atlantic Highlands (an estimated 1950 km of stream length) and in the Mid-Atlantic Coastal Plain (1250 km). Acidic streams affected primarily by acidic deposition but also influenced by naturally occurring organic anions accounted for another 1180 km of acidic stream length and were located in the New Jersey Pine Barrens, plateau tops in the Mid-Atlantic and Southeast Highlands, and the Florida Panhandle. The total length of streams acidic due to acid mine drainage in the NSS (4590 km) was about the same as the total length of acidic streams likely affected by acidic deposition (4380 km). Acidic streams whose acid anion composition was dominated by organics were located in Florida and the Mid-Atlantic Coastal Plain. In Florida, most of the acidic streams were organic dominated, whereas about half of the streams in the Mid-Atlantic Coastal Plain were organic dominated. Organic-dominated acidic streams were not observed in the Mid-Atlantic and Southeast Highlands.

  1. Stream chemistry in the eastern United States, 2, Current sources of acidity in acidic and low acid-neutralizing capacity streams

    NASA Astrophysics Data System (ADS)

    Herlihy, Alan T.; Kaufmann, Philip R.; Mitch, Mark E.

    1991-04-01

    We examined anion composition in National Stream Survey (NSS) data in order to evaluate the most probable sources of current acidity in acidic and low acid-neutralizing capacity (ANC) streams in the eastern United States. Acidic streams that had almost no organic influence (less than 10% of total anions) and sulfate and nitrate concentrations indicative of evaporative concentration of atmospheric deposition were classified as acidic due to acidic deposition. These acidic streams were located in small (<30 km2) forested watersheds in the Mid-Atlantic Highlands (an estimated 1950 km of stream length) and in the Mid-Atlantic Coastal Plain (1250 km). Acidic streams affected primarily by acidic deposition but also influenced by naturally occurring organic anions accounted for another 1180 km of acidic stream length and were located in the New Jersey Pine Barrens, plateau tops in the Mid-Atlantic and Southeast Highlands, and the Florida Panhandle. The total length of streams acidic due to acid mine drainage in the NSS (4590 km) was about the same as the total length of acidic streams likely affected by acidic deposition (4380 km). Acidic streams whose acid anion composition was dominated by organics were located in Florida and the Mid-Atlantic Coastal Plain. In Florida, most of the acidic streams were organic dominated, whereas about half of the streams in the Mid-Atlantic Coastal Plain were organic dominated. Organic-dominated acidic streams were not observed in the Mid-Atlantic and Southeast Highlands.

  2. Evolutionary diversification of the avian fatty acid-binding proteins.

    PubMed

    Hughes, Austin L; Piontkivska, Helen

    2011-12-15

    Phylogenetic analysis of avian and other vertebrate fatty acid binding proteins (FABPs) supported the hypothesis that several gene duplications within this family occurred prior to the most recent common ancestor (MRCA) of tetrapods and bony fishes. The chicken genome encodes two liver-expressed FABPs: (1) L-FABP or FABP1; and (2) Lb-FABP. We propose that the latter be designated FABP10, because in our phylogenetic analysis it clustered with zebrafish FABP10. Bioinformatic analysis of across-tissue gene expression patterns in the chicken showed some congruence with phylogenetic relationships. On the basis of expression, chicken FABP genes seemed to form two major groups: (1) a cluster of genes many of which showed predominant expression in the digestive system (FABP1, FABP2, FABP6, FABP10, RBP1, and CRABP1); and (2) a cluster of genes most of which had predominant expression in tissues other than those of the digestive system, including muscle and the central nervous system (FABP3, FABP4, FABP5, FABP7, and PMP2). Since these clusters corresponded to major clusters in the phylogenetic tree as well, it seems a plausible hypothesis that the earliest duplication in the vertebrate FABP family led to the divergence of a gut-specialized gene from a gene expressed mainly in nervous and muscular systems. Data on gene expression in livers of two lines of chickens selected for high growth and low growth showed differences between FABP1 and FABP10 expressions in the liver, supporting the hypothesis of functional divergence between the two chicken liver-expressed FABPs related to food intake.

  3. Expression of liver fatty acid binding protein in hepatocellular carcinoma☆

    PubMed Central

    Cho, Soo-Jin; Ferrell, Linda D.; Gill, Ryan M.

    2017-01-01

    Summary Loss of expression of liver fatty acid binding protein (LFABP) by immunohistochemistry has been shown to be characteristic of a subset of hepatocellular adenomas (HCAs) in which HNF1A is inactivated. Transformation to hepatocellular carcinoma is thought to be a very rare phenomenon in the HNF1A-inactivated variant of HCA. However, we recently observed 2 cases at our institution, 1 definite hepatocellular carcinoma and 1 possible hepatocellular carcinoma, with loss of LFABP staining, raising the possibility that LFABP down-regulation may be associated with hepatocellular carcinogenesis. Our aim was to evaluate hepatocellular carcinomas arising in various backgrounds and with varying degrees of differentiation for loss of LFABP staining. Twenty total cases of hepatocellular carcinoma were examined. Thirteen cases arose in a background of cirrhosis due to hepatitis C (n = 8) or steatohepatitis (n = 5); 7 cases arose in a noncirrhotic background, with 2 cases arising within HNF1A-inactivated variant HCA and 2 cases arising within inflammatory variant HCA. Complete loss of expression of LFABP was seen in 6 of 20 cases, including 2 cases of hepatocellular carcinoma arising within HNF1A-inactivated variant HCA. Thus, loss of staining for LFABP appears to be common in hepatocellular carcinoma and may be seen in well-differentiated hepatocellular carcinoma. Therefore, LFABP loss should not be interpreted as evidence for hepatocellular adenoma over carcinoma, when other features support a diagnosis of hepatocellular carcinoma. The findings raise consideration for a role of HNF1A inactivation in hepatocellular carcinogenesis, particularly in less differentiated tumors. PMID:26997447

  4. Construction of an S-layer protein exhibiting modified self-assembling properties and enhanced metal binding capacities.

    PubMed

    Pollmann, Katrin; Matys, Sabine

    2007-07-01

    The functional S-layer protein gene slfB of the uranium mining waste pile isolate Bacillus sphaericus JG-A12 was cloned as a polymerase chain reaction product into the expression vector pET Lic/Ek 30 and heterologously expressed in Escherichia coli Bl21(DE3). The addition of His tags to the N and C termini enabled the purification of the recombinant protein by Ni-chelating chromatography. The Ni binding capacity of the His-tagged recombinant S-layer protein was compared with that of the wild-type S layer. The inductively coupled plasma mass spectrometry analyses demonstrate a significantly enhanced Ni binding capability of the recombinant protein. In addition, the self-assembling properties of the purified modified S-layer proteins were studied by light microscopy and scanning electron microscopy. Whereas the wild-type S-layer proteins re-assembled into regular cylindric structures, the recombinant S-layer proteins reassembled into regular sheets that formed globular agglomerating structures. The nanoporous structure of the protein meshwork, together with its enhanced Ni binding capacity, makes the recombinant S-layer attractive as a novel self-assembling biological template for the fabrication of metal nanoclusters and construction of nanomaterials that are of technical interest.

  5. Dynamics of palmitic acid complexed with rat intestinal fatty acid binding protein.

    PubMed

    Zhu, L; Kurian, E; Prendergast, F G; Kemple, M D

    1999-02-02

    Dynamics of palmitic acid (PA), isotopically enriched with 13C at the second, seventh, or terminal methyl position, were investigated by 13C NMR. Relaxation measurements were made on PA bound to recombinant rat intestinal fatty acid binding protein (I-FABP) at pH 5.5 and 23 degreesC, and, for comparison, on PA incorporated into 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (MPPC) micelles, and dissolved in methanol. The 13C relaxation data, T1, and steady-state nuclear Overhauser effect (NOE) obtained at two different magnetic fields were interpreted using the model-free approach [Lipari, G., and Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559]. The overall rotational correlation time of the fatty acid.protein complex was 2.5 +/- 0.4 ns, which is substantially less than the value expected for the protein itself (>6 ns). Order parameters (S2), which are a measure of the amplitude of the internal motion of individual C-H vectors with respect to the PA molecule, while largest for C-2 and smallest for the methyl carbon, were relatively small (<0.4) in the protein complex. S2 values for given C-H vectors also were smaller for PA in the MPPC micelles and in methanol than in the protein complex. Correlation times reflective of the time scale of the internal motion of the C-H vectors were in all cases <60 ps. These results support the view that the fatty acid is not rigidly anchored within the I-FABP binding pocket, but rather has considerable freedom to move within the pocket.

  6. Physicochemical aspects of the energetics of binding of sulphanilic acid with bovine serum albumin

    NASA Astrophysics Data System (ADS)

    Banipal, Tarlok S.; Kaur, Amandeep; Banipal, Parampaul K.

    2017-01-01

    The thermodynamic study of the binding of sulphanilic acid with model transport protein bovine serum albumin is a promising approach in the area of synthesizing new sulfa drugs with improved therapeutic effect. Thus, such binding studies play an important role in the rational drug design process. The binding between sulphanilic acid and bovine serum albumin has been studied using calorimetry, light scattering in combination with spectroscopic and microscopic techniques. The calorimetric data reveals the presence of two sequential nature of binding sites where the first binding site has stronger affinity ( 104 M- 1) and second binding site has weaker affinity ( 103 M- 1). However, the spectroscopic (absorption and fluorescence) results suggest the presence of single low affinity binding site ( 103 M- 1) on protein. The contribution of polar and non-polar interactions to the binding process has been explored in the presence of various additives. It is found that sulphanilic acid binds with high affinity at Sudlow site II and with low affinity at Sudlow site I of protein. Light scattering and circular dichroism measurements have been used to study the effect on the molecular topology and conformation of protein, respectively. Thus these studies provide important insights into the binding of sulphanilic acid with bovine serum albumin both quantitatively and qualitatively.

  7. Amino acid composition of cadmium-binding protein induced in a marine diatom

    SciTech Connect

    Maita, Y.; Kawaguchi, S. )

    1989-09-01

    Organisms living in environments polluted with heavy metals develop tolerance against these contaminants. The tolerance has been attributed to the ability to synthesize metal binding substances. These recent findings imply metal binding complexes from animals and plants, although having very similar functional properties, may have entirely different amino acid compositions. Researchers reported that cadystin from fission yeast, Schizosaccharomyces pombe was composed of only glutamic acid, cysteine, and glycine. A year later, a heavy metal binding substance was isolated from Rauwolfia serpetina which contains only Glu, Cys, and Gly. Heavy metal binding complexes isolated from the water hyacinth and morning glory Datura innoxia also showed an amino acid composition similar to cadystin or phytochelatin. In this study, the cadmium binding protein induced in the marine diatom, Phaeodactylum tricornutum, was isolated and purified and its amino acid composition determined.

  8. Low capacity of erythrocytes to bind with immune complexes via C3b receptor in patients with systemic lupus erythematosus: correlation with pathological proteinuria

    SciTech Connect

    Nojima, Y.; Terai, C.; Minota, S.; Takano, K.; Miyakawa, Y.; Takaku, F.

    1985-01-01

    Erythrocytes from 51 patients with systemic lupus erythematosus and 75 controls were tested for the capacity to bind aggregated human gamma-globulin labeled with radioiodine in the presence of complement. Both in patients and controls, a trimodal distribution of binding capacity was observed. Low (less than 9% of the added radioactivity), intermediate (9-17%), and high binding (more than 17%) were observed in 13, 58, and 29% in controls and in 49, 43 and 8% in lupus patients. The low binding capacity of erythrocytes persisted even after patients entered remission following steroid therapy. A genetic control of binding capacity was supported by familial surveys. Prevalence of pathological proteinuria was significantly higher in patients with low binding capacity than those with intermediate or high binding capacity (16/25 vs 7/26, P less than 0.01). These results indicate that an impaired physiological disposal of immune complexes via the erythrocyte C3b receptor in lupus patients may contribute to the development of renal involvement.

  9. Phase Diagram and Heat Capacities of the Malonic Acid/Water System

    NASA Astrophysics Data System (ADS)

    Hansen, A.; Beyer, K. D.

    2003-12-01

    Malonic acid is one of the more ubiquitous dicarboxylic acids found in the atmosphere and is quite soluble in water. Therefore, its impact on particle/cloud droplet formation needs to be better understood through the study of the thermodynamics of its aqueous solutions. The liquid/solid phase diagram and solution heat capacities of the malonic acid/water binary system have been investigated using differential scanning calorimetry and infrared spectroscopy of thin films. We report here the first measurement of the ice melting envelope as well as the ice/malonic acid eutectic temperature and composition in this binary system. Evidence from both thermal analysis and infrared spectroscopy is shown for a malonic acid hydrate, possibly C3H4O4ṡ6H2O. We have observed the formation of this hydrate over a large range of concentrations, and have found it is a major fraction of samples within that region. We have also determined the enthalpy of fusion of malonic acid as well as the constant pressure heat capacities of solutions in the concentration range 5 - 55 wt% malonic acid from 323 K to the freezing point of each solution.

  10. Characterization of Naphthaleneacetic Acid Binding to Receptor Sites on Cellular Membranes of Maize Coleoptile Tissue 1

    PubMed Central

    Ray, Peter M.; Dohrmann, Ulrike; Hertel, Rainer

    1977-01-01

    Characteristics of and optimum conditions for saturable (“specific”) binding of [14C]naphthaleneacetic acid to sites located on membranous particles from maize (Zea mays L.) coleoptiles are described. Most, if not all, of the specific binding appears to be due to a single kinetic class of binding sites having a KD of 5 to 7 × 10−7m for naphthalene-1-acetic acid (NAA). Binding of NAA is insensitive to high monovalent salt concentrations, indicating that binding is not primarily ionic. However, specific binding is inhibited by Mg2+ or Ca2+ above 5 mm. Specific binding is improved by organic acids, especially citrate. Binding is heat-labile and is sensitive to agents that act either on proteins or on lipids. Specific binding is reversibly inactivated by reducing agents such as dithioerythritol; a reducible group, possibly a disulfide group, may be located at the binding site and required for its function. The affinity of the specific binding sites for auxins is modified by an unidentified dialyzable, heat-stable, apparently amphoteric, organic factor (“supernatant factor”) found in maize tissue. PMID:16659851

  11. Investigation of salt properties with electro-acoustic measurements and their effect on dynamic binding capacity in hydrophobic interaction chromatography.

    PubMed

    Müller, Egbert; Faude, Alexander

    2008-01-11

    The pH dependence in hydrophobic interaction chromatography (HIC) is usually discussed exclusively in terms of protein dependence and there are no clear defined trends. Many of the deviations from an ideal solution are caused solely by the high salt concentration, as protein concentration is usually negligible. So pH dependency in hydrophobic interaction chromatography could also be the result of pH dependent changes of ion properties from the salt solution. The possibility that pH dependent ion hydration or ion association in highly concentrated salt solutions may influence the dynamic protein binding capacity onto HIC resins was investigated. In buffer solutions commonly used in HIC e.g. sodium chloride, ammonium sulphate and sodium citrate pH dependent maxima in the electro-acoustic signals were found. These maxima are related to an increase of the ion sizes by hydration or ion association. At low ionic strength the maxima are in the range between 4.5 and 6 and they increased in concentrated electrolyte solutions to values between 6 and 8. The range of these maxima is in the same region as dynamic protein binding capacity maxima often observed in HIC. For a qualitative interpretation of this phenomenon of increased protein stabilization by volume exclusion effect extended scaling theory can be used. This theory predicts a maximum of protein stabilization if the ratio of salt ion diameter to water is 1.8. According to the hypothesis raised here, if the pH dependent ratio of salt ion diameter to water approaches this value the transport of the protein in the pore system is less restricted and an increase in binding capacity can be produced.

  12. Partial purification of fatty-acid binding protein by ammonium sulphate fractionation.

    PubMed

    Avanzati, B; Catalá, A

    1983-07-01

    By fractionation of rat liver cytosol with 70% saturation ammonium sulphate, a soluble fraction showing high affinity for oleic acid was obtained. The binding of oleic acid to this fraction was inhibited by flavaspidic acid. The molecular weight of the main protein present in this fraction was 12 000 as determined by SDS-poly-acrylamide-gel electrophoresis. This soluble fraction stimulated the transfer of oleic acid from microsomes to phosphatidylcholine liposomes as demonstrated by a transfer assay in vitro. The behaviour of this fraction is similar to that described for fatty-acid binding protein.

  13. Fatty Acid-Binding Protein 5 Facilitates the Blood-Brain Barrier Transport of Docosahexaenoic Acid.

    PubMed

    Pan, Yijun; Scanlon, Martin J; Owada, Yuji; Yamamoto, Yui; Porter, Christopher J H; Nicolazzo, Joseph A

    2015-12-07

    The brain has a limited ability to synthesize the essential polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA) from its omega-3 fatty acid precursors. Therefore, to maintain brain concentrations of this PUFA at physiological levels, plasma-derived DHA must be transported across the blood-brain barrier (BBB). While DHA is able to partition into the luminal membrane of brain endothelial cells, its low aqueous solubility likely limits its cytosolic transfer to the abluminal membrane, necessitating the requirement of an intracellular carrier protein to facilitate trafficking of this PUFA across the BBB. As the intracellular carrier protein fatty acid-binding protein 5 (FABP5) is expressed at the human BBB, the current study assessed the putative role of FABP5 in the brain endothelial cell uptake and BBB transport of DHA in vitro and in vivo, respectively. hFAPB5 was recombinantly expressed and purified from Escherichia coli C41(DE3) cells and the binding affinity of DHA to hFABP5 assessed using isothermal titration calorimetry. The impact of FABP5 siRNA on uptake of (14)C-DHA into immortalized human brain microvascular endothelial (hCMEC/D3) cells was assessed. An in situ transcardiac perfusion method was optimized in C57BL/6 mice and subsequently used to compare the BBB influx rate (Kin) of (14)C-DHA between FABP5-deficient (FABP5(-/-)) and wild-type (FABP5(+/+)) C57BL/6 mice. DHA bound to hFABP5 with an equilibrium dissociation constant of 155 ± 8 nM (mean ± SEM). FABP5 siRNA transfection decreased hCMEC/D3 mRNA and protein expression of FABP5 by 53.2 ± 5.5% and 44.8 ± 13.7%, respectively, which was associated with a 14.1 ± 2.7% reduction in (14)C-DHA cellular uptake. By using optimized conditions for the in situ transcardiac perfusion (a 1 min preperfusion (10 mL/min) followed by perfusion of (14)C-DHA (1 min)), the Kin of (14)C-DHA was 0.04 ± 0.01 mL/g/s. Relative to FABP5(+/+) mice, the Kin of (14)C-DHA decreased 36.7 ± 12.4% in FABP5(-/-) mice

  14. Purification of antifreeze protein from wheat bran (Triticum aestivum L.) based on its hydrophilicity and ice-binding capacity.

    PubMed

    Zhang, Chao; Zhang, Hui; Wang, Li; Zhang, Junhui; Yao, Huiyuan

    2007-09-19

    Wheat-bran ( Triticum aestivum L.) antifreeze protein ( TaAFP) was purified 323-fold to electrophoretic homogeneity with an overall yield of 1.64% from wheat-bran protein by a specific three-step procedure. The three-step procedure was quicker, cheaper, and more effective than the five-step procedure we used earlier. First, TaAFP was concentrated by a phosphate buffer, on the basis of its strong hydrophilicity that was validated by thermal gravimetric analyses and a surface hydrophobicity analysis. Second, TaAFP was trapped in ice crystals for its specific ice-binding capacity, which was proved by ice-binding protocols. Remarkably, the ice-binding step was the most effective step, and the purification factor of this step was up to 270-fold. Finally, TaAFP was purified by HPLC purification, a complementary step for the specific ice-binding protocol, to electrophoretic homogeneity. Our protocols provide peers a novel and effective way for the search and purification of potential AFPs.

  15. A gravimetric adaptation of the filter paper press method for the determination of water-binding capacity.

    PubMed

    Karmas, E; Turk, K

    1975-07-18

    A gravimetric adaptation of the filter paper press method for the determination of water-binding capacity in meat was developed and its sensitivity was compared to that of the conventional planimetric technique of the method. Both the gravimetric and planimetric techniques were applied to samples of cooked fish treated with various water binders. The mean results of the samples were grouped and compared using an analysis of variance. In all comparisons, the gravimetric data produced higher F-values than did the planimetric data for the same samples. This indicated greater senstivity for the gravimetric technique.

  16. Effect of d-amino acids on IgE binding to peanut allergens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    D-amino acids are formed when L-amino acids are exposed to heat. The objective was to determine the existence of D-amino acids in roasted peanut and their effect on IgE binding. Raw and roasted peanut protein extracts were hydrolyzed in 6 N HCL under vacuum. The hydrolysates were then analyzed for D...

  17. Structure and nucleic acid binding activity of the nucleoporin Nup157

    PubMed Central

    Seo, Hyuk-Soo; Blus, Bartlomiej J.; Janković, Nina Z.; Blobel, Günter

    2013-01-01

    At the center of the nuclear pore complex (NPC) is a uniquely versatile central transport channel. Structural analyses of distinct segments (“protomers”) of the three “channel” nucleoporins yielded a model for how this channel is constructed. Its principal feature is a midplane ring that can undergo regulated diameter changes of as much as an estimated 30 nm. To better understand how a family of “adaptor” nucleoporins—concentrically surrounding this channel—might cushion these huge structural changes, we determined the crystal structure of one adaptor nucleoporin, Nup157. Here, we show that a recombinant Saccharomyces cerevisiae Nup157 protomer, representing two-thirds of Nup157 (residues 70–893), folds into a seven-bladed β-propeller followed by an α-helical domain, which together form a C-shaped architecture. Notably, the structure contains a large patch of positively charged residues, most of which are evolutionarily conserved. Consistent with this surface feature, we found that Nup15770–893 binds to nucleic acids, although in a sequence-independent manner. Nevertheless, this interaction supports a previously reported role of Nup157, and its paralogue Nup170, in chromatin organization. Based on its nucleic acid binding capacity, we propose a dual location and function of Nup157. Finally, modeling the remaining C-terminal portion of Nup157 shows that it projects as a superhelical stack from the compact C-shaped portion of the molecule. The predicted four hinge regions indicate an intrinsic flexibility of Nup157, which could contribute to structural plasticity within the NPC. PMID:24062435

  18. BindML/BindML+: Detecting Protein-Protein Interaction Interface Propensity from Amino Acid Substitution Patterns.

    PubMed

    Wei, Qing; La, David; Kihara, Daisuke

    2017-01-01

    Prediction of protein-protein interaction sites in a protein structure provides important information for elucidating the mechanism of protein function and can also be useful in guiding a modeling or design procedures of protein complex structures. Since prediction methods essentially assess the propensity of amino acids that are likely to be part of a protein docking interface, they can help in designing protein-protein interactions. Here, we introduce BindML and BindML+ protein-protein interaction sites prediction methods. BindML predicts protein-protein interaction sites by identifying mutation patterns found in known protein-protein complexes using phylogenetic substitution models. BindML+ is an extension of BindML for distinguishing permanent and transient types of protein-protein interaction sites. We developed an interactive web-server that provides a convenient interface to assist in structural visualization of protein-protein interactions site predictions. The input data for the web-server are a tertiary structure of interest. BindML and BindML+ are available at http://kiharalab.org/bindml/ and http://kiharalab.org/bindml/plus/ .

  19. Elucidating the Influence of Gold Nanoparticles on the Binding of Salvianolic Acid B and Rosmarinic Acid to Bovine Serum Albumin

    PubMed Central

    Peng, Xin; Qi, Wei; Huang, Renliang; Su, Rongxin; He, Zhimin

    2015-01-01

    Salvianolic acid B and rosmarinic acid are two main water-soluble active ingredients from Salvia miltiorrhiza with important pharmacological activities and clinical applications. The interactions between salvianolic acid B (or rosmarinic acid) and bovine serum albumin (BSA) in the presence and absence of gold nanoparticles (Au NPs) with three different sizes were investigated by using biophysical methods for the first time. Experimental results proved that two components quenched the fluorescence of BSA mainly through a static mechanism irrespective of the absence or presence of Au NPs. The presence of Au NPs decreased the binding constants of salvianolic acid B with BSA from 27.82% to 10.08%, while Au NPs increased the affinities of rosmarinic acid for BSA from 0.4% to 14.32%. The conformational change of BSA in the presence of Au NPs (caused by a noncompetitive binding between Au NPs and drugs at different albumin sites) induced changeable affinity and binding distance between drugs and BSA compared with no Au NPs. The competitive experiments revealed that the site I (subdomain IIA) of BSA was the primary binding site for salvianolic acid B and rosmarinic acid. Additionally, two compounds may induce conformational and micro-environmental changes of BSA. The results would provide valuable binding information between salvianolic acid B (or rosmarinic acid) and BSA, and also indicated that the Au NPs could alter the interaction mechanism and binding capability of drugs to BSA, which might be beneficial to understanding the pharmacokinetics and biological activities of the two drugs. PMID:25861047

  20. Calreticulin is a microbial-binding molecule with phagocytosis-enhancing capacity.

    PubMed

    Liu, Xuemei; Xu, Na; Zhang, Shicui

    2013-09-01

    Calreticulin (CRT) is a highly conserved calcium-binding protein mainly involved in directing proper conformation of proteins and controlling calcium level. Accumulating data also show that CRT is emerging as an immune-relevant molecule. In this study, we demonstrated that the CRT gene from the amphioxus Branchiostoma japonicum, named Bjcrt, consisted of a signal peptide, three domains (N-, P-, C-domains) and an ER retrieval signal sequence (KDEL), which appears to be the ancient form of vertebrate CRTs, and Bjcrt was expressed in a tissue-specific manner, with the most abundant expression in the notochord. We also demonstrated for the first time that the recombinant BjCRT (rBjCRT) was able to bind the Gram-negative bacterium Escherichia coli and the Gram-positive bacterium Staphylococcus aureus. Moreover, both BjCRT as well as human recombinant calreticulin were able to promote the phagocytosis of E. coli and S. aureus by sea bass macrophages. These results indicate that CRT is a microbial-binding molecule and possesses an ability to enhance phagocytosis, a novel function assigned to CRT, reenforcing the notion that CRT is an immune-relevant molecule associated with host immune responses.

  1. Bordetella dermonecrotic toxin binds to target cells via the N-terminal 30 amino acids.

    PubMed

    Fukui-Miyazaki, Aya; Ohnishi, Shinya; Kamitani, Shigeki; Abe, Hiroyuki; Horiguchi, Yasuhiko

    2011-03-01

    Bordetella dermonecrotic toxin (DNT) affects the biological function of host cells by activating intracellular Rho GTPases. The toxin binds to unidentified receptor(s) via 54 N-terminal amino acids, undergoes intramolecular cleavage on the C-terminal side of Arg(44) by furin or furin-like protease, and eventually enters the cytoplasm where the Rho GTPases reside. The binding to the receptor(s) and intramolecular cleavage are essential for DNT to intoxicate cells, and the 54 amino-acid binding domain encompasses the cleavage site, however, it is unclear whether these two events are related. In this study, we could narrow down the cell-binding domain to the N-terminal amino acids 2-30. The region does not contain the furin-recognition site, indicating that the cell binding and the intramolecular cleavage are independent events.

  2. Changes in erythrocyte sodium, sodium transport and /sup 3/H ouabain binding capacity during digoxin administration in the pig

    SciTech Connect

    Whittaker, J.; Hawkins, M.; Swaminathan, R.

    1983-02-14

    Time course of the changes in erthrocyte sodium content, sodium transport, /sup 3/H ouabain binding capacity and Na/sup +/, K/sup +/-ATPase activity were measured for 14 weeks, in 6 young pigs treated with digoxin and in 6 control pigs. After one week of treatment the erythrocyte sodium content increased from 5.4 mmol/kg cells and the efflux rate constant of sodium decreased. With prolonged treatment the erythrocyte sodium content returned to normal and the /sup 3/H ouabain binding capacity increased by week 5. The plasma digoxin concentration decreased from 1.1 ng/ml at week 5 to 0.6 ng/ml at week 8 probably due to the decline in dose (..mu..g/kg) of digoxin with age. The efflux rate constant of sodium and Na/sup +/, K/sup +/-ATPase activity were higher towards the end of treatment. It is concluded that with prolonged administration of digoxin there is an increase in erythrocyte sodium pump units.

  3. Characterization of the cation-binding capacity of a potassium-adsorption filter used in red blood cell transfusion.

    PubMed

    Suzuki, Takao; Muto, Shigeaki; Miyata, Yukio; Maeda, Takao; Odate, Takayuki; Shimanaka, Kimio; Kusano, Eiji

    2015-06-01

    A K(+) -adsorption filter was developed to exchange K(+) in the supernatant of stored irradiated red blood cells with Na(+) . To date, however, the filter's adsorption capacity for K(+) has not been fully evaluated. Therefore, we characterized the cation-binding capacity of this filter. Artificial solutions containing various cations were continuously passed through the filter in 30 mL of sodium polystyrene sulfonate at 10 mL/min using an infusion pump at room temperature. The cation concentrations were measured before and during filtration. When a single solution containing K(+) , Li(+) , H(+) , Mg(2+) , Ca(2+) , or Al(3+) was continuously passed through the filter, the filter adsorbed K(+) and the other cations in exchange for Na(+) in direct proportion to the valence number. The order of affinity for cation adsorption to the filter was Ca(2+) >Mg(2+) >K(+) >H(+) >Li(+) . In K(+) -saturated conditions, the filter also adsorbed Na(+) . After complete adsorption of these cations on the filter, their concentration in the effluent increased in a sigmoidal manner over time. Cations that were bound to the filter were released if a second cation was passed through the filter, despite the different affinities of the two cations. The ability of the filter to bind cations, especially K(+) , should be helpful when it is used for red blood cell transfusion at the bedside. The filter may also be useful to gain a better understanding of the pharmacological properties of sodium polystyrene sulfonate.

  4. Origin of the low temperature excess heat capacity of isotopically substituted acetylsalicylic acid

    NASA Astrophysics Data System (ADS)

    Schröder, F.; Winkler, B.; Bauer, J. D.; Haussühl, E.; Rivera Escoto, B.; Tristan López, F.; Avalos Borja, M.; Richter, C.; Ferner, J.

    2011-09-01

    The low temperature heat capacities of single crystals of fully protonated acetylsalicylic acid, measured between 2 K < T < 80 K, have been compared to samples in which the methyl-group was replaced by CD3, CH2D and 13CH3. For the partially methyl-deuterated crystal (CH2D) a significant excess heat capacity was found below T < 40 K with a broad maximum around T ≈ 14 K. The thermodynamic data are explained on the basis of a Schottky model using results obtained in earlier NMR and neutron spectroscopic experiments. In contrast, the excess heat capacity of the fully deuterated compound can be explained by a change of the phonon density of states.

  5. Molecular Dynamic Simulations Reveal the Structural Determinants of Fatty Acid Binding to Oxy-Myoglobin

    PubMed Central

    Chintapalli, Sree V.; Bhardwaj, Gaurav; Patel, Reema; Shah, Natasha; Patterson, Randen L.; van Rossum, Damian B.; Anishkin, Andriy; Adams, Sean H.

    2015-01-01

    The mechanism(s) by which fatty acids are sequestered and transported in muscle have not been fully elucidated. A potential key player in this process is the protein myoglobin (Mb). Indeed, there is a catalogue of empirical evidence supporting direct interaction of globins with fatty acid metabolites; however, the binding pocket and regulation of the interaction remains to be established. In this study, we employed a computational strategy to elucidate the structural determinants of fatty acids (palmitic & oleic acid) binding to Mb. Sequence analysis and docking simulations with a horse (Equus caballus) structural Mb reference reveals a fatty acid-binding site in the hydrophobic cleft near the heme region in Mb. Both palmitic acid and oleic acid attain a “U” shaped structure similar to their conformation in pockets of other fatty acid-binding proteins. Specifically, we found that the carboxyl head group of palmitic acid coordinates with the amino group of Lys45, whereas the carboxyl group of oleic acid coordinates with both the amino groups of Lys45 and Lys63. The alkyl tails of both fatty acids are supported by surrounding hydrophobic residues Leu29, Leu32, Phe33, Phe43, Phe46, Val67, Val68 and Ile107. In the saturated palmitic acid, the hydrophobic tail moves freely and occasionally penetrates deeper inside the hydrophobic cleft, making additional contacts with Val28, Leu69, Leu72 and Ile111. Our simulations reveal a dynamic and stable binding pocket in which the oxygen molecule and heme group in Mb are required for additional hydrophobic interactions. Taken together, these findings support a mechanism in which Mb acts as a muscle transporter for fatty acid when it is in the oxygenated state and releases fatty acid when Mb converts to deoxygenated state. PMID:26030763

  6. Murine protein which binds preferentially to oligo-C-rich single-stranded nucleic acids.

    PubMed Central

    Goller, M; Funke, B; Gehe-Becker, C; Kröger, B; Lottspeich, F; Horak, I

    1994-01-01

    Two single-stranded nucleic acid binding proteins mCBP and mCTBP were identified by means of their binding to a potential recombination hotspot in LTRs of mouse retro-transposons. Both are nuclear proteins of 35 and 55 kDa respectively. mCBP binds preferentially to oligo dC, mCTBP to oligo dCdT. mCBP was purified and its cDNA was isolated and sequenced. Images PMID:8208614

  7. Recombinant canine single chain insulin analogues: insulin receptor binding capacity and ability to stimulate glucose uptake.

    PubMed

    Adams, Jamie P; Holder, Angela L; Catchpole, Brian

    2014-12-01

    Virtually all diabetic dogs require exogenous insulin therapy to control their hyperglycaemia. In the UK, the only licensed insulin product currently available is a purified porcine insulin preparation. Recombinant insulin is somewhat problematic in terms of its manufacture, since the gene product (preproinsulin) undergoes substantial post-translational modification in pancreatic β cells before it becomes biologically active. The aim of the present study was to develop recombinant canine single chain insulin (SCI) analogues that could be produced in a prokaryotic expression system and which would require minimal processing. Three recombinant SCI constructs were developed in a prokaryotic expression vector, by replacing the insulin C-peptide sequence with one encoding a synthetic peptide (GGGPGKR), or with one of two insulin-like growth factor (IGF)-2 C-peptide coding sequences (human: SRVSRRSR; canine: SRVTRRSSR). Recombinant proteins were expressed in the periplasmic fraction of Escherichia coli and assessed for their ability to bind to the insulin and IGF-1 receptors, and to stimulate glucose uptake in 3T3-L1 adipocytes. All three recombinant SCI analogues demonstrated preferential binding to the insulin receptor compared to the IGF-1 receptor, with increased binding compared to recombinant canine proinsulin. The recombinant SCI analogues stimulated glucose uptake in 3T3-L1 adipocytes compared to negligible uptake using recombinant canine proinsulin, with the canine insulin/cIGF-2 chimaeric SCI analogue demonstrating the greatest effect. Thus, biologically-active recombinant canine SCI analogues can be produced relatively easily in bacteria, which could potentially be used for treatment of diabetic dogs.

  8. Multispectroscopic and docking studies on the binding of chlorogenic acid isomers to human serum albumin: Effects of esteryl position on affinity.

    PubMed

    Tang, Bin; Huang, Yanmei; Ma, Xiangling; Liao, Xiaoxiang; Wang, Qing; Xiong, Xinnuo; Li, Hui

    2016-12-01

    Structural differences among various dietary polyphenols affect their absorption, metabolism, and bioactivities. In this work, chlorogenic acid (CA) and its two positional isomers, neochlorogenic acid (NCA) and cryptochlorogenic acid (CCA), were investigated for their binding reactions with human serum albumin (HSA) using fluorescence, ultraviolet-visible, Fourier transform infrared and circular dichroism spectroscopies, as well as molecular docking. All three isomers were bound to HSA at Sudlow's site I and affected the protein secondary structure. CCA presented the strongest ability of hydrogen-bond formation, and both CA and NCA generated more electrostatic interactions with HSA. The albumin-binding capacity of these compounds decreased in the order CCA>NCA>CA. The compound with 4-esteryl structure showed higher binding affinity and larger conformational changes to HSA than that with 3- or 5-esteryl structures. These comparative studies on structure-affinity relationship contributed to the structural modification and design of phenolic food additives or new polyphenol-like drugs.

  9. Treatment with oleic acid reduces IgE binding to peanut and cashew allergens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Oleic acid (OA) is known to bind and change the bioactivities of proteins, such as a-lactalbumin and ß-lactoglobulin in vitro. The objective of this study was to determine if OA binds to allergens from a peanut extract or cashew allergen and changes their allergenic properties. Peanut extract or c...

  10. Chemical treatment of olive pomace: effect on acid-basic properties and metal biosorption capacity.

    PubMed

    Martín-Lara, M A; Pagnanelli, F; Mainelli, S; Calero, M; Toro, L

    2008-08-15

    In this study, olive pomace, an agricultural waste that is very abundant in Mediterranean area, was modified by two chemical treatments in order to improve its biosorption capacity. Potentiometric titrations and IR analyses were used to characterise untreated olive pomace (OP), olive pomace treated by phosphoric acid (PAOP) and treated by hydrogen peroxide (HPOP). Acid-base properties of all investigated biosorbents were characterised by two main kinds of active sites, whose nature and concentration were determined by a mechanistic model assuming continuous distribution for the proton affinity constants. Titration modelling denoted that all investigated biosorbents (OP, PAOP and HPOP) were characterised by the same kinds of active sites (carboxylic and phenolic), but with different total concentrations with PAOP richer than OP and HPOP. Single metal equilibrium studies in batch reactors were carried out to determine the capacity of these sorbents for copper and cadmium ions at constant pH. Experimental data were analysed and compared using the Langmuir isotherm. The order of maximum uptake capacity of copper and cadmium ions on different biosorbents was PAOP>HPOP>OP. The maximum adsorption capacity of copper and cadmium, was obtained as 0.48 and 0.10 mmol/g, respectively, for PAOP. Metal biosorption tests in presence of Na(+) in solution were also carried out in order to evaluate the effect of chemical treatment on biomass selectivity. These data showed that PAOP is more selective for cadmium than the other sorbents, while similar selectivity was observed for copper.

  11. Quest for the binding mode of malachite green with humic acid

    NASA Astrophysics Data System (ADS)

    Zhang, Hongmei; Yin, Mingxing; Shi, Jinghua; Wang, Yanqing

    2015-02-01

    The association of malachite green (MG) with humic acid (HA) was investigated by using fluorescence, UV-vis spectroscopy and molecular Modelling method. The fluorescence spectral results indicated that the binding between MG and HA occurred by mainly hydrophobic and electrostatic forces with association constants of KA (298 K) = 6.24 × 105 L/mol and KA (310 K) = 10.20 × 105 L/mol. There were more than one binding sites on HA to bind with MG. The binding sites of MG with HA primarily located at the aromatic rings of HA. MG could enter into the hydrophobic cavities of HA to quench the fluorescence of HA. On the contrary, HA binding caused MG to a coplanar conformation with more extended π bond distribution by π-π stacking interactions. The experiment and calculation data both showed that the hydrophobic binding cavities in HA played a key role in its binding with MG.

  12. Sex Steroid Modulation of Fatty Acid Utilization and Fatty Acid Binding Protein Concentration in Rat Liver

    PubMed Central

    Ockner, Robert K.; Lysenko, Nina; Manning, Joan A.; Monroe, Scott E.; Burnett, David A.

    1980-01-01

    The mechanism by which sex steroids influence very low density hepatic lipoprotein triglyceride production has not been fully elucidated. In previous studies we showed that [14C]oleate utilization and incorporation into triglycerides were greater in hepatocyte suspensions from adult female rats than from males. The sex differences were not related to activities of the enzymes of triglyceride biosynthesis, whereas fatty acid binding protein (FABP) concentration in liver cytosol was greater in females. These findings suggested that sex differences in lipoprotein could reflect a sex steroid influence on the availability of fatty acids for hepatocellular triglyceride biosynthesis. In the present studies, sex steroid effects on hepatocyte [14C]oleate utilization and FABP concentration were investigated directly. Hepatocytes from immature (30-d-old) rats exhibited no sex differences in [14C]oleate utilization. With maturation, total [14C]oleate utilization and triglyceride biosynthesis increased moderately in female cells and decreased markedly in male cells; the profound sex differences in adults were maximal by age 60 d. Fatty acid oxidation was little affected. Rats were castrated at age 30 d, and received estradiol, testosterone, or no hormone until age 60 d, when hepatocyte [14C]oleate utilization was studied. Castration virtually eliminated maturational changes and blunted the sex differences in adults. Estradiol or testosterone largely reproduced the appropriate adult pattern of [14C]oleate utilization regardless of the genotypic sex of the treated animal. In immature females and males, total cytosolic FABP concentrations were similar. In 60-d-old animals, there was a striking correlation among all groups (females, males, castrates, and hormone-treated) between mean cytosolic FABP concentration on the one hand, and mean total [14C]oleate utilization (r = 0.91) and incorporation into triglycerides (r = 0.94) on the other. In 30-d-old animals rates of [14C

  13. Stream chemistry in the eastern United States. 2. Current sources of acidity in acidic and low acid-neutralizing capacity streams

    SciTech Connect

    Herlihy, A.T.; Kaufmann, P.R.; Mitch, M.E. )

    1991-04-01

    The authors examined anion composition in National Stream Survey (NSS) data in order to evaluate the most probably sources of current acidity in acidic and low acid-neutralizing capacity (ANC) streams in the eastern US. Acidic streams that had almost no organic influence (less than 10% of total anions) and sulfate and nitrate concentrations indicative of evaporative concentration of atmospheric deposition were classified as acidic due to acidic deposition. These acidic streams were located in small (<30 km{sup 2}) forested watersheds in the Mid-Atlantic Highlands (an estimated 1,950 km of stream length) and in the Mid-Atlantic Coastal Plain (1,250 km). Acidic streams affected primarily by acidic deposition but also influenced by naturally occurring organic anions accounted for another 1,180 km of acidic stream length, and were located in the New Jersey Pine Barrens, plateau tops in the Mid-Atlantic and Southeast Highlands, and the Florida Panhandle. The total length of streams acidic due to acid mine drainage in the NSS (4,590 km) was about the same as the total length of acidic streams likely affected by acidic deposition (4,380 km). Acidic streams whose acid anion composition was dominated by organics were located in Florida and the Mid-Atlantic Coastal Plain. In Florida, most of the acidic streams were organic dominated, whereas about half of the streams in the Mid-Atlantic Coastal Plain were organic dominated. Organic-dominated acidic streams were not observed in the Mid-Atlantic and Southeast Highlands.

  14. Functionalization of magnetic nanoparticles with high-binding capacity for affinity separation of therapeutic proteins

    NASA Astrophysics Data System (ADS)

    Masthoff, Ingke-Christine; David, Florian; Wittmann, Christoph; Garnweitner, Georg

    2014-01-01

    Magnetic nanoparticles with immobilized metal ligands were prepared for the separation of antibody fragments. First, iron oxide nanoparticles were produced in a solvothermal synthesis using triethylene glycol as solvent and iron(III) acetylacetonate as organic precursor. Via functionalization of the particles with priorly reacted 3-glycidoxypropyltrimethoxysilane and N α, N α-bis(carboxymethyl)- l-lysine (NTA), and charging with Ni2+, magnetic affinity adsorbents were obtained. The particles were applied to separate a His-tagged antibody fragment from a heterogeneous protein mixture of a microbial cultivation supernatant. Binding properties and specificity for purification of the target product ABF D1.3 scFv were optimized regarding the GNTA concentration and were found superior as compared to commercially available systems. A molar ratio of 1:2 Fe2O3:GNTA was most beneficial for the specific purification of the antibody fragment.

  15. Structural Conservation of Ligand Binding Reveals a Bile Acid-like Signaling Pathway in Nematodes*

    PubMed Central

    Zhi, Xiaoyong; Zhou, X. Edward; Melcher, Karsten; Motola, Daniel L.; Gelmedin, Verena; Hawdon, John; Kliewer, Steven A.; Mangelsdorf, David J.; Xu, H. Eric

    2012-01-01

    Bile acid-like molecules named dafachronic acids (DAs) control the dauer formation program in Caenorhabditis elegans through the nuclear receptor DAF-12. This mechanism is conserved in parasitic nematodes to regulate their dauer-like infective larval stage, and as such, the DAF-12 ligand binding domain has been identified as an important therapeutic target in human parasitic hookworm species that infect more than 600 million people worldwide. Here, we report two x-ray crystal structures of the hookworm Ancylostoma ceylanicum DAF-12 ligand binding domain in complex with DA and cholestenoic acid (a bile acid-like metabolite), respectively. Structure analysis and functional studies reveal key residues responsible for species-specific ligand responses of DAF-12. Furthermore, DA binds to DAF-12 mechanistically and is structurally similar to bile acids binding to the mammalian bile acid receptor farnesoid X receptor. Activation of DAF-12 by cholestenoic acid and the cholestenoic acid complex structure suggest that bile acid-like signaling pathways have been conserved in nematodes and mammals. Together, these results reveal the molecular mechanism for the interplay between parasite and host, provide a structural framework for DAF-12 as a promising target in treating nematode parasitism, and provide insight into the evolution of gut parasite hormone-signaling pathways. PMID:22170062

  16. Calcium ion binding to a soil fulvic acid using a donnan potential model

    USGS Publications Warehouse

    Marinsky, J.A.; Mathuthu, A.; Ephraim, J.H.; Reddy, M.M.

    1999-01-01

    Calcium ion binding to a soil fulvic acid (Armadale Bh Horizon) was evaluated over a range of calcium ion concentrations, from pH 3.8 to 7.3, using potentiometric titrations and calcium ion electrode measurements. Fulvic acid concentration was constant (100 milligrams per liter) and calcium ion concentration varied up to 8 X 10-4 moles per liter. Experiments discussed here included: (1) titrations of fulvic acid-calcium ion containing solutions with sodium hydroxide; and (2) titrations of fully neutralized fulvic acid with calcium chloride solutions. Apparent binding constants (expressed as the logarithm of the value, log ??app) vary with solution pH, calcium ion concentration, degree of acid dissociation, and ionic strength (from log ??app = 2.5 to 3.9) and are similar to those reported by others. Fulvic acid charge, and the associated Donnan Potential, influences calcium ion-fulvic acid ion pair formation. A Donnan Potential corrrection term allowed calculation of intrinsic calcium ion-fulvic acid binding constants. Intrinsic binding constants vary from 1.2 to 2.5 (the average value is about log??= 1.6) and are similar to, but somewhat higher than, stability constants for calcium ion-carboxylic acid monodentate complexes. ?? by Oldenbourg Wissenschaftsverlag, Mu??nchen.

  17. A Siglec-like sialic-acid-binding motif revealed in an adenovirus capsid protein

    PubMed Central

    Rademacher, Christoph; Bru, Thierry; McBride, Ryan; Robison, Elizabeth; Nycholat, Corwin M; Kremer, Eric J; Paulson, James C

    2012-01-01

    Sialic-acid-binding immunoglobulin-like lectins (Siglecs) are a family of transmembrane receptors that are well documented to play roles in regulation of innate and adaptive immune responses. To see whether the features that define the molecular recognition of sialic acid were found in other sialic-acid-binding proteins, we analyzed 127 structures with bound sialic acids found in the Protein Data Bank database. Of these, the canine adenovirus 2-fiber knob protein showed close local structural relationship to Siglecs despite low sequence similarity. The fiber knob harbors a noncanonical sialic-acid recognition site, which was then explored for detailed specificity using a custom glycan microarray comprising 58 diverse sialosides. It was found that the adenoviral protein preferentially recognizes the epitope Neu5Acα2-3[6S]Galβ1-4GlcNAc, a structure previously identified as the preferred ligand for Siglec-8 in humans and Siglec-F in mice. Comparison of the Siglec and fiber knob sialic-acid-binding sites reveal conserved structural elements that are not clearly identifiable from the primary amino acid sequence, suggesting a Siglec-like sialic-acid-binding motif that comprises the consensus features of these proteins in complex with sialic acid. PMID:22522600

  18. Phosphate ester hydrolysis and DNA binding capacity of two new dinuclear Ni(II) complexes

    NASA Astrophysics Data System (ADS)

    Wang, Y.; Xiao, W.; Mao, J. W.; Zhou, H.; Pan, Z. Q.

    2013-03-01

    Two new dinuclear Ni(II) complexes, [Ni2L1(OAc)2]ClO4 (1) and [Ni2L2(OAc)2] Br·0.5H2O (2) (where HL is the condensation product of 2,6-diformyl-4-X-phenol (X = CH3, Br) and N1-(2-aminoethyl)-N2-(4-nitrobenzyl)ethane-1,2-diamine), were synthesized and characterized. The coordination polyhedron of each metal ion in the complexes can be approximately described as a distorted octahedron. The Ni-Ni distances in 1 and 2 are 3.306 Å and 3.369 Å, respectively. The phosphate hydrolysis promoted by the complexes was examined using 4-nitrophenyl phosphate disodium salt hexahydrate (pNPP) as the substrate. The catalytic rate constants (kcat) are 1.29 × 10-5 s-1 for 1 and 0.47 × 10-5 s-1 for 2 at 25 °C in physiological pH condition. The binding activities of the complexes toward calf thymus (CT-DNA) were analyzed by spectroscopic and voltammetric methods, and the corresponding binding constants are 7.75 × 105 and 6.51 × 104 M-1 for 1 and 8.59 × 105 and 9.18 × 104 M-1 for 2, respectively. The linear Stern-Volmer quenching constants obtained in the fluorescent spectroscopic study of 1 and 2 are 0.7 × 103 and 1.5 × 103 M-1, respectively.

  19. Structural and functional analysis of fatty acid-binding proteins

    PubMed Central

    Storch, Judith; McDermott, Lindsay

    2009-01-01

    The mammalian FA-binding proteins (FABPs) bind long-chain FA with high affinity. The large number of FABP types is suggestive of distinct functions in specific tissues. Multiple experimental approaches have shown that individual FABPs possess both unique and overlapping functions, some of which are based on specific elements in the protein structure. Although FA binding affinities for all FABPs tend to correlate directly with FA hydrophobicity, structure-function studies indicate that subtle three-dimensional changes that occur upon ligand binding may promote specific protein-protein or protein-membrane interactions that ultimately determine the function of each FABP. The conformational changes are focused in the FABP helical/portal domain, a region that was identified by in vitro studies to be vital for the FA transport properties of the FABPs. Thus, the FABPs modulate intracellular lipid homeostasis by regulating FA transport in the nuclear and extra-nuclear compartments of the cell; in so doing, they also impact systemic energy homeostasis. PMID:19017610

  20. Capture and release of mixed acid gasses with binding organic liquids

    DOEpatents

    Heldebrant, David J.; Yonker, Clement R.

    2010-09-21

    Reversible acid-gas binding organic liquid systems that permit separation and capture of one or more of several acid gases from a mixed gas stream, transport of the liquid, release of the acid gases from the ionic liquid and reuse of the liquid to bind more acid gas with significant energy savings compared to current aqueous systems. These systems utilize acid gas capture compounds made up of strong bases and weak acids that form salts when reacted with a selected acid gas, and which release these gases when a preselected triggering event occurs. The various new materials that make up this system can also be included in various other applications such as chemical sensors, chemical reactants, scrubbers, and separators that allow for the specific and separate removal of desired materials from a gas stream such as flue gas.

  1. Fulvic acid-sulfide ion competition for mercury ion binding in the Florida everglades

    USGS Publications Warehouse

    Reddy, M.M.; Aiken, G.R.

    2001-01-01

    Negatively charged functional groups of fulvic acid compete with inorganic sulfide ion for mercury ion binding. This competition is evaluated here by using a discrete site-electrostatic model to calculate mercury solution speciation in the presence of fulvic acid. Model calculated species distributions are used to estimate a mercury-fulvic acid apparent binding constant to quantify fulvic acid and sulfide ion competition for dissolved inorganic mercury (Hg(II)) ion binding. Speciation calculations done with PHREEQC, modified to use the estimated mercury-fulvic acid apparent binding constant, suggest that mercury-fulvic acid and mercury-sulfide complex concentrations are equivalent for very low sulfide ion concentrations (about 10-11 M) in Everglades' surface water. Where measurable total sulfide concentration (about 10-7 M or greater) is present in Everglades' surface water, mercury-sulfide complexes should dominate dissolved inorganic mercury solution speciation. In the absence of sulfide ion (for example, in oxygenated Everglades' surface water), fulvic acid binding should dominate Everglades' dissolved inorganic mercury speciation.

  2. Endogenous fatty acids in olfactory hairs influence pheromone binding protein structure and function in Lymantria dispar.

    PubMed

    Nardella, Jason; Terrado, Mailyn; Honson, Nicolette S; Plettner, Erika

    2015-08-01

    The gypsy moth utilizes a pheromone, (7R,8S)-2-methyl-7,8-epoxyoctadecane, for mate location. The pheromone is detected by sensory hairs (sensilla) on the antennae of adult males. Sensilla contain the dendrites of olfactory neurons bathed in lymph, which contains pheromone binding proteins (PBPs). We have extracted and identified free fatty acids from lymph of sensory hairs, and we demonstrate that these function as endogenous ligands for gypsy moth PBP1 and PBP2. Homology modeling of both PBPs, and docking of fatty acids reveal multiple binding sites: one internal, the others external. Pheromone binding assays suggest that these fatty acids increase PBP-pheromone binding affinity. We show that fatty acid binding causes an increase in α-helix content in the N-terminal domain, but not in the C-terminal peptide of both proteins. The C-terminal peptide was shown to form a α-helix in a hydrophobic, homogeneous environment, but not in the presence of fatty acid micelles. Through partition assays we show that the fatty acids prevent adsorption of the pheromone on hydrophobic surfaces and facilitate pheromone partition into an aqueous phase. We propose that lymph is an emulsion of fatty acids and PBP that influence each other and thereby control the partition equilibria of hydrophobic odorants.

  3. Simultaneous determination of Ca, Cu, Ni, Zn and Cd binding strengths with fulvic acid fractions by Schubert's method

    USGS Publications Warehouse

    Brown, G.K.; MacCarthy, P.; Leenheer, J.A.

    1999-01-01

    The equilibrium binding of Ca2+, Ni2+, Cd2+, Cu2+ and Zn2+ with unfractionated Suwannee river fulvic acid (SRFA) and an enhanced metal binding subfraction of SRFA was measured using Schubert's ion-exchange method at pH 6.0 and at an ionic strength (??) of 0.1 (NaNO3). The fractionation and subfractionation were directed towards obtaining an isolate with an elevated metal binding capacity or binding strength as estimated by Cu2+ potentiometry (ISE). Fractions were obtained by stepwise eluting an XAD-8 column loaded with SRFA with water eluents of pH 1.0 to pH 12.0. Subfractions were obtained by loading the fraction eluted from XAD-8 at pH 5.0 onto a silica gel column and eluting with solvents of increasing polarity. Schuberts ion exchange method was rigorously tested by measuring simultaneously the conditional stability constants (K) of citric acid complexed with the five metals at pH 3.5 and 6.0. The logK of SRFA with Ca2+, Ni2+, Cd2+, Cu2+ and Zn2+ determined simultaneously at pH 6.0 follow the sequence of Cu2+>Cd2+>Ni2+>Zn2+>Ca2+ while all logK values increased for the enhanced metal binding subfraction and followed a different sequence of Cu2+>Cd2+>Ca2+>Ni2+>Zn2+. Both fulvic acid samples and citric acid exhibited a 1:1 metal to ligand stochiometry under the relatively low metal loading conditions used here. Quantitative 13C nuclear magnetic resonance spectroscopy showed increases in aromaticity and ketone content and decreases in aliphatic carbon for the elevated metal binding fraction while the carboxyl carbon, and elemental nitrogen, phosphorus, and sulfur content did not change. The more polar, elevated metal binding fraction did show a significant increase in molecular weight over the unfractionated SRFA. Copyright (C) 1999 Elsevier Science B.V.

  4. Binding affinity and capacities for ytterbium(3+) and hafinum(4+) by chemical entities of plant tissue fragments.

    PubMed

    Worley, R; Clearfield, A; Ellis, W C

    2002-12-01

    The binding affinity of ytterbium (Yb3+) and hafinum (Hf4+) to ligands of chemical entities of fragments of bermudagrass tissues and their resistance to exchanging Yb with other ligands and to displacement by protons were investigated. Chemical entities of acid resistant NDF (ARNDF), 0.1 N acid detergent fiber (0.1 N ADF), and permanganate cellulose (CELL) were prepared from fragments of bermudagrass hay (Cynodon dactylon [L.] Pers.) obtained by grinding to pass a 2-mm sieve. 175Ytterbium and Yb, as YbCl3, were initially bound to each preparation by soaking for 12 h in pH 5.5 borate buffer to obtain Yb bound onto ligands having affinity constants for Yb equal to or greater than that for the weakly stable borate ligand, Yb > or = borate. The fraction of Yb > or = borate was measured and fragments then sequentially exposed to acetate, citrate, nitrotriacetate (NTA), and EDTA ions to allow exchange of Yb from Yb > or = borate with ligands having affinity constants for Yb equal to or greater than acetate (Yb > or = acetate), citrate (Yb > or = citrate), NTA (Yb > or = NTA), and EDTA (Yb > or = EDTA) ions. Binding of Yb > or = borate indicated the existence of two species of ligands: strong ligands binding essentially 100% of added Yb at levels of 1 to 1,300 ppm (0.1 N ADF) and at 1 to 7,000 ppm (ARNDF); and weaker ligands binding 4 and 8% of the Yb, respectively, at levels of added Yb greater than 1,300 ppm and 7,000 ppm. Ytterbium > or = acetate of ARNDF, but not 0.1 N ADF, was as resistant to exchange as Yb > or = citrate. Ytterbium > or = borate was exchanged extensively (85% or greater) with soluble ligands having affinity constants > or = NTA. Ytterbium resistance to proton displacement at pH of 1.5 increased with Yb > or = EDTA > Yb > or = NTA > Yb > or = citrate > Yb > or = acetate. Very efficient binding of Yb to CELL suggested that such chemical preparations are not representative of native cellulose. Hafnium (4+) was strongly bound to plant tissues rendering

  5. Corn starch granules with enhanced load-carrying capacity via citric acid treatment.

    PubMed

    Kim, Jong-Yea; Huber, Kerry C

    2013-01-02

    This research investigated conditions by which maize starch granule porosity and load-carrying capacity (LCC) might be enhanced via treatment with varying citric acid concentrations (0.5-1.5 M), temperatures (40-60 °C), and lengths of treatment (1-8 h). At the lowest temperatures (40 and 50 °C), citric acid treatment induced minimal physicochemical changes to granules. In contrast, both aqueous and oil LCCs of starches treated at 60 °C (0.5 M citric acid, 2 h) were almost doubled (15.69 and 14.48 mL/10 g starch, respectively), recovering 92% of the granular starch after treatment. Such treatment increased starch hydration capacity (0.97-1.91) and reduced gelatinization enthalpy (10.6-7.4 J/g). More severe treatment conditions adversely impacted aqueous LCC (due to excessive granule swelling), but improved oil absorption. The basis for LCC enhancement by citric acid treatment was ascribed to leaching of starch material from granules and partial disruption of the granule crystalline structure, as opposed to starch hydrolysis or chemical substitution.

  6. Physiochemical studies on achatininH, a novel sialic acid-binding lectin.

    PubMed Central

    Mandal, C; Basu, S; Mandal, C

    1989-01-01

    We have purified a sialic acid-binding lectin, achatininH, in a single step by affinity chromatography, having high affinity for 9-O-acetylneuraminic acid. The physicochemical characterization of the interaction of achatininH with bivalent metal ions and sialic acid derivatives by the use of spectrofluorimetry, spectropolarimetry and precipitin reaction is reported. From fluorescence quenching studies the binding of Ca2+ (Ka = 251 +/- 9 M-1) and of Mn2+ (Ka = 86 +/- 5 M-1) was found to be weak, but their presence is absolutely necessary for sugar binding as well as biological activity. The nature and position of the substituent group play a very important role in the binding affinity. AchatininH shows a high affinity for 9-O-acetylneuraminic acid (Ka = 1.20 x 10(3) +/- 0.07 x 10(3) M-1) compared with that for the 4-O-acetyl derivative. In oligomers the binding strength increases in the order monosaccharide less than disaccharide less than trisaccharide. The binding affinity of achatininH for the disaccharide was found to reach a peak around pH 8. From c.d. spectral studies achatininH was found to have a high beta-sheet content (46%) and a low alpha-helix content (24%). From precipitin analysis at least one sugar-binding site on each of the 16 monomer subunits of the protein is indicated. PMID:2920028

  7. The LIMP-2/SCARB2 Binding Motif on Acid β-Glucosidase

    PubMed Central

    Liou, Benjamin; Haffey, Wendy D.; Greis, Kenneth D.; Grabowski, Gregory A.

    2014-01-01

    The acid β-glucosidase (glucocerbrosidase (GCase)) binding sequence to LIMP-2 (lysosomal integral membrane protein 2), the receptor for intracellular GCase trafficking to the lysosome, has been identified. Heterologous expression of deletion constructs, the available GCase crystal structures, and binding and co-localization of identified peptides or mutant GCases were used to identify and characterize a highly conserved 11-amino acid sequence, DSPIIVDITKD, within human GCase. The binding to LIMP-2 is not dependent upon a single amino acid, but the interactions of GCase with LIMP-2 are heavily influenced by Asp399 and the di-isoleucines, Ile402 and Ile403. A single alanine substitution at any of these decreases GCase binding to LIMP-2 and alters its pH-dependent binding as well as diminishing the trafficking of GCase to the lysosome and significantly increasing GCase secretion. Enterovirus 71 also binds to LIMP-2 (also known as SCARB2) on the external surface of the plasma membrane. However, the LIMP-2/SCARB2 binding sequences for enterovirus 71 and GCase are not similar, indicating that LIMP-2/SCARB2 may have multiple or overlapping binding sites with differing specificities. These findings have therapeutic implications for the production of GCase and the distribution of this enzyme that is delivered to various organs. PMID:25202012

  8. Fluorescence studies on binding of pyrene and its derivatives to humic acid.

    PubMed

    Nakashima, K; Maki, M; Ishikawa, F; Yoshikawa, T; Gong, Y-K; Miyajima, T

    2007-07-01

    Binding of pyrene (PyH) and its derivatives to humic acid (HA) has been studied by fluorescence spectroscopy. The nature of the interaction between HA and pyrene derivatives are extensively investigated by employing three derivatives ranging from anionic to cationic compounds: 1-pyrenebutylic acid (PyA), 1-pyrenemethanol (PyM), and 1-pyrenebutyltrimethylammonium bromide (PyB). Binding constants between HA and PyX (X=H, A, M, B) are obtained by steady-state fluorescence quenching techniques, and it is found that PyB has a markedly large binding constant among the pyrene family. This is attributed to a strong electrostatic interaction between cationic PyB and anionic HA. The result suggests that an electrostatic interaction plays a dominant role in binding of pyrenes to humic acid. The importance of electrostatic interaction was also confirmed by a salt effect on the binding constant. Influence of collisional quenching on the binding constant, which causes overestimation of the binding constant, was examined by time-resolved fluorescence spectroscopy as well as temperature effect in steady-state fluorescence measurements. It is elucidated that collisional quenching does not much bring overestimation into the binding constants.

  9. The consequences of fruit and vegetable fibre fermentation on their binding capacity for MeIQx and the effects of soluble fibre sources on the binding affinity of wheat bran preparations.

    PubMed

    Ryden, P; Robertson, J A

    1995-08-01

    Fruits and vegetables provide dietary fibre some of which is partly soluble in the upper gut; in the colon it is highly fermentable. Using alcohol-insoluble residues prepared from a range of fruits and vegetables the effects of fermentation on the changes in composition and binding capacity have been assessed for the hydrophobic mutagen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). Fermentation was extensive and resulted in destruction of most of the pectic polysaccharides. Of the unfermented vegetable fibre only cabbage had a measurable hydrophobic binding capacity. The binding capacities of unfermented apple, carrot and sugar beet were negligible. After fermentation, binding capacities, (per mg of fermented residue), increased. Although fermented cabbage was found to have the highest capacity of the fruit and vegetable fibres this remained less than the least effective of unfermented wheat bran samples which had a relatively high affinity for MeIQx. Mucin inhibited the binding of MeIQx to wheat bran fibre but apple fibre did not. The results show that the contribution of fruit and vegetable fibre to a hydrophobic binding matrix in the colon is insignificant and the suggested harmful effect of fruit and vegetable fibre, maintaining hydrophobic mutagens in solution, can be prevented by the presence of wheat bran fibre.

  10. Enterocyte Fatty Acid Binding Proteins (FABPs): Different Functions of Liver- and Intestinal- FABPs in the Intestine

    PubMed Central

    Gajda, Angela M.; Storch, Judith

    2014-01-01

    SUMMARY Fatty acid binding proteins (FABP) are highly abundant cytosolic proteins that are expressed in most mammalian tissues. In the intestinal enterocyte, both Liver- (LFABP; FABP1) and Intestinal-fatty acid binding proteins (IFABP; FABP2) are expressed. These proteins display high affinity binding for long chain fatty acids (FA) and other hydrophobic ligands, thus they are believed to be involved with uptake and trafficking of lipids in the intestine. In vitro studies have identified differences in ligand binding stoichiometry and specificity, and in mechanisms of FA transfer to membranes, and it has been hypothesized that LFABP and IFABP have difference functions in the enterocyte. Studies directly comparing LFABP- and IFABP-null mice have revealed markedly different phenotypes, indicating that these proteins indeed have different functions in intestinal lipid metabolism and whole body energy homeostasis. In this review, we discuss the evolving knowledge of the functions of LFABP and IFABP in the intestinal enterocyte. PMID:25458898

  11. Computing Optimal Binding of Two Nucleic Acid Chains

    NASA Astrophysics Data System (ADS)

    Hodas, Nathan O.; Aalberts, Daniel P.

    2004-03-01

    We present an algorithm to calculate the optimal binding conformation and free energy of two RNA molecules, one or both oligomeric. This algorithm has applications to modeling DNA microarrays, RNA splice-site recognitions, and other anti-sense problems. While other recent algorithms perform the same calculation in time proportional to the sum of the lengths cubed O((N_1+N_2)^3), our uc(bindigo) algorithm scales as the product of the sequence lengths O(N1 ot N_2). To demonstrate its speed and utility, we use uc(bindigo) to screen vast sequence data to investigate the binding proclivities of U1 snRNA to donor splice sites.

  12. In vitro bile acid binding and short-chain fatty acid profile of flax fiber and ethanol co-products.

    PubMed

    Fodje, Adele M L; Chang, Peter R; Leterme, Pascal

    2009-10-01

    Fibers from flaxseed and co-products from ethanol production could be potential sources of dietary fiber in human diet. In vitro fermentation and bile acid binding models were used to investigate the metabolic effects of lignaMax (Bioriginal Food and Science Corp., Saskatoon, SK, Canada) flax meal, spent flax meal, soluble flax gum, wheat insoluble fiber (WIF), and rye insoluble fiber (RIF). Wheat and rye bran were used as reference samples. Bile acid binding of substrates was analysed at taurocholate ([(14)C]taurocholate) concentration of 12.5 mM. Soluble flax gum showed the highest bile acid binding (0.57 micromol/mg of fiber) (P acid binding between wheat bran (0.2 micromol/mg of fiber) and WIF (0.26 micromol/mg of fiber). RIF had higher (P acid binding (0.20 micromol/mg of fiber) than rye bran (0.13 micromol/mg of fiber). Substrates were hydrolyzed and incubated with pig fecal samples. Short-chain fatty acid (SCFA) profile and gas accumulation (G(f)) were compared. Soluble flax gum generated the highest amount of acetic and propionic acids. SCFA profiles of wheat/rye brans and WIF/RIF were similar (except for butyric acid). G(f) for soluble flax gum was greater (P < .001) than that of spent flax meal. G(f) values of the wheat samples were similar, whereas the G(f) of the rye bran was higher (P < .001) than that of RIF. Fractional degradation rate (micro(t = T/2)) (P < .001) was also recorded. The highest mu(t = T/2) was observed for the soluble flax gum. Oil-depleted flaxseed fractions and WIF/RIF (co-products from ethanol production) could be potential sources of dietary fiber in human nutrition.

  13. Lanthanum carbonate reduces urine phosphorus excretion: evidence of high-capacity phosphate binding.

    PubMed

    Pennick, Michael; Poole, Lynne; Dennis, Kerry; Smyth, Michael

    2012-01-01

    The effectiveness of phosphate binders can be assessed by evaluating urinary phosphorus excretion in healthy volunteers, which indicates the ability of the phosphate binder to reduce gastrointestinal phosphate absorption. Healthy volunteers were enrolled into one of five separate randomized trials; four were open label and one double blind. Following a screening period of ≤28 days, participants received differing tablets containing lanthanum carbonate [LC, 3000 mg/day of elemental lanthanum (in one study other doses were also used)]. Participants received a standardized phosphate diet and remained in the relevant study center throughout the duration of each treatment period. The end point in all studies was the reduction in urinary phosphorus excretion. Reductions in mean 24-h urinary phosphorus excretion in volunteers receiving a lanthanum dose of 3000 mg/day were between 236 and 468 mg/day over the five separate studies. These data in healthy volunteers can be used to estimate the amount of reduction of dietary phosphate absorption by LC. The reduction in 24-h urinary phosphorus excretion per tablet was compared with published data on other phosphate binders. Although there are limitations, evidence suggests that LC is a very effective phosphate binder in terms of binding per tablet.

  14. High-capacity hollow porous dummy molecular imprinted polymers using ionic liquid as functional monomer for selective recognition of salicylic acid.

    PubMed

    Xiang, Haiyan; Peng, Mijun; Li, Hui; Peng, Sheng; Shi, Shuyun

    2017-01-30

    The existence of strong intramolecular hydrogen bond in salicylic acid (SA) weakens its intermolecular hydrogen bonding with functional monomer, then it is a challenge work to fabricate molecularly imprinted polymers (MIPs) for SA recognition with high capacity and good selectivity. Here, hollow porous dummy MIPs (HPDMIPs) were prepared using benzoic acid (BA) as dummy template, ionic liquid (i.e. 1-vinyl-3-methylimidazolium chloride) as functional monomer, and MCM-48 as sacrificial support. Factors that affected adsorption, such as type of template and porogen, mole ratio of template-functional monomer-cross-linker and type of binding solvent, were optimized in detail. Multiple strong interactions between SA and ionic liquid in HPDMIPs deduced higher binding capacity (29.75mg/g), imprinting factor (5.61) and selectivity than any previously reported MIPs by traditional or surface imprinting technology. The large surface area (543.9m(2)/g) with hollow porous structure resulted in faster kinetic binding (25min). The equilibrium data fitted well to Freundlich equation and the adsorption process could be described by pseudo-second order model. Finally, HPDMIPs were successfully applied to selectively extract and enrich SA from Actinidia chinensis with a relatively high recovery (84.6-94.5%).

  15. Zirconium oxide aerogel for effective enrichment of phosphopeptides with high binding capacity.

    PubMed

    Zhang, Liyuan; Xu, Jin; Sun, Liangliang; Ma, Junfeng; Yang, Kaiguang; Liang, Zhen; Zhang, Lihua; Zhang, Yukui

    2011-04-01

    In this study, zirconium oxide (ZrO(2)) aerogel was synthesized via a green sol-gel approach, with zirconium oxychloride, instead of the commonly used alkoxide with high toxicity, as the precursor. With such material, phosphopeptides from the digests of 4 pmol of β-casein with the coexistence of 100 times (mol ratio) BSA could be selectively captured, and identified by MALDI-TOF MS. Due to the large surface area (416.0 m(2) g(-1)) and the mesoporous structure (the average pore size of 10.2 nm) of ZrO(2) aerogel, a 20-fold higher loading capacity for phosphopeptide, YKVPQLEIVPN[pS]AEER (MW 1952.12), was obtained compared to that of commercial ZrO(2) microspheres (341.5 vs. 17.87 mg g(-1)). The metal oxide aerogel was further applied in the enrichment of phosphopeptides from 100 ng nonfat milk, and 17 phosphopeptides were positively identified, with a 1.5-fold improvement in phosphopeptide detection compared with previously reported results. These results demonstrate that ZrO(2) aerogel can be a powerful enrichment material for phosphoproteome study.

  16. Evaluation of buffering capacity and acid neutralizing-pH time profile of antacids.

    PubMed

    Lin, M S; Sun, P; Yu, H Y

    1998-10-01

    The antacid properties of seven antacids listed in the hospital formulary of a medical center were evaluated with in vitro tests. These included not only the preliminary antacid test and acid-neutralizing capacity test as described in the United States Pharmacopeia (USP XXIII), but also a buffering pH profile test. The preliminary antacid test measured the final pH of a 10-mL solution of 0.5 N HCl 10 minutes after addition of the minimum recommended dose of an antacid, while the neutralizing capacity test measured the amount (mEq) of HCl neutralized by the minimum recommended dose in 15 minutes. The buffering pH profile recorded the pH time course of dynamic simulated gastric fluid neutralization by a dose of an antacid. In the preliminary antacid test, magnesium oxide showed the highest pH (9.52 +/- 0.14, mean +/- standard deviation, n = 3); aluminum phosphate gel yielded a final pH of 2.51 +/- 0.01, thus failing to meet the criteria of an antacid (pH > 3.5). In the acid-neutralizing capacity test, hydrotalcite had the highest neutralizing capacity (28.26 +/- 0.3 mEq), while sodium bicarbonate had the lowest (7.40 +/- 0.12 mEq). In the buffering pH profile test, aluminum-magnesium hydroxide suspensions and hydrotalcite tablets maintained a steady optimum pH (3-5) for around 1.5 hours. One tablet of calcium carbonate, sodium bicarbonate or magnesium oxide could not raise the gastric pH to above 3, but two tablets increased the pH excessively (5.3 to 8.6). The higher dose (two tablets) of aluminum hydroxide hexitol complex could not raise the pH to the optimal level. These findings demonstrate that there is disparity in the antacid effectiveness estimated by the neutralizing capacity test and the buffering pH profile test and suggest that the efficacy of an antacid cannot be accurately predicted from its acid-neutralizing capacity. The dose of antacids greatly influences the neutralizing pH profiles. Aluminum-magnesium compounds appear to provide steadier buffering

  17. Effect of malondialdehyde treatment on the IgE binding capacity and conformational structure of shrimp tropomyosin.

    PubMed

    Song, Yongna; Li, Zhenxing; Lin, Hong; Du, Shuyuan; Hao, Zina; Lin, Haixin; Zhu, Zhen

    2015-05-15

    The aim of this work was to determine the effect of malondialdehyde (MDA) treatment of shrimp tropomyosin (TM) with respect to IgE binding capacity and conformational structure. Following treatment with MDA, changes in TM molecular weight were characterized by SDS-PAGE and TM cross-linking was observed, then the aggregates were recognized by IgG/IgE in immunoblot analysis. Meanwhile, TM allergenicity slowly decreased following MDA treatment. These data show a correlation between the loss of TM structure and allergenic potential. TM secondary structure became more disordered following treatment with increasing concentrations of MDA. Moreover, the enhancement of surface hydrophobicity was also in accordance with the effect caused by MDA. Additionally, MDA treatment resulted in an increase in carbonyl content and a decrease in free amine groups and available lysine. The results suggest that MDA-induced conformational changes in TM can significantly influence the antigenicity and allergenicity of TM.

  18. [Method of determining serum iron and iron-binding capacity without protein precipitation in very young children].

    PubMed

    Chenusha, V P; Voronka, G Sh

    1978-01-01

    A modification of bathophenanthroline method is described. The method was used for diagnostics of anemia and in order to control the treatment of anemia in children of early ages. The method was checked by estimation of known concentrations of iron in solutions as well as by addition of known amounts of iron to blood serum. Standard deviations ranged from +/- 1.04 to +/- 6.5 and variation coefficient--from 0.5% to 10%. Normal values of iron content and of iron-binding capacity of blood serum, estimated by means of the modified method in 30 healthy children of early ages, did not differ from the corresponding data published in the literature.

  19. Binding affinities of amino acid analogues at the charged aqueous titania interface: implications for titania-binding peptides.

    PubMed

    Sultan, Anas M; Hughes, Zak E; Walsh, Tiffany R

    2014-11-11

    Despite the extensive utilization of biomolecule-titania interfaces, biomolecular recognition and interactions at the aqueous titania interface remain far from being fully understood. Here, atomistic molecular dynamics simulations, in partnership with metadynamics, are used to calculate the free energy of adsorption of different amino acid side chain analogues at the negatively-charged aqueous rutile TiO2 (110) interface, under conditions corresponding with neutral pH. Our calculations predict that charged amino acid analogues have a relatively high affinity to the titania surface, with the arginine analogue predicted to be the strongest binder. Interactions between uncharged amino acid analogues and titania are found to be repulsive or weak at best. All of the residues that bound to the negatively-charged interface show a relatively stronger adsorption compared with the charge-neutral interface, including the negatively-charged analogue. Of the analogues that are found to bind to the titania surface, the rank ordering of the binding affinities is predicted to be "arginine" > "lysine" ≈ aspartic acid > "serine". This is the same ordering as was found previously for the charge-neutral aqueous titania interface. Our results show very good agreement with available experimental data and can provide a baseline for the interpretation of peptide-TiO2 adsorption data.

  20. Computational scheme for the prediction of metal ion binding by a soil fulvic acid

    USGS Publications Warehouse

    Marinsky, J.A.; Reddy, M.M.; Ephraim, J.H.; Mathuthu, A.S.

    1995-01-01

    The dissociation and metal ion binding properties of a soil fulvic acid have been characterized. Information thus gained was used to compensate for salt and site heterogeneity effects in metal ion complexation by the fulvic acid. An earlier computational scheme has been modified by incorporating an additional step which improves the accuracy of metal ion speciation estimates. An algorithm is employed for the prediction of metal ion binding by organic acid constituents of natural waters (once the organic acid is characterized in terms of functional group identity and abundance). The approach discussed here, currently used with a spreadsheet program on a personal computer, is conceptually envisaged to be compatible with computer programs available for ion binding by inorganic ligands in natural waters.

  1. Development of porcine ficolin-alpha monoclonal and polyclonal antibodies for determining the binding capacity of multiple GlcNAc-binding proteins to bacterial danger components.

    PubMed

    Nahid, M Abu; Ross, Steven J; Umiker, Benjamin R; Li, Huapeng; Sugii, Sunji; Bari, Latiful

    2016-02-01

    Ficolins are a group of oligomeric defense proteins assembled from collagen-like stalks and fibrinogen-like domains that have common biochemical specificity for N-acetyl-d-glucose amine (GlcNAc) and can function as opsonins. In this report, GlcNAc-binding protein (GBP) purified from porcine nonimmune serum was biochemically characterized as ficolin-α. Ficolin-α was used as an immunogen to generate both rabbit polyclonal and murine monoclonal anti-ficolin-α antibodies, which are not yet commercially available. GBPs have been shown to be present in many animals, including humans; however, their functions are largely unknown. GBPs from chicken, dog, horse, bovine, and human sera were isolated using various chromatography methods. Interestingly, anti-ficolin-α antibody showed cross-reaction with those animal sera GBPs. Furthermore, anti-ficolin-α antibody was reactive with the GlcNAc eluate of Escherichia coli O26-bound and Salmonella-bound porcine serum proteins. Functionally, GBPs and bacteria-reactive pig serum proteins were able to bind with pathogen-associated molecular patterns such as lipopolysaccharides and lipoteichoic acids. Our studies demonstrate that ficolin-α specific antibody was reactive with GBPs from many species as well as bacteria-reactive serum proteins. These proteins may play important roles in innate immunity by sensing danger components that can lead to antibacterial activity.

  2. Method for nucleic acid hybridization using single-stranded DNA binding protein

    DOEpatents

    Tabor, Stanley; Richardson, Charles C.

    1996-01-01

    Method of nucleic acid hybridization for detecting the presence of a specific nucleic acid sequence in a population of different nucleic acid sequences using a nucleic acid probe. The nucleic acid probe hybridizes with the specific nucleic acid sequence but not with other nucleic acid sequences in the population. The method includes contacting a sample (potentially including the nucleic acid sequence) with the nucleic acid probe under hybridizing conditions in the presence of a single-stranded DNA binding protein provided in an amount which stimulates renaturation of a dilute solution (i.e., one in which the t.sub.1/2 of renaturation is longer than 3 weeks) of single-stranded DNA greater than 500 fold (i.e., to a t.sub.1/2 less than 60 min, preferably less than 5 min, and most preferably about 1 min.) in the absence of nucleotide triphosphates.

  3. Fluctuating capacity and advance decision-making in Bipolar Affective Disorder — Self-binding directives and self-determination

    PubMed Central

    Gergel, Tania; Owen, Gareth S.

    2015-01-01

    For people with Bipolar Affective Disorder, a self-binding (advance) directive (SBD), by which they commit themselves to treatment during future episodes of mania, even if unwilling, can seem the most rational way to deal with an imperfect predicament. Knowing that mania will almost certainly cause enormous damage to themselves, their preferred solution may well be to allow trusted others to enforce treatment and constraint, traumatic though this may be. No adequate provision exists for drafting a truly effective SBD and efforts to establish such provision are hampered by very valid, but also paralysing ethical, clinical and legal concerns. Effectively, the autonomy and rights of people with bipolar are being ‘protected’ through being denied an opportunity to protect themselves. From a standpoint firmly rooted in the clinical context and experience of mania, this article argues that an SBD, based on a patient-centred evaluation of capacity to make treatment decisions (DMC-T) and grounded within the clinician–patient relationship, could represent a legitimate and ethically coherent form of self-determination. After setting out background information on fluctuating capacity, mania and advance directives, this article proposes a framework for constructing such an SBD, and considers common objections, possible solutions and suggestions for future research. PMID:25939286

  4. 180 Ah kg-1 specific capacity positive tubular electrodes for lead acid batteries

    NASA Astrophysics Data System (ADS)

    de Andrade, J.; Impinnisi, P. R.; do Vale, D. L.

    2011-05-01

    Two disadvantages of lead acid batteries are poor power and energy densities and the necessity of relatively long recharging times. In this paper it is presented the results of ongoing work aimed at increasing both the positive active material (PAM) specific capacity and the positive plate charge acceptability. The experimental results show that adequate curing processes can be used to develop an interconnected structure among nanometric PbO2 particles to produce tubular electrodes with specific capacity higher than 180 Ah kg-1 and maintain this value for 130 cycles with deep discharges. These PbO2 positive plates are expected to exhibit higher charge acceptability due to their larger PAM surface area as compared to conventional ones, but the results indicate that the high internal ohmic resistance of the grid/PAM zone limits the fast charge efficiency.

  5. Vitamins, fatty acids, and antioxidant capacity stability during storage of freeze-dried human milk.

    PubMed

    Lozano, Blanca; Castellote, Ana Isabel; Montes, Rosa; López-Sabater, M Carmen

    2014-09-01

    Although freezing is the most common method used to preserve human milk, nutritional and immunological components may be lost during storage. Freeze-drying could increase the shelf life of human milk, while preserving its original characteristics. Seventy-two samples of freeze-dried human milk were stored for different periods of time, up to a maximum of 3 months, at 4 °C or 40 °C. Vitamin C, tocopherols, antioxidant capacity, and fatty acids composition were analyzed. A new HILIC-UHPLC method improving vitamin C determination was also validated. Ascorbic acid and total vitamin C concentrations significantly decreased at both temperatures, while antioxidant capacity only decreased at 40 °C. Fatty acids composition and both γ-tocopherol and δ-tocopherol contents remained unaltered. The stability after storage of freeze-dried milk was higher than that reported for frozen or fresh milk indicating that freeze-drying is a promising option to improve the preservation of human milk in banks.

  6. Serum fatty acid binding protein 4, free fatty acids and metabolic risk markers

    PubMed Central

    Karakas, Sidika E.; Almario, Rogelio U.; Kim, Kyoungmi

    2009-01-01

    Fatty acid binding protein (FABP) 4 chaperones free fatty acids (FFA) in the adipocytes during lipolysis. Serum FFA relates to Metabolic Syndrome (METS) and serum FABP4 is emerging as a novel risk marker. In 36 overweight/obese women, serum FABP4 and FFA were measured hourly during 5-hour oral glucose tolerance test (OGTT). Insulin resistance was determined using frequently sampled intravenous GTT (FS-IVGTT). Serum lipids and inflammation markers were measured at fasting. During OGTT, serum FABP4 decreased by 40%, reaching its nadir at 3h (from 45.3±3.1 to 31.9±1.6 ng/mL) and stayed below the baseline at 5 h (35.9±2.2 ng/mL) (p < 0.0001 for both, compared to the baseline). Serum FFA decreased by 10 fold, reaching a nadir at 2h (from 0.611±0.033 to 0.067±0.004 mmol/L), then rebounded to 0.816±0.035 mmol/ L at 5h (p < 0.001 for both, compared to baseline). Both fasting-FABP4 and nadir-FABP4 correlated with obesity. Nadir-FABP4 correlated also with insulin resistance parameters from FS-IVGTT and with inflammation. Nadir-FFA, but not fasting-FFA, correlated with the METS-parameters. In conclusion, fasting-FABP4 related to metabolic risk markers more strongly than fasting-FFA. Nadir-FABP4 and nadir-FFA measured after glucose loading may provide better risk assessment than the fasting values. PMID:19394980

  7. Binding of Polycarboxylic Acids to Cationic Mixed Micelles: Effects of Polymer Counterion Binding and Polyion Charge Distribution.

    PubMed

    Yoshida; Sokhakian; Dubin

    1998-09-15

    Mixed micelles of cetyltrimethylammonium chloride (CTAC) and n-dodecyl hexaoxyethylene glycol monoether (C12E8) bind to polyanions when the mole fraction of the cationic surfactant exceeds a critical value (Yc). Yc corresponds to a critical micelle surface charge density at which polyelectrolyte will bind to this colloidal particle. Turbidimetric titrations were used to determine Yc for such cationic-nonionic micelles in the presence of acrylic acid and acrylamido-2-methylpropane sulfonate homopolymers (PAA and PAMPS, respectively) and their copolymers with acrylamide, as function of pH, ionic strength, and polyelectrolyte counterion. In 0.20 M NaCl, Yc for PAA is found to be remarkably insensitive to pH, i.e., virtually independent of the apparent polymer charge density xiapp. On the other hand, the expected inverse relationship between Yc and xiapp is observed either for PAA when NaCl is replaced by TMACl (tetramethylammonium chloride), or when xiapp is manipulated using acrylic acid/acrylamide copolymers at high pH. The effective charge density of PAA is thus seen to be suppressed by specific sodium ion binding, indicating that the influence of salts on the interaction of polycarboxylic acids with colloidal particles may differ qualitatively from their effect on the analogous behavior of strong polyanions. Comparisons between homo- and copolymers of acrylic acid were carried out also to test the hypothesis that the "mobility" of charges on PAA at moderate pH (degree of ionization less than unity) could make this "annealed" polymer exhibit the behavior of a more highly charged one. The results, while consistent with this expectation, were obscured by the likely effect of copolymer sequence distributions. Copyright 1998 Academic Press.

  8. Influence of organic matter decomposition on soluble carbon and its copper-binding capacity.

    PubMed

    Merritt, Karen A; Erich, M Susan

    2003-01-01

    Bulk and low molecular weight (LMW) (<1 kDa) water-extractable carbon were collected from fresh and microbially degraded wheat straw (Triticum aestivum L.) and crimson clover (Trifolium incarnatum L.) residues to monitor early-stage humification over an 8-wk incubation. Copper complexation parameters were determined for both bulk and LMW water-extractable C for both plant materials in a separate 1-wk incubation. Humification progressed through increasing molar absorptivity (A285) and phenolic and total acidity (TA), and through an increase in average molecular size and degree of polymerization as determined by ultrafiltration and changes in fluorescence peak locations. Such dynamic transformations demonstrate that while humification is a bulk property, with C breakdown and stabilization occurring simultaneously and continuously in soil, its early stages can be effectively monitored for fresh plant residues. Significant changes consistently occurred during the first 7 d of the incubation and were more pronounced for LMW fractions than bulk extracts. For both residues, water-extractable C extracted initially and following a 7-d incubation desorbed and complexed 0.11 to 0.55 mmol resin-bound Cu g(-1) C. Low molecular weight water-extractable C generated the higher values within this range, and values increased consistently following incubation. Potential concerns regarding LMW soluble Cu complexes include percolation through soils or runoff into adjacent water bodies as well as effects on plant root development.

  9. Differential binding of thyroxine and triiodothyronine to acidic isoforms of thyroid hormone binding globulin in human serum

    SciTech Connect

    Terasaki, T.; Pardridge, W.M.

    1988-05-17

    The differential availability of thyroxine (T/sub 4/) and 3,5,3'-triiodothyronine (T/sub 3/) to liver from the circulating thyroid hormone binding globulin (TBG)-bound pool suggests that the two thyroid hormones may bind to different TBG isoforms in human serum. In the present study, the binding of (/sup 125/I)T/sub 4/ and (/sup 125/I)T/sub 3/ to human serum proteins was investigated by using slab gel isoelectric focusing and chromatofocusing. In normal human male serum, (/sup 125/I)T/sub 4/ was localized to four isoforms of TBG called TBG-I, -II, -III, and -IV, with isoelectric points (pI's) of 4.30, 4.35, 4.45, and 4.55, respectively. (/sup 125/I)T/sub 3/ was localized to only two isoforms of TBG, TBG-III, and -IV, with pI's that were identical with those for (/sup 125/I)T/sub 4/. In normal female serum, (/sup 125/I)T/sub 4/ was localized to the same four isoforms of TBG as those of normal male serum, while (/sup 125/I)T/sub 3/ was localized to TBG-II, -III, -IV, and -V (pI = 4.65). In pregnant female serum, (/sup 125/I)T/sub 4/ was localized to five isoforms, whereas (/sup 125/I)T/sub 3/ was localized to four. IEF was also performed with male serum loaded with various concentrations of unlabeled T/sub 3/. The K/sub i/ values of T/sub 3/ binding to TBG-I, -II, -III, and -IV were 5.0, 2.4, 0.86, and 0.46 nM, respectively. The TBG isoforms in normal male serum were also separated by sequential concanavalin A-Sepharose affinity chromatography and the chromatofocusing (pH range of 3.5-5.0). T/sub 4/ preferentially bound to the most acidic isoforms of TBG in the pI range of 3.8-4.0, whereas the less acidic fractions (pH 4.0-4.2) bound both T/sub 4/ and T/sub 3/. In conclusion, this study shows that T/sub 4/ and T/sub 3/ do not bind to a single competitive binding site on TBG. Instead, T/sub 4/ is preferentially bound by the most acidic TBG isoforms owing to a 10-fold lower affinity of T/sub 3/ for these proteins.

  10. Zinc-induced oligomerization of zinc α2 glycoprotein reveals multiple fatty acid-binding sites.

    PubMed

    Zahid, Henna; Miah, Layeque; Lau, Andy M; Brochard, Lea; Hati, Debolina; Bui, Tam T T; Drake, Alex F; Gor, Jayesh; Perkins, Stephen J; McDermott, Lindsay C

    2016-01-01

    Zinc α2 glycoprotein (ZAG) is an adipokine with a class I MHC protein fold and is associated with obesity and diabetes. Although its intrinsic ligand remains unknown, ZAG binds the dansylated C11 fatty acid 11-(dansylamino)undecanoic acid (DAUDA) in the groove between the α1 and α2 domains. The surface of ZAG has approximately 15 weak zinc-binding sites deemed responsible for precipitation from human plasma. In the present study the functional significance of these metal sites was investigated. Analytical ultracentrifugation (AUC) and CD showed that zinc, but not other divalent metals, causes ZAG to oligomerize in solution. Thus ZAG dimers and trimers were observed in the presence of 1 and 2 mM zinc. Molecular modelling of X-ray scattering curves and sedimentation coefficients indicated a progressive stacking of ZAG monomers, suggesting that the ZAG groove may be occluded in these. Using fluorescence-detected sedimentation velocity, these ZAG-zinc oligomers were again observed in the presence of the fluorescent boron dipyrromethene fatty acid C16-BODIPY (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-hexadecanoic acid). Fluorescence spectroscopy confirmed that ZAG binds C16-BODIPY. ZAG binding to C16-BODIPY, but not to DAUDA, was reduced by increased zinc concentrations. We conclude that the lipid-binding groove in ZAG contains at least two distinct fatty acid-binding sites for DAUDA and C16-BODIPY, similar to the multiple lipid binding seen in the structurally related immune protein CD1c. In addition, because high concentrations of zinc occur in the pancreas, the perturbation of these multiple lipid-binding sites by zinc may be significant in Type 2 diabetes where dysregulation of ZAG and zinc homoeostasis occurs.

  11. Cu(II) binding by a pH-fractionated fulvic acid

    USGS Publications Warehouse

    Brown, G.K.; Cabaniss, S.E.; MacCarthy, P.; Leenheer, J.A.

    1999-01-01

    The relationship between acidity, Cu(II) binding and sorption to XAD resin was examined using Suwannee River fulvic acid (SRFA). The work was based on the hypothesis that fractions of SRFA eluted from an XAD column at various pH's from 1.0 to 12.0 would show systematic variations in acidity and possibly aromaticity which in turn would lead to different Cu(II) binding properties. We measured equilibrium Cu(II) binding to these fractions using Cu2+ ion-selective electrode (ISE) potentiometry at pH 6.0. Several model ligands were also examined, including cyclopentane-1,2,3,4-tetracarboxylic acid (CP-TCA) and tetrahydrofuran-2,3,4,5-tetracarboxylic acid (THF-TCA), the latter binding Cu(II) much more strongly as a consequence of the ether linkage. The SRFA Cu(II) binding properties agreed with previous work at high ionic strength, and binding was enhanced substantially at lower ionic strength, in agreement with Poisson-Boltzmann predictions for small spheres. Determining Cu binding constants (K(i)) by non-linear regression with total ligand concentrations (L(Ti)) taken from previous work, the fractions eluted at varying pH had K(i) similar to the unfractionated SRFA, with a maximum enhancement of 0.50 log units. We conclude that variable-pH elution from XAD does not isolate significantly strong (or weak) Cu(II)-binding components from the SRFA mixture. Copyright (C) 1999 Elsevier Science B.V.

  12. The presence of high-affinity, low-capacity estradiol-17β binding in rainbow trout scale indicates a possible endocrine route for the regulation of scale resorption

    USGS Publications Warehouse

    Persson, Petra; Shrimpton, J.M.; McCormick, S.D.; Bjornsson, Bjorn Thrandur

    2000-01-01

    High-affinity, low-capacity estradiol-17β (E2) binding is present in rainbow trout scale. The Kd and Bmax of the scale E2 binding are similar to those of the liver E2 receptor (Kd is 1.6 ± 0.1 and 1.4 ± 0.1 nM, and Bmax is 9.1 ± 1.2 and 23.1 ± 2.2 fmol x mg protein-1, for scale and liver, respectively), but different from those of the high-affinity, low-capacity E2 binding in plasma (Kd is 4.0 ± 0.4 nM and Bmax is 625.4 ± 63.1 fmol x mg protein-1). The E2 binding in scale was displaced by testosterone, but not by diethylstilbestrol. Hence, the ligand binding specificity is different from that of the previously characterized liver E2 receptor, where E2 is displaced by diethylstilbestrol, but not by testosterone. The putative scale E2 receptor thus appears to bind both E2 and testosterone, and it is proposed that the increased scale resorption observed during sexual maturation in both sexes of several salmonid species may be mediated by this receptor. No high-affinity, low-capacity E2 binding could be detected in rainbow trout gill or skin.

  13. Comparative inhibition by hard and soft metal ions of steroid-binding capacity of renal mineralocorticoid receptor cross-linked to the 90-kDa heat-shock protein heterocomplex.

    PubMed Central

    Galigniana, M D; Piwien-Pilipuk, G

    1999-01-01

    We analysed the inhibitory effects in vitro and in vivo of several metal ions on aldosterone binding to the rat kidney mineralocorticoid receptor with the purpose of assessing possible toxic effects of those ions on sodium retention, as well as to obtain information on receptor structural requirements for ligand binding. For the assays in vitro, the inhibitory effects of 20 metal ions were analysed on steroid-binding capacity for renal receptor cross-linked to 90-kDa heat-shock protein (hsp90) by pretreatment with dimethyl pimelimidate. Cross-linking prevented the artifactual dissociation of hsp90 (and, consequently, the loss of steroid binding) from the mineralocorticoid receptor due to the presence of high concentrations of salt in the incubation medium. Cross-linked heterocomplex showed no difference in ligand specificity and affinity with respect to native receptor, but increased stability upon thermal- or ionic-strength-induced destabilization was observed. Treatments in vitro with metal ions in the range 10(-8)-10(-1) M resulted in a differential inhibitory effect for each particular ion on aldosterone binding. Using the negative logarithm of metal concentration for 50% inhibition, the ions could be correlated with their Klopman hardness constants. The analysis of this relationship led us to postulate three types of reaction: with thiol, imidazole and carboxyl groups. The essential role played by these residues in steroid binding was confirmed by chemical modification of cysteines with dithionitrobenzoic acid, histidines with diethyl pyrocarbonate and acidic amino acids with Woodward's reagent (N-ethyl-5-phenylisoxazolium-3'-sulphonate). Importantly, the toxic effects of some metal ions were also observed by treatments in vivo of adrenalectomized rats on both steroid-binding capacity and aldosterone-dependent sodium-retaining properties. We suggest that those amino acid residues are involved in the activation process of the mineralocorticoid receptor upon

  14. Binding and cleavage of nucleic acids by the "hairpin" ribozyme.

    PubMed

    Chowrira, B M; Burke, J M

    1991-09-03

    The "hairpin" ribozyme derived from the minus strand of tobacco ringspot virus satellite RNA [(-)sTRSV] efficiently catalyzes sequence-specific RNA hydrolysis in trans (Feldstein et al., 1989; Hampel & Triz, 1989; Haseloff & Gerlach, 1989). The ribozyme does not cleave DNA. An RNA substrate analogue containing a single deoxyribonucleotide residue 5' to the cleavage site (A-1) binds to the ribozyme efficiently but cannot be cleaved. A DNA substrate analogue with a ribonucleotide at A-1 is cleaved; thus A-1 provides the only 2'-OH required for cleavage. These results support cleavage via a transphosphorylation mechanism initiated by attack of the 2'-OH of A-1 on the scissile phosphodiester. The ribozyme discriminates between DNA and RNA in both binding and cleavage. Results indicate that the 2'-OH of A-1 functions in complex stabilization as well as cleavage. The ribozyme efficiently cleaves a phosphorothioate diester linkage, suggesting that the pro-Rp oxygen at the scissile phosphodiester does not coordinate Mg2+.

  15. Affinity regression predicts the recognition code of nucleic acid binding proteins

    PubMed Central

    Pelossof, Raphael; Singh, Irtisha; Yang, Julie L.; Weirauch, Matthew T.; Hughes, Timothy R.; Leslie, Christina S.

    2016-01-01

    Predicting the affinity profiles of nucleic acid-binding proteins directly from the protein sequence is a major unsolved problem. We present a statistical approach for learning the recognition code of a family of transcription factors (TFs) or RNA-binding proteins (RBPs) from high-throughput binding assays. Our method, called affinity regression, trains on protein binding microarray (PBM) or RNA compete experiments to learn an interaction model between proteins and nucleic acids, using only protein domain and probe sequences as inputs. By training on mouse homeodomain PBM profiles, our model correctly identifies residues that confer DNA-binding specificity and accurately predicts binding motifs for an independent set of divergent homeodomains. Similarly, learning from RNA compete profiles for diverse RBPs, our model can predict the binding affinities of held-out proteins and identify key RNA-binding residues. More broadly, we envision applying our method to model and predict biological interactions in any setting where there is a high-throughput ‘affinity’ readout. PMID:26571099

  16. Magnetic properties, acid neutralization capacity, and net acid production of rocks in the Animas River Watershed Silverton, Colorado

    USGS Publications Warehouse

    McCafferty, Anne E.; Yager, Douglas B.; Horton, Radley M.; Diehl, Sharon F.

    2006-01-01

    Federal land managers along with local stakeholders in the Upper Animas River watershed near Silverton, Colorado are actively designing and implementing mine waste remediation projects to mitigate the effects of acid mine drainage from several abandoned hard rock metal mines and mills. Local source rocks with high acid neutralization capacity (ANC) within the watershed are of interest to land managers for use in these remediation projects. A suite of representative samples was collected from propylitic to weakly sericitic-altered volcanic and plutonic rocks exposed in outcrops throughout the watershed. Acid-base accounting laboratory methods coupled with mineralogic and geochemical characterization provide insight into lithologies that have a range of ANC and net acid production (NAP). Petrophysical lab determinations of magnetic susceptibility converted to estimates for percent magnetite show correlation with the environmental properties of ANC and NAP for many of the lithologies. A goal of our study is to interpret watershed-scale airborne magnetic data for regional mapping of rocks that have varying degrees of ANC and NAP. Results of our preliminary work are presented here.

  17. Molecular Switch Controlling the Binding of Anionic Bile Acid Conjugates to Human Apical Sodium-dependent Bile Acid Transporter

    PubMed Central

    Rais, Rana; Acharya, Chayan; Tririya, Gasirat; MacKerell, Alexander D.; Polli, James E.

    2010-01-01

    The human apical sodium-dependent bile acid transporter (hASBT) may serve as a prodrug target for oral drug absorption. Synthetic, biological, NMR and computational approaches identified the structure-activity relationships of mono- and dianionic bile acid conjugates for hASBT binding. Experimental data combined with a conformationally-sampled pharmacophore/QSAR modeling approach (CSP-SAR) predicted that dianionic substituents with intramolecular hydrogen bonding between hydroxyls on the cholane skeleton and the acid group on the conjugate's aromatic ring increased conjugate hydrophobicity and improved binding affinity. Notably, the model predicted the presence of a conformational molecular switch, where shifting the carboxylate substituent on an aromatic ring by a single position controlled binding affinity. Model validation was performed by effectively shifting the spatial location of the carboxylate by inserting a methylene adjacent to the aromatic ring, resulting in the predicted alteration in binding affinity. This work illustrates conformation as a determinant of ligand binding affinity to a biological transporter. PMID:20504026

  18. Chapter A6. Section 6.6. Alkalinity and Acid Neutralizing Capacity

    USGS Publications Warehouse

    Rounds, Stewart A.; Wilde, Franceska D.

    2002-01-01

    Alkalinity (determined on a filtered sample) and Acid Neutralizing Capacity (ANC) (determined on a whole-water sample) are measures of the ability of a water sample to neutralize strong acid. Alkalinity and ANC provide information on the suitability of water for uses such as irrigation, determining the efficiency of wastewater processes, determining the presence of contamination by anthropogenic wastes, and maintaining ecosystem health. In addition, alkalinity is used to gain insights on the chemical evolution of an aqueous system. This section of the National Field Manual (NFM) describes the USGS field protocols for alkalinity/ANC determination using either the inflection-point or Gran function plot methods, including calculation of carbonate species, and provides guidance on equipment selection.

  19. Location and binding mechanism of an ESIPT probe 3-hydroxy-2-naphthoic acid in unsaturated fatty acid bound serum albumins.

    PubMed

    Ghorai, Shyamal Kr; Tripathy, Debi Ranjan; Dasgupta, Swagata; Ghosh, Sanjib

    2014-02-05

    The binding site and the binding mechanism of 3-hydroxy-2-naphthoic acid (3HNA) in oleic acid (OA) bound serum albumins (bovine serum albumin (BSA) and human serum albumin (HSA)) have been determined using steady state and time resolved emission of tryptophan residues (Trp) in proteins and the ESIPT emission of 3HNA. Time resolved anisotropy of the probe 3HNA and low temperature phosphorescence of Trp residues of BSA in OA bound BSA at 77K reveals a drastic change of the binding site of 3HNA in the ternary system compared to that in the free protein. 3HNA binds near Trp213 in the ternary system whereas 3HNA binds near Trp134 in the free protein. The structure of OA bound BSA generated using docking methodology exhibits U-bend configuration of all bound OA. The docked pose of 3HNA in the free protein and in OA bound albumins (ternary systems) and the concomitant perturbation of the structure of proteins around the binding region of 3HNA corroborate the enhanced ESIPT emission of 3HNA and the energy transfer efficiency from the donor Trp213 of BSA to 3HNA acceptor in 3HNA-OA-BSA system.

  20. Reduction and Reoxidation of Humic Acid: Influence on Spectroscopic Properties and Proton Binding

    SciTech Connect

    Maurer, F.; Christl, I; Kretzschmar, R

    2010-01-01

    Previous studies on proton and metal binding to humic substances have not considered a potential influence of reduction and oxidation of functional groups. Therefore, we investigated how proton binding of a purified soil humic acid was affected by reduction. Reduction of the humic acid was carried out using an electrochemical cell that allowed us to measure the amounts of electrons and protons involved in reduction reactions. We further applied spectroscopic methods (UV-vis, fluorescence, FT-IR, C-1s NEXAFS) to detect possible chemical changes in the humic acid induced by reduction and reoxidation. The effect of reduction on proton binding was determined with acid-base titrations in the pH range 4-10 under controlled redox conditions. During reduction, 0.54 mol kg{sup -1} protons and 0.55 mol kg{sup -1} electrons were transferred to humic acid. NICA-Donnan modeling revealed an equivalent increase in proton-reactive sites (0.52 mol kg{sup -1}) in the alkaline pH-range. Our results indicate that reduction of humic acid increased the amount of proton-reactive sites by 15% compared to the untreated state. Spectroscopic differences between the untreated and reduced humic acid were minor, apart from a lower UV-vis absorption of the reduced humic acid between 400 and 700 nm.

  1. Saturated fatty-acids regulate retinoic acid signaling and suppress tumorigenesis by targeting fatty-acid-binding protein 5

    PubMed Central

    Levi, Liraz; Wang, Zeneng; Doud, Mary Kathryn; Hazen, Stanley L.; Noy, Noa

    2015-01-01

    Long chain fatty acids (LCFA) serve as energy sources, components of cell membranes, and precursors for signalling molecules. Here we show that these biological compounds also regulate gene expression and that they do so by controlling the transcriptional activities of the retinoic acid (RA)-activated nuclear receptors RAR and PPARβ/δ. The data indicate that these activities of LCFA are mediated by FABP5 which delivers ligands from the cytosol to nuclear PPARβ/δ. Both saturated and unsaturated LCFA (SLCFA, ULCFA) bind to FABP5, thereby displacing RA and diverting it to RAR. However, while SLCFA inhibit, ULCFA activate the FABP5/PPARβ/δ pathway. We show further that, by concomitantly promoting activation of RAR and inhibiting the activation of PPARβ/δ, SLCFA suppress the oncogenic properties of FABP5-expressing carcinoma cells in cultured cells and in vivo. The observations suggest that compounds that inhibit FABP5 may constitute a new class of drugs for therapy of certain types of cancer. PMID:26592976

  2. Saturated fatty acids regulate retinoic acid signalling and suppress tumorigenesis by targeting fatty acid-binding protein 5.

    PubMed

    Levi, Liraz; Wang, Zeneng; Doud, Mary Kathryn; Hazen, Stanley L; Noy, Noa

    2015-11-23

    Long chain fatty acids (LCFA) serve as energy sources, components of cell membranes and precursors for signalling molecules. Here we show that these biological compounds also regulate gene expression and that they do so by controlling the transcriptional activities of the retinoic acid (RA)-activated nuclear receptors RAR and PPARβ/δ. The data indicate that these activities of LCFA are mediated by FABP5, which delivers ligands from the cytosol to nuclear PPARβ/δ. Both saturated and unsaturated LCFA (SLCFA, ULCFA) bind to FABP5, thereby displacing RA and diverting it to RAR. However, while SLCFA inhibit, ULCFA activate the FABP5/PPARβ/δ pathway. We show further that, by concomitantly promoting the activation of RAR and inhibiting the activation of PPARβ/δ, SLCFA suppress the oncogenic properties of FABP5-expressing carcinoma cells in cultured cells and in vivo. The observations suggest that compounds that inhibit FABP5 may constitute a new class of drugs for therapy of certain types of cancer.

  3. Cytotoxic Activity of Salicylic Acid-Containing Drug Models with Ionic and Covalent Binding.

    PubMed

    Egorova, Ksenia S; Seitkalieva, Marina M; Posvyatenko, Alexandra V; Khrustalev, Victor N; Ananikov, Valentine P

    2015-11-12

    Three different types of drug delivery platforms based on imidazolium ionic liquids (ILs) were synthesized in high preparative yields, namely, the models involving (i) ionic binding of drug and IL; (ii) covalent binding of drug and IL; and (iii) dual binding using both ionic and covalent approaches. Seven ionic liquids containing salicylic acid (SA-ILs) in the cation or/and in the anion were prepared, and their cytotoxicity toward the human cell lines CaCo-2 (colorectal adenocarcinoma) and 3215 LS (normal fibroblasts) was evaluated. Cytotoxicity of SA-ILs was significantly higher than that of conventional imidazolium-based ILs and was comparable to the pure salicylic acid. It is important to note that the obtained SA-ILs dissolved in water more readily than salicylic acid, suggesting benefits of possible usage of traditional nonsoluble active pharmaceutical ingredients in an ionic liquid form.

  4. Docking simulations suggest that all- trans retinoic acid could bind to retinoid X receptors

    NASA Astrophysics Data System (ADS)

    Tsuji, Motonori; Shudo, Koichi; Kagechika, Hiroyuki

    2015-10-01

    Retinoid X receptors (RXRs) are ligand-controlled transcription factors which heterodimerize with other nuclear receptors to regulate gene transcriptions associated with crucial biological events. 9- cis retinoic acid (9cRA), which transactivates RXRs, is believed to be an endogenous RXR ligand. All- trans retinoic acid (ATRA) is a natural ligand for retinoic acid receptors (RARs), which heterodimerize with RXRs. Although the concentration of 9cRA in tissues is very low, ATRA is relatively abundant and some reports show that ATRA activates RXRs. We computationally studied the possibility of ATRA binding to RXRs using two different docking methods with our developed programs to assess the binding affinities of naturally occurring retinoids. The simulations showed good correlations to the reported binding affinities of these molecules for RXRs and RARs.

  5. High capacity for extracellular acid-base regulation in the air-breathing fish Pangasianodon hypophthalmus.

    PubMed

    Damsgaard, Christian; Gam, Le Thi Hong; Tuong, Dang Diem; Thinh, Phan Vinh; Huong Thanh, Do Thi; Wang, Tobias; Bayley, Mark

    2015-05-01

    The evolution of accessory air-breathing structures is typically associated with reduction of the gills, although branchial ion transport remains pivotal for acid-base and ion regulation. Therefore, air-breathing fishes are believed to have a low capacity for extracellular pH regulation during a respiratory acidosis. In the present study, we investigated acid-base regulation during hypercapnia in the air-breathing fish Pangasianodon hypophthalmus in normoxic and hypoxic water at 28-30°C. Contrary to previous studies, we show that this air-breathing fish has a pronounced ability to regulate extracellular pH (pHe) during hypercapnia, with complete metabolic compensation of pHe within 72 h of exposure to hypoxic hypercapnia with CO2 levels above 34 mmHg. The high capacity for pHe regulation relies on a pronounced ability to increase levels of HCO3(-) in the plasma. Our study illustrates the diversity in the physiology of air-breathing fishes, such that generalizations across phylogenies may be difficult.

  6. Binding of retinoic acid receptor heterodimers to DNA. A role for histones NH2 termini.

    PubMed

    Lefebvre, P; Mouchon, A; Lefebvre, B; Formstecher, P

    1998-05-15

    The retinoic acid signaling pathway is controlled essentially through two types of nuclear receptors, RARs and RXRs. Ligand dependent activation or repression of retinoid-regulated genes is dependent on the binding of retinoic acid receptor (RAR)/9-cis-retinoic acid receptor (RXR) heterodimers to retinoic acid response element (RARE). Although unliganded RXR/RAR heterodimers bind constitutively to DNA in vitro, a clear in vivo ligand-dependent occupancy of the RARE present in the RARbeta2 gene promoter has been reported (Dey, A., Minucci, S., and Ozato, K. (1994) Mol. Cell. Biol. 14, 8191-8201). Nucleosomes are viewed as general repressors of the transcriptional machinery, in part by preventing the access of transcription factors to DNA. The ability of hRXRalpha/hRARalpha heterodimers to bind to a nucleosomal template in vitro has therefore been examined. The assembly of a fragment from the RARbeta2 gene promoter, which contains a canonical DR5 RARE, into a nucleosome core prevented hRXRalpha/hRARalpha binding to this DNA, in conditions where a strong interaction is observed with a linear DNA template. However, histone tails removal by limited proteolysis and histone hyperacetylation yielded nucleosomal RAREs able to bind to hRXRalpha/hRARalpha heterodimers. These data establish therefore the role of histones NH2 termini as a major impediment to retinoid receptors access to DNA, and identify histone hyperacetylation as a potential physiological regulator of retinoid-induced transcription.

  7. Effect of limited enzymatic hydrolysis on linoleic acid binding properties of β-lactoglobulin.

    PubMed

    Sponton, Osvaldo E; Perez, Adrián A; Carrara, Carlos; Santiago, Liliana G

    2014-03-01

    β-Lactoglobulin (BLG) is a member of lipocalin family, proteins with ability to bind small hydrophobic ligands, such as retinol, vitamins and fatty acids. Moreover, BLG is susceptible to protease action producing a wide range of polypeptides depending on the hydrolysis degree (HD). In the present work, the effect of limited enzymatic hydrolysis on fatty acid binding properties of BLG was studied. Linoleic acid (LA) was used as a model fatty acid. Limited enzymatic hydrolysis was performed using α-chymotrypsin immobilised on agarose microparticles. BLG hydrolysates were produced at HD: 1%, 3% and 5%. In order to determine the influence of HD on BLG molecular weight SDS-PAGE was used. BLG structural modification and LA binding properties were monitored by means of fluorescence spectroscopic techniques. The increase in HD produced: (i) a BLG degradation and a molecular weight distribution of BLG hydrolysates and (ii) an increased exposition of buried hydrophobic residues, however it was observed a decrease in surface hydrophobicity possibly due to a deterioration of hydrophobic protein domains. It was observed that enzymatic hydrolysis treatment produced a decrease in BLG ability for binding LA. It was concluded that limited enzymatic hydrolysis could deteriorate the specific site on BLG structure necessary for binding LA.

  8. Effects of exogenous abscisic acid on yield, antioxidant capacities, and phytochemical contents of greenhouse grown lettuces.

    PubMed

    Li, Zheng; Zhao, Xin; Sandhu, Amandeep K; Gu, Liwei

    2010-05-26

    Antioxidants and phytochemicals in vegetables are known to provide health benefits. Strategies that enhance these properties are expected to increase the nutritional values of vegetables. The objective of this research is to assess the effects of exogenous abscisic acid (ABA) on yield, antioxidant capacities, and phytochemical content of lettuces grown in a greenhouse. Red loose leaf lettuce (cv. Galactic) and green loose leaf lettuce (cv. Simpson Elite) were cultivated using a randomized complete block design. Three concentrations of ABA in water [0 (control), 150, 300 ppm] were sprayed on the 30th and 39th days after sowing, and lettuces were harvested on the 46th day. Exogenous ABA significantly decreased yield of green and red lettuces. Total phenolic and total anthocyanin contents in red lettuce treated with ABA were significantly higher than in controls, whereas no significant differences were observed in green lettuce. ABA significantly induced the accumulation of chlorophyll b and total carotenoids in lettuces. The phenolic compounds identified and quantified in red and green lettuces included caffeoyltartaric acid, 5-O-caffeoylquinic acid, dicaffeoyltartaric acid, 3,5-dicaffeoylquinic acid, and quercetin 3-(6''-malonyl)-glucoside. Additionally, cyanidin 3-glucoside, cyanidin 3-(3''-malonoyl)-glucoside, and cyanidin 3-(6''-malonoyl)-glucoside in red lettuces were quantified. No significant effects of ABA on these individual phytochemicals were observed in green lettuces, whereas ABA significantly elevated the content of individual phytochemicals in red lettuces except for 5-O-caffeoylquinic acid. Differences among red lettuces with or without exogenous ABA were visualized on the score plots of principal component analyses. Loading plot indicated that multiple phenolic compounds contributed to the observed differences in red lettuces.

  9. Chemical modification of human albumin at cys34 by ethacrynic acid: structural characterisation and binding properties.

    PubMed

    Bertucci, C; Nanni, B; Raffaelli, A; Salvadori, P

    1998-10-01

    Derivatization of the free cys3,4 in human albumin, which is reported to occur under physiological conditions, has been performed in vitro by reaction of the protein with ethacrynic acid. This modification has been investigated by mass spectrometry and circular dichroism. Ethacrynic acid has been proven to bind human albumin either covalently and non-covalently. This post-translational modification does not determine significant changes in the secondary structure of the protein, as shown by the comparable circular dichroism spectra of the native and the modified proteins. Furthermore, the binding properties of the human albumin samples have been investigated by circular dichroism and equilibrium dialysis. The affinity to the higher affinity binding sites does not change either for drugs binding to site I, like phenylbutazone, or to site II, like diazepam, while a small but significant increase has been observed for bilirubin, known to bind to site III. Nevertheless significant decreases of the affinity at the lower affinity binding sites of the modified protein were observed for both drugs binding to site I or to site II.

  10. Tight binding of NAP-22 with acidic membrane lipids.

    PubMed

    Maekawa, Shohei; Kobayashi, Yuumi; Morita, Mitsuhiro; Suzaki, Toshinobu

    2015-07-23

    Recovery of various signal transduction molecules in the detergent-resistant membrane microdomain (DRM) fraction suggests the importance of this region in cellular functions. Insolubility of the outer leaflet of DRM to the non-ionic detergent is ascribed to the tight association of cholesterol and sphingolipid. Since, poor localization of sphingolipid is observed in the inner leaflet, the physicochemical background of the insolubility of the inner leaflet is hence still an enigma. NAP-22 (also called BASP1 or CAP-23) is a neuron-enriched calmodulin-binding protein and one of the major proteins in the DRM of the neuronal cell membrane. A previous study showed the presence of several lipids in a NAP-22 fraction after the process of extraction and column chromatography. In this study, the effect of lipid extraction on NAP-22 was studied through native-gel electrophoresis, ultracentrifugation, and electron microscopic observation. The mobility of NAP-22 in native-PAGE was shifted from low to high after delipidation. Delipidated NAP-22 bound phosphatidylserine (PS), phosphatidylinosotol, and ganglioside. Some part of the mixture of PS and NAP-22 was recovered in the insoluble fraction after Triton X-100 treatment and the addition of cholesterol enhanced the amount of NAP-22 in the insoluble fraction.

  11. Binding of caffeine with caffeic acid and chlorogenic acid using fluorescence quenching, UV/vis and FTIR spectroscopic techniques.

    PubMed

    Belay, Abebe; Kim, Hyung Kook; Hwang, Yoon-Hwae

    2016-03-01

    The interactions of caffeine (CF) with chlorogenic acid (CGA) and caffeic acid (CFA) were investigated by fluorescence quenching, UV/vis and Fourier transform infrared (FTIR) spectroscopic techniques. The results of the study indicated that the fluorescence quenching between caffeine and hydroxycinnamic acids could be rationalized in terms of static quenching or the formation of non-fluorescent CF-CFA and CF-CGA complexes. From fluorescence quenching spectral analysis, the quenching constant (KSV), quenching rate constant (kq), number of binding sites (n), thermodynamic properties and conformational changes of the interaction were determined. The quenching constants (KSV) between CF and CGA, CFA are 1.84 × 10(4) and 1.04 × 10(4) L/mol at 298 K and their binding site n is ~ 1. Thermodynamic parameters determined using the Van't Hoff equation indicated that hydrogen bonds and van der Waal's forces have a major role in the reaction of caffeine with caffeic acid and chlorogenic acid. The 3D fluorescence, UV/vis and FTIR spectra also showed that the binding of CF with CFA and CGA induces conformational changes in CFA and CGA.

  12. Nucleic acid binding proteins in highly purified Creutzfeldt-Jakob disease preparations.

    PubMed Central

    Sklaviadis, T; Akowitz, A; Manuelidis, E E; Manuelidis, L

    1993-01-01

    The nature of the infectious agent causing human Creutzfeldt-Jakob disease (CJD), a slowly progressive dementia, is controversial. As in scrapie, no agent-specific proteins or nucleic acids have been identified. However, biological features of exponential replication and agent strain variation, as well as physical size and density data, are most consistent with a viral structure--i.e., a nucleic acid-protein complex. It is often assumed that nuclease treatment, which does not reduce infectious titer, leaves no nucleic acids of > 50 bp. However, nucleic acids of 500-6000 bp can be extracted from highly purified infectious complexes with a mass of approximately 1.5 x 10(7) daltons. It was therefore germane to search for nucleic acid binding proteins that might protect an agent genome. We here use Northwestern blotting to show that there are low levels of nonhistone nucleic acid binding proteins in highly purified infectious 120S gradient fractions. Several nucleic acid binding proteins were clearly host encoded, whereas others were apparent only in CJD, but not in parallel preparations from uninfected brain. Small amounts of residual host Gp34 (prion protein) did not bind any 32P-labeled nucleic acid probes. Most of the minor "CJD-specific" proteins had an acidic pI, a characteristic of many viral core proteins. Such proteins deserve further study, as they probably contribute to unique properties of resistance described for these agents. It remains to be seen if any of these proteins are agent encoded. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8516321

  13. Sugar-nucleotide-binding and autoglycosylating polypeptide(s) from nasturtium fruit: biochemical capacities and potential functions.

    PubMed

    Faik, A; Desveaux, D; MacLachlan, G

    2000-05-01

    Polypeptide assemblies cross-linked by S-S bonds (molecular mass>200 kDa) and single polypeptides folded with internal S-S cross-links (<41 kDa) have been detected by SDS/PAGE in particulate membranes and soluble extracts of developing cotyledons of nasturtium (Tropaeolum majus L.). When first prepared from fruit homogenates, these polypeptides were found to bind reversibly to UDP-Gal (labelled with [(14)C]Gal or [(3)H]uridine), and to co-precipitate specifically with added xyloglucan from solutions made with 67% ethanol. Initially, the bound UDP-[(14)C]Gal could be replaced (bumped) by adding excess UDP, or exchanged (chased) with UDP-Gal, -Glc, -Man or -Xyl. However, this capacity for turnover was lost during incubation in reaction media, or during SDS/PAGE under reducing conditions, even as the glycone moiety was conserved by autoglycosylation to form a stable 41 kDa polypeptide. Polyclonal antibodies raised to a similar product purified from Arabidopsis bound to all the labelled nasturtium polypeptides in immunoblotting tests. The antibodies also inhibited the binding of nasturtium polypeptides to UDP-Gal, the uptake of UDP-[(14)C]Gal into intact nasturtium membrane vesicles and the incorporation of [(14)C]Gal into nascent xyloglucan within these vesicles. This is the first direct evidence that these polypeptides facilitate the channelling of UDP-activated sugars from the cytoplasm through Golgi vesicle membranes to lumenal sites, where they can be used as substrates for glycosyltransferases to synthesize products such as xyloglucan.

  14. Identification of amino acids essential for DNA binding and dimerization in p67SRF: implications for a novel DNA-binding motif.

    PubMed Central

    Sharrocks, A D; Gille, H; Shaw, P E

    1993-01-01

    The serum response factor (p67SRF) binds to a palindromic sequence in the c-fos serum response element (SRE). A second protein, p62TCF binds in conjunction with p67SRF to form a ternary complex, and it is through this complex that growth factor-induced transcriptional activation of c-fos is thought to take place. A 90-amino-acid peptide, coreSRF, is capable for dimerizing, binding DNA, and recruiting p62TCF. By using extensive site-directed mutagenesis we have investigated the role of individual coreSRF amino acids in DNA binding. Mutant phenotypes were defined by gel retardation and cross-linking analyses. Our results have identified residues essential for either DNA binding or dimerization. Three essential basic amino acids whose conservative mutation severely reduced DNA binding were identified. Evidence which is consistent with these residues being on the face of a DNA binding alpha-helix is presented. A phenylalanine residue and a hexameric hydrophobic box are identified as essential for dimerization. The amino acid phasing is consistent with the dimerization interface being presented as a continuous region on a beta-strand. A putative second alpha-helix acts as a linker between these two regions. This study indicates that p67SRF is a member of a protein family which, in common with many DNA binding proteins, utilize an alpha-helix for DNA binding. However, this alpha-helix is contained within a novel domain structure. Images PMID:8417320

  15. Diminished GABA(A) receptor-binding capacity and a DNA base substitution in a patient with treatment-resistant depression and anxiety.

    PubMed

    Kosel, Markus; Rudolph, Uwe; Wielepp, Peter; Luginbühl, Martin; Schmitt, Wolfgang; Fisch, Hans U; Schlaepfer, Thomas E

    2004-02-01

    In this report, we describe the case of a caucasian male patient, aged 42 years, suffering from severe treatment-resistant generalized anxiety disorder with panic attacks and from severe major depression, for which he was treated with a course of electroconvulsive therapy. During electroconvulsive treatment, anesthesia was difficult to induce with etomidate and, once, propofol. Bispectral indices recordings (assessing the depth of anesthesia) revealed a much shorter duration of loss of responsiveness compared to a control patient receiving also a course of electroconvulsive therapy. Since GABA receptor-mediated regulation of cortical excitability is important with respect to general anesthesia, we investigated the density of GABA(A) receptors with (123)I-iomazenil SPECT and found a clearly diminished binding of the radiotracer in the right frontal and orbitotemporal regions compared to the recordings in a 38-year-old healthy male control. Genetic analysis of the exons 7 and 8 of the GABRB1-3 genes coding for the beta1-3 subunits of the GABA(A) receptors revealed a silent G to A substitution in the third position of amino acid 257 of the beta1-subunit. To our knowledge, this is the first report of a link between insensitivity to anesthetic agents and altered GABA(A) receptor function in a clinical case. Whereas reduced GABA(A) receptor-binding capacity has been investigated in anxiety disorders, this has not been the case in depressive disorders. This case illustrates how clinical observations in psychiatry can prompt investigation by modern techniques and potentially link clinics and basic sciences. No conclusion can, however, be made about casual links in this single case [corrected].

  16. Fatty Acid-Binding Protein in Small Intestine IDENTIFICATION, ISOLATION, AND EVIDENCE FOR ITS ROLE IN CELLULAR FATTY ACID TRANSPORT

    PubMed Central

    Ockner, Robert K.; Manning, Joan A.

    1974-01-01

    A soluble fatty acid-binding protein (FABP), mol wt ∼ 12,000 is present in intestinal mucosa and other tissues that utilize fatty acids, including liver, myocardium, adipose, and kidney. This protein binds long chain fatty acids both in vivo and in vitro. FABP was isolated from rat intestine by gel filtration and isoelectric focusing. It showed a reaction of complete immunochemical identity with proteins in the 12,000 mol wt fatty acid-binding fractions of liver, myocardium, and adipose tissue supernates. (The presence of immunochemically nonidentical 12,000 mol wt FABP in these tissues is not excluded.) By quantitative radial immunodiffusion, supernatant FABP concentration in mucosa from proximal and middle thirds of jejuno-ileum significantly exceeded that in distal third, duodenum, and liver, expressed as micrograms per milligram soluble protein, micrograms per gram DNA, and micrograms per gram tissue. FABP concentration in villi was approximately three times greater than in crypts. Small quantities of FABP were present in washed nuclei-cell membrane, mitochondrial and microsomal fractions. However, the amount of FABP solubilized per milligram membrane protein was similar for all particulate fractions, and total membrane-associated FABP was only about 16% of supernatant FABP. Intestinal FABP concentration was significantly greater in animals maintained on high fat diets than on low fat; saturated and unsaturated fat diets did not differ greatly in this regard. The preponderance of FABP in villi from proximal and middle intestine, its ability to bind fatty acids in vivo as well as in vitro, and its response to changes in dietary fat intake support the concept that this protein participates in cellular fatty acid transport during fat absorption. Identical or closely related 12,000 mol wt proteins may serve similar functions in other tissues. Images PMID:4211161

  17. Lead binding to soil fulvic and humic acids: NICA-Donnan modeling and XAFS spectroscopy.

    PubMed

    Xiong, Juan; Koopal, Luuk K; Tan, WenFeng; Fang, LinChuan; Wang, MingXia; Zhao, Wei; Liu, Fan; Zhang, Jing; Weng, LiPing

    2013-10-15

    Binding of lead (Pb) to soil fulvic acid (JGFA), soil humic acids (JGHA, JLHA), and lignite-based humic acid (PAHA) was investigated through binding isotherms and XAFS. Pb binding to humic substances (HS) increased with increasing pH and decreasing ionic strength. The NICA-Donnan model described Pb binding to the HS satisfactorily. The comparison of the model parameters showed substantial differences in median Pb affinity constants among JGFA, PAHA, and the soil HAs. Milne's "generic" parameters did not provide an adequate prediction for the soil samples. The Pb binding prediction with generic parameters for the soil HAs was improved significantly by using the value n(Pb1) = 0.92 instead of the generic value n(Pb1) = 0.60. The n(Pb1)/n(H1) ratios obtained were relatively high, indicating monodentate Pb binding to the carboxylic-type groups. The nPb2/nH2 ratios depended somewhat on the method of optimization, but the values were distinctly lower than the n(Pb1)/nH1 ratios, especially when the optimization was based on Pb bound vs log [Pb(2+)]. These low values indicate bidentate binding to the phenolic-type groups at high Pb concentration. The NICA-Donnan model does not consider bidentate binding of Pb to a carboxylic- and a phenolic-type group. The EXAFS results at high Pb loading testified that Pb was bound in bidentate complexes of one carboxylic and one phenolic group (salicylate-type) or two phenolic groups (catechol-type) in ortho position.

  18. A bidentate Lewis acid with a telluronium ion as an anion-binding site

    NASA Astrophysics Data System (ADS)

    Zhao, Haiyan; Gabbaï, François P.

    2010-11-01

    The search for receptors that can selectively capture small and potentially toxic anions in protic media has sparked a renewed interest in the synthesis and anion-binding properties of polydentate Lewis acids. Seeking new paradigms to enhance the anion affinities of such systems, we synthesized a bidentate Lewis acid that contains a boryl and a telluronium moiety as Lewis acidic sites. Anion-complexation studies indicate that this telluronium borane displays a high affinity for fluoride in methanol. Structural and computational studies show that the unusual fluoride affinity of this bidentate telluronium borane can be correlated with the formation of a B-F --> Te chelate motif supported by a strong lone-pair(F) --> σ*(Te-C) donor-acceptor interaction. These results, which illustrate the viability of heavier chalcogenium centres as anion-binding sites, allow us to introduce a novel strategy for the design of polydentate Lewis acids with enhanced anion affinities.

  19. A bidentate Lewis acid with a telluronium ion as an anion-binding site.

    PubMed

    Zhao, Haiyan; Gabbaï, François P

    2010-11-01

    The search for receptors that can selectively capture small and potentially toxic anions in protic media has sparked a renewed interest in the synthesis and anion-binding properties of polydentate Lewis acids. Seeking new paradigms to enhance the anion affinities of such systems, we synthesized a bidentate Lewis acid that contains a boryl and a telluronium moiety as Lewis acidic sites. Anion-complexation studies indicate that this telluronium borane displays a high affinity for fluoride in methanol. Structural and computational studies show that the unusual fluoride affinity of this bidentate telluronium borane can be correlated with the formation of a B-F → Te chelate motif supported by a strong lone-pair(F) → σ*(Te-C) donor-acceptor interaction. These results, which illustrate the viability of heavier chalcogenium centres as anion-binding sites, allow us to introduce a novel strategy for the design of polydentate Lewis acids with enhanced anion affinities.

  20. Fatty acid binding proteins have the potential to channel dietary fatty acids into enterocyte nuclei[S

    PubMed Central

    Esteves, Adriana; Knoll-Gellida, Anja; Canclini, Lucia; Silvarrey, Maria Cecilia; André, Michèle; Babin, Patrick J.

    2016-01-01

    Intracellular lipid binding proteins, including fatty acid binding proteins (FABPs) 1 and 2, are highly expressed in tissues involved in the active lipid metabolism. A zebrafish model was used to demonstrate differential expression levels of fabp1b.1, fabp1b.2, and fabp2 transcripts in liver, anterior intestine, and brain. Transcription levels of fabp1b.1 and fabp2 in the anterior intestine were upregulated after feeding and modulated according to diet formulation. Immunofluorescence and electron microscopy immunodetection with gold particles localized these FABPs in the microvilli, cytosol, and nuclei of most enterocytes in the anterior intestinal mucosa. Nuclear localization was mostly in the interchromatin space outside the condensed chromatin clusters. Native PAGE binding assay of BODIPY-FL-labeled FAs demonstrated binding of BODIPY-FLC12 but not BODIPY-FLC5 to recombinant Fabp1b.1 and Fabp2. The binding of BODIPY-FLC12 to Fabp1b.1 was fully displaced by oleic acid. In vivo experiments demonstrated, for the first time, that intestinal absorption of dietary BODIPY-FLC12 was followed by colocalization of the labeled FA with Fabp1b and Fabp2 in the nuclei. These data suggest that dietary FAs complexed with FABPs are able to reach the enterocyte nucleus with the potential to modulate nuclear activity. PMID:26658423

  1. Phenanthrene binding by humic acid-protein complexes as studied by passive dosing technique.

    PubMed

    Zhao, Jian; Wang, Zhenyu; Ghosh, Saikat; Xing, Baoshan

    2014-01-01

    This work investigated the binding behavior of phenanthrene by humic acids (HA-2 and HA-5), proteins (bovine serum albumin (BSA)), lysozyme and pepsin), and their complexes using a passive dosing technique. All sorption isotherms were fitted well with Freundlich model and the binding capability followed an order of HA-5 > HA-2 > BSA > pepsin > lysozyme. In NaCl solution, phenanthrene binding to HA-BSA complexes was much higher than the sum of binding to individual HA and BSA, while there was no enhancement for HA-pepsin. Positively charged lysozyme slightly lowered phenanthrene binding on both HAs due to strong aggregation of HA-lysozyme complexes, leading to reduction in the number of binding sites. The binding enhancement by HA-BSA was observed under all tested ion species and ionic strengths. This enhancement can be explained by unfolding of protein, reduction of aggregate size and formation of HA-BSA complexes with favorable conformations for binding phenanthrene.

  2. Total iron binding capacity

    MedlinePlus

    ... GM. Disorders of iron homeostasis: iron deficiency and overload. In: Hoffman R, Benz EJ Jr, Silberstein LE, ... to achieve this important distinction for online health information and services. Learn more about A.D.A. ...

  3. Towards the elucidation of molecular determinants of cooperativity in the liver bile acid binding protein.

    PubMed

    Pedò, Massimo; D'Onofrio, Mariapina; Ferranti, Pasquale; Molinari, Henriette; Assfalg, Michael

    2009-11-15

    Bile acid binding proteins (BABPs) are cytosolic lipid chaperones contributing to the maintenance of bile acid homeostasis and functional distribution within the cell. Liver BABPs act in parallel with ileal transporters to ensure vectorial transport of bile salts in hepatocytes and enterocytes, respectively. We describe the investigation of ligand binding to liver BABP, an essential step in the understanding of intracellular bile salt transport. Binding site occupancies were monitored in NMR titration experiments using (15)N-labelled ligand, while the relative populations of differently bound BABP forms were assessed by mass spectrometry. This site-specific information allowed the determination of intrinsic thermodynamic parameters and the identification of an extremely high cooperativity between two binding sites. Protein-observed NMR experiments revealed a global structural rearrangement which suggests an allosteric mechanism at the basis of the observed cooperativity. The view of a molecular tool capable of buffering against significant concentrations of free bile salts in a large range of solution conditions emerges from the observed pH-dependence of binding. We set to determine the molecular determinants of cooperativity by analysing the binding properties of a protein containing a mutated internal histidine. Both mass spectrometry and NMR experiments are consistent with an overall decreased binding affinity of the mutant, while the measured diffusion coefficients of ligand species reveal that the affinity loss concerns essentially one of the two binding sites. We therefore identified a mutation able to disrupt energetic communication functional to efficient binding and conclude that the buried histidine establishes contacts that stabilize the ternary complex.

  4. Sialic acid mediates the initial binding of positively charged inorganic particles to alveolar macrophage membranes.

    PubMed

    Gallagher, J E; George, G; Brody, A R

    1987-06-01

    Pulmonary macrophages phagocytize inhaled particles and are postulated to play a role in the development of pulmonary interstitial fibrogenesis. The basic biologic mechanisms through which inhaled particles bind to macrophage membranes and subsequently are phagocytized remain unclear. We hypothesize that positively charged particles bind to negatively charged sialic acid (SA) residues on macrophage membranes. Alveolar Macrophages (AM) were collected by saline lavage from normal rat lungs. The cells adhered to plastic coverslips in serum-free phosphate buffered saline at 37 degrees C for 45 min and then were maintained at 4 degrees C for the binding experiments. Even distribution of SA groups on AM surfaces was demonstrated by scanning electron microscopy of wheat germ agglutinin (WGA) conjugated to 50 nm gold spheres. The WGA is a lectin that binds specifically to sialic acid, and pretreatment of AM with this lectin prevented the binding of positively charged carbonyl iron (C-Fe) spheres, aluminum (Al) spheres, and chrysotile asbestos fibers to AM surfaces. Limulus protein, another lectin with binding specificity for SA, similarly blocked the binding of positively charged spheres and chrysotile asbestos fibers but not negatively charged glass spheres or crocidolite asbestos fibers. Con A and ricin, lectins that bind to mannose and galactose residues, respectively, did not block particle binding. When both positively charged iron spheres and negatively charged glass spheres were prebound to AM membranes, subsequent treatment with WGA displaced only the positively charged spheres from macrophage surfaces. Con A and ricin had no effect on prebound positively charged C-Fe and Al spheres.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. Serum iron level, ferritin and total iron binding capacity level among nonpregnant women with and without melasma

    PubMed Central

    Behrangi, Elham; Baniasadi, Farzaneh; Esmaeeli, Shooka; Hedayat, Kosar; Goodarzi, Azade; Azizian, Zahra

    2015-01-01

    Background: Melasma is a common acquired disorder characterized by symmetric, hyperpigmented patches with an irregular outline, occurring most commonly on the face. It is most prevalent among young to middle-aged women. Although iron overload affects skin pigmentation, effect of iron deficiency on skin is not clear. So, we evaluated serum iron level, ferritin and total iron binding capacity (TIBC) level among nonpregnant women with and without melasma. Materials and Methods: A cross-sectional case study was conducted in 2012 at university dermatologic department on 33 nonpregnant women with melasma (case) and 33 nonpregnant women without melasma (control). Serum iron level, TIBC and ferritin in the two groups was measured and compared. Results: Serum iron level was lower in the case group (85 ± 11) in comparison with control group (102 ± 9), but the difference was not significant (P: 0.9). Mean TIBC and Ferritin were higher in the case group (TIBC: 329.4 ± 29, ferritin: 6 ± 18) than the control group (TIBC: 329.3 ± 29, ferritin: 33 ± 6) without significant difference. Conclusion: Although the serum iron level was lower in nonpregnant women with mealsma, it was not significant compared with those without melasma. PMID:26109976

  6. Stemflow Acid Neutralization Capacity in a Broadleaved Deciduous Forest: The Role of Edge Effects

    NASA Astrophysics Data System (ADS)

    Levia, D. F., Jr.; Shiklomanov, A.

    2014-12-01

    The fragmentation of forests is occurring at an accelerated rate in parts of the United States. Forest fragmentation creates edge habitat that affects the biogeochemistry of forests. Atmospheric deposition is known to increase at the forest edge in comparison to the forest interior. Past research has demonstrated the critical role of edge effects on throughfall chemistry but no known work has examined the relationship between stemflow chemistry and edge effects. To fill this data gap, we quantified the stemflow acid neutralization capacity (ANC) of nineteen Liriodendron tulipifera L. (yellow poplar) trees between forest edge and interior locations in the Piedmont of the mid-Atlantic USA. ANC was measured directly by potentiometric titration. Both stemflow pH and ANC were higher for L. tulipifera trees on the forest edge as opposed to those in interior locations (p < 0.01), although marked variability was observed among individual trees. It is critical to note that the ANC of stemflow of edge trees is almost certainly contextual, depending on geographic locality. This is to say that stemflow from edge trees may neutralize acid inputs in some locations (as in our case) but lead to enhanced acidification of aqueous inputs to forest soils in other locales where the dry deposition of acid anions is high. The experimental results have ramifications for forest management schema seeking to increase or decrease the extent of edge habitat in forest fragments.

  7. Probing the General Time Scale Question of Boronic Acid Binding with Sugars in Aqueous Solution at Physiological pH

    PubMed Central

    Ni, Nanting; Laughlin, Sarah; Wang, Yingji; Feng, You; Zheng, Yujun

    2012-01-01

    The boronic acid group is widely used in chemosensor design due to its ability to reversibly bind diol-containing compounds. The thermodynamic properties of the boronic acid-diol binding process have been investigated extensively. However, there are few studies of the kinetic properties of such binding processes. In this report, stopped-flow method was used for the first time to study the kinetic properties of the binding between three model arylboronic acids, 4-, 5-, and 8-isoquinolinylboronic acids, and various sugars. With all the boronic acid-diol pair sexamined, reactions were complete within seconds. The kon values with various sugars follow the order of D-fructose >D-tagatose>D-mannose >D-glucose. This trend tracks the thermodynamic binding affinities for these sugars and demonstrates that the “on” rate is the key factor determining the binding constant. PMID:22464680

  8. Research on the fiber reflecting sensor for detecting the residual capacity of the lead-acid battery

    NASA Astrophysics Data System (ADS)

    Zhao, Mingfu; Zhong, Nianbing; Chen, Yan; Luo, Yuwei

    2006-11-01

    According to the Lambert-Bee law, we can see that the photic absorption coefficient is related to the matter's concentration, the distance of the light through the absorption medium and the transmitted light intensity. The paper just according to the physical phenomena and the theory make the reflex energy relate to the concentration testing of the electrolyte, at the same time the electrolyte's concentration is related to the capacity of lead-acid battery on a corresponding function relation, so we can know the capacity state of the lead-acid battery according to the measurement on the electrolyte's concentration. According to the experiment and research the author deeply discussed how the temperature change affects the capacity of lead-acid battery and the concentration's changing relation, according to the analyses of the thermo-optic effect, we made a new reflecting fiber sensor based on the comparative temperature testing theory and absorption which can eliminate the temperature effect on the tested signal namely the output signal just related to the concentration, so really reflects the change of the capacity of the lead-acid battery when it is in the charge and discharge process. The results of the experiment and theory analyses show that this method is easy to realize the online testing of the capacity of lead-acid battery. This sensor has many merits such as precise measurement, sensitive reaction, long-life use etc. It can be widely used in the electric capacity testing of the automobile lead-acid battery, the electric capacity testing of the industry lead-acid battery, liquor's concentration testing and salinity testing of the sea and have a bright future.

  9. Binding and solubility of oleic acid to laboratory materials: A possible artifact

    SciTech Connect

    Mailman, D.; Rose, C. )

    1990-01-01

    The possibility that significant amounts of fatty acids were dissolved in or bound to the surfaces of common laboratory materials was examined. The uptake or adsorption of radioisotopically labeled oleic acid and cholic acid by plastic tubing of Tygon{trademark}, Teflon{trademark}, and polyethylene, and Pyrex{trademark}, and borosilicate glass, and steel was measured. {sup 3}H-oleic acid and {sup 14}C-cholic acid were used in the presence of different concentration of unlabeled oleic acid, cholic acid, and/or bovine serum albumin. Concentrations, composition, pH, and perfusion rates were varied. Relatively large amounts of oleic acid were lost by dissolving in plastic and adsorption to glass or metal. The degree of losses decreased in the presence of compounds in the perfusion solution which could bind or dissolve oleic acid. In contrast, cholic acid was not lost to plastic, glass or metal. The magnitude of and influence of perfusion rate, composition, pH, and sequence of perfusion solutions on oleic acid losses were sufficiently large that the results of certain studies, such as those of unstirred water layers or albumin-stimulated fatty acid uptake by hepatocytes may need to be reexamined.

  10. Identification of amino acids in the Dr adhesin required for binding to decay-accelerating factor.

    PubMed

    Van Loy, Cristina P; Sokurenko, Evgeni V; Samudrala, Ram; Moseley, Steve L

    2002-07-01

    Members of the Dr family of adhesins of Escherichia coli recognize as a receptor the Dr(a) blood-group antigen present on the complement regulatory and signalling molecule, decay-accelerating factor (DAF). One member of this family, the Dr haemagglutinin, also binds to a second receptor, type IV collagen. Structure/function information regarding these adhesins has been limited and domains directly involved in the interaction with DAF have not been determined. We devised a strategy to identify amino acids in the Dr haemagglutinin that are specifically involved in the interaction with DAF. The gene encoding the adhesive subunit, draE, was subjected to random mutagenesis and used to complement a strain defective for its expression. The resulting mutants were enriched and screened to obtain those that do not bind to DAF, but retain binding to type IV collagen. Individual amino acid changes at positions 10, 63, 65, 75, 77, 79 and 131 of the mature DraE sequence significantly reduced the ability of the DraE adhesin to bind DAF, but not collagen. Over half of the mutants obtained had substitutions within amino acids 63-81. Analysis of predicted structures of DraE suggest that these proximal residues may cluster to form a binding domain for DAF.

  11. Biochemical and Structural Characterization of Lysophosphatidic Acid Binding by a Humanized Monoclonal Antibody

    SciTech Connect

    J Fleming; J Wojciak; M Campbell; T Huxford

    2011-12-31

    Lysophosphatidic acid (LPA) is a common product of glycerophospholipid metabolism and an important mediator of signal transduction. Aberrantly high LPA concentrations accompany multiple disease states. One potential approach for treatment of these diseases, therefore, is the therapeutic application of antibodies that recognize and bind LPA as their antigen. We have determined the X-ray crystal structure of an anti-LPA antibody (LT3015) Fab fragment in its antigen-free form to 2.15 {angstrom} resolution and in complex with two LPA isotypes (14:0 and 18:2) to resolutions of 1.98 and 2.51 {angstrom}, respectively. The variable CDR (complementarity-determining region) loops at the antigen binding site adopt nearly identical conformations in the free and antigen-bound crystal structures. The crystallographic models reveal that the LT3015 antibody employs both heavy- and light-chain CDR loops to create a network of eight hydrogen bonds with the glycerophosphate head group of its LPA antigen. The head group is almost completely excluded from contact with solvent, while the hydrocarbon tail is partially solvent-exposed. In general, mutation of amino acid residues at the antigen binding site disrupts LPA binding. However, the introduction of particular mutations chosen strategically on the basis of the structures can positively influence LPA binding affinity. Finally, these structures elucidate the exquisite specificity demonstrated by an anti-lipid antibody for binding a structurally simple and seemingly unconstrained target molecule.

  12. Distribution of dissolved organic carbon and metal-binding capacity among ultrafilterable fractions isolated from selected surface waters of the southeastern United States

    NASA Astrophysics Data System (ADS)

    Alberts, James J.; Giesy, John P.; Evans, David W.

    1984-06-01

    The binding capacities of surface waters for Cd, Cu, and Pb were determined for eight water samples representing four rivers and two swamps from Florida and Georgia in the southeastern United States The binding capacity ranges were CdBC=0 04 to 0 79 μg atm/L, CuBC=1 0 to 5 4 μg atm/L, and PbBC=5 0 to 17 8 μg atm/L Binding capacity values from the southeastern United States are shown to be in good agreement with values reported from the northeastern part of the country and northern Europe The CdBC was due primarily to inorganic ligand binding, while PbBC was predominantly a result of organic matter The CuBC was due to a complex function of both organic and inorganic binding Significant portions of the CuBC and PbBC could be removed from the waters by ultrafiltration of particles between 0·45 μm and 52 Å in diameter Ultrafiltration, even to removing particles > 13 Å diameter, did not affect the CdBC Distributional studies of the dissolved organic carbon in these systems reveal that significant fractions of the DOC are present in the ultrafilterable fraction which contains significant portions of the CuBC and PbBC

  13. Calcium Binding to Amino Acids and Small Glycine Peptides in Aqueous Solution: Toward Peptide Design for Better Calcium Bioavailability.

    PubMed

    Tang, Ning; Skibsted, Leif H

    2016-06-01

    Deprotonation of amino acids as occurs during transfer from stomach to intestines during food digestion was found by comparison of complex formation constants as determined electrochemically for increasing pH to increase calcium binding (i) by a factor of around 6 for the neutral amino acids, (ii) by a factor of around 4 for anions of the acidic amino acids aspartic and glutamic acid, and (iii) by a factor of around 5.5 for basic amino acids. Optimized structures of the 1:1 complexes and ΔHbinding for calcium binding as calculated by density functional theory (DFT) confirmed in all complexes a stronger calcium binding and shorter calcium-oxygen bond length in the deprotonated form. In addition, the stronger calcium binding was also accompanied by a binding site shift from carboxylate binding to chelation by α-amino group and carboxylate oxygen for leucine, aspartate, glutamate, alanine, and asparagine. For binary amino acid mixtures, the calcium-binding constant was close to the predicted geometric mean of the individual amino acid binding constants indicating separate binding of calcium to two amino acids when present together in solution. At high pH, corresponding to conditions for calcium absorption, the binding affinity increased in the order Lys < Arg < Cys < Gln < Gly ∼ Ala < Asn < His < Leu < Glu< Asp. In a series of glycine peptides, calcium-binding affinity was found to increase in the order Gly-Leu ∼ Gly-Gly < Ala-Gly < Gly-His ∼ Gly-Lys-Gly < Glu-Cys-Gly < Gly-Glu, an ordering confirmed by DFT calculations for the dipeptides and which also accounted for large synergistic effects in calcium binding for up to 6 kJ/mol when compared to the corresponding amino acid mixtures.

  14. Studies on the binding of fulvic acid with transferrin by spectroscopic analysis

    NASA Astrophysics Data System (ADS)

    Zhang, Xiu-feng; Yang, Guang; Dong, Yu; Zhao, Yan-qin; Sun, Xiao-ran; Chen, Lei; Chen, Hong-bo

    2015-02-01

    Transferrin has shown potential in the delivery of anticancer drugs into primarily proliferating cancer cells that over-express transferrin receptors. Fulvic acid has a wide range of biological and pharmacological activities which caused widespread concerns, the interaction of fulvic acid with human serum transferrin (Tf) has great significance for gaining a deeper insight about anticancer activities of fulvic acid. In this study, the mechanism of interaction between fulvic acid and Tf, has been investigated by using fluorescence quenching, thermodynamics, synchronous fluorescence and circular dichroism (CD) under physiological condition. Our results have shown that fulvic acid binds to Tf and form a new complex, and the calculated apparent association constants are 5.04 × 108 M-1, 5.48 × 107 M-1, 7.38 × 106 M-1 from the fluorescence quenching at 288 K, 298 K, and 310 K. The thermodynamic parameters indicate that hydrogen bonding and weak van der Waals are involved in the interaction between fulvic acid and Tf. The binding of fulvic acid to Tf causes the α-helix structure content of the protein to reduce, and resulting that peptide chains of Tf become more stretched. Our results have indicated a mechanism of the interaction between fulvic acid and Tf, which may provide information for possible design of methods to deliver drug molecules via transferrin to target tissues and cells effectively.

  15. Folate binding protein: therapeutic natural nanotechnology for folic acid, methotrexate, and leucovorin.

    PubMed

    Merzel, Rachel L; Boutom, Sarah M; Chen, Junjie; Frey, Carolina; Shedden, Kerby; Marsh, E Neil G; Banaszak Holl, Mark M

    2017-02-16

    Blood serum proteins play a critical role in the transport, biodistribution, and efficacy of systemically-delivered therapeutics. Here, we have investigated the concentration- and ligand-dependent aggregation of folate binding protein (FBP), focusing in particular on folic acid, an important vitamin and targeting agent; methotrexate, an antifolate drug used to treat cancer and rheumatoid arthritis; and leucovorin which is used to decrease methotrexate toxicity. We employed atomic force microscopy to characterize, on a particle-by-particle basis, the volumes of the FBP nanoparticles that form upon ligand binding. We measured the distribution of FBP nanoparticle volumes as a function of ligand concentration over physiologically- and therapeutically-relevant ranges. At physiologically-relevant concentrations, significant differences in particle volume distributions exist that we hypothesize are consistent with different trafficking mechanisms for folic acid and methotrexate. In addition, we hypothesize leucovorin is trafficked and delivered like folic acid at therapeutically-relevant concentrations. We propose that changes in dosing procedures could improve the delivery and therapeutic index for methotrexate and other folic acid-targeted drug conjugates and imaging agents. Specifically, we suggest pre-binding the drugs to FBP may provide a better formulation for drug delivery of methotrexate for both cancer and rheumatoid arthritis. This would be analogous to pre-binding paclitaxel to albumin, which is already used in the clinic.

  16. Ferulic acid enhances IgE binding to peanut allergens in western blots.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Because phenolic compounds can precipitate or complex with proteins, we postulated that interactions of phenolics with IgE antibodies help enhance IgE binding to peanut allergens in Western blots. Three different phenolics, such as, ferulic, caffeic and chlorogenic acids were examined. Each was mixe...

  17. Breast Cancer Prevention by Fatty Acid Binding Protein MRG-Induced Pregnancy Like Mammary Gland Differentiation

    DTIC Science & Technology

    2005-08-01

    Annual Summary 3. DATES COVERED (From - To) 1 AUG 2004 - 31 JUL 2005 4. TITLE AND SUBTITLE Breast Cancer Prevention by Fatty Acid Binding Protein...differentiation. Overexpression of MRG in human breast cancer cells induced differentiation with changes in cellular morphology and a significant increase

  18. Affi-Gel Blue for nucleic acid removal and early enrichment of nucleotide binding proteins.

    PubMed

    Deutscher, Murray P

    2009-01-01

    Passage of an extract or supernatant fraction through a column of Affi-Gel Blue and batchwise elution can be a rapid and effective early procedure for removal of nucleic acid, concentration of the sample and purification of nucleotide binding proteins.

  19. The influence of fatty acids on theophylline binding to human serum albumin. Comparative fluorescence study

    NASA Astrophysics Data System (ADS)

    Maciążek-Jurczyk, M.; Sułkowska, A.; Bojko, B.; Równicka-Zubik, J.; Szkudlarek-Haśnik, A.; Zubik-Skupień, I.; Góra, A.; Dubas, M.; Korzonek-Szlacheta, I.; Wielkoszyński, T.; Żurawiński, W.; Sosada, K.

    2012-04-01

    Theophylline, popular diuretic, is used to treat asthma and bronchospasm. In blood it forms complexes with albumin, which is also the main transporter of fatty acids. The aim of the present study was to describe the influence of fatty acids (FA) on binding of theophylline (Th) to human serum albumin (HSA) in the high affinity binding sites. Binding parameters have been obtained on the basis of the fluorescence analysis. The data obtained for the complex of Th and natural human serum albumin (nHSA) obtained from blood of obese patients qualified for surgical removal of stomach was compared with our previous studies on the influence of FA on the complex of Th and commercially available defatted human serum albumin (dHSA).

  20. H-binding groups in lignite vs. soil humic acids: NICA-Donnan and spectroscopic parameters

    SciTech Connect

    Drosos, M.; Jerzykiewicz, M.; Deligiannakis, Y.

    2009-04-15

    A comparative study has been carried out for two sets of humic acids isolated from lignites and soils. H-binding data were analyzed using the NICA-Donnan model, for three Greek lignite humic acids (HA) plus IHSS Leonardite reference HA, and five Greek soil HAs plus a commercial peat HA. {sup 13}C-CP-MAS NMR and H-binding data provide quantitative estimates for functional groups, showing that lignite HAs of diverse origin have strikingly homogeneous properties, while the H-binding structural units of soil HAs are characterized by a large degree of variability. Consistent differences between soil HA vs. lignite HA are revealed at the level of functional groups' concentrations. In the pH range 4 to 10, soil HA showed a charge variation < 3 (equiv kg{sup -1}) while lignite HAs showed a higher charge variation > 3.5 (equiv kg{sup -1}).

  1. Characterization of the Adeno-Associated Virus 1 and 6 Sialic Acid Binding Site

    PubMed Central

    Huang, Lin-Ya; Patel, Ami; Ng, Robert; Miller, Edward Blake; Halder, Sujata; McKenna, Robert; Asokan, Aravind

    2016-01-01

    ABSTRACT The adeno-associated viruses (AAVs), which are being developed as gene delivery vectors, display differential cell surface glycan binding and subsequent tissue tropisms. For AAV serotype 1 (AAV1), the first viral vector approved as a gene therapy treatment, and its closely related AAV6, sialic acid (SIA) serves as their primary cellular surface receptor. Toward characterizing the SIA binding site(s), the structure of the AAV1-SIA complex was determined by X-ray crystallography to 3.0 Å. Density consistent with SIA was observed in a pocket located at the base of capsid protrusions surrounding icosahedral 3-fold axes. Site-directed mutagenesis substitution of the amino acids forming this pocket with structurally equivalent residues from AAV2, a heparan sulfate binding serotype, followed by cell binding and transduction assays, further mapped the critical residues conferring SIA binding to AAV1 and AAV6. For both viruses five of the six binding pocket residues mutated (N447S, V473D, N500E, T502S, and W503A) abolished SIA binding, whereas S472R increased binding. All six mutations abolished or decreased transduction by at least 50% in AAV1. Surprisingly, the T502S substitution did not affect transduction efficiency of wild-type AAV6. Furthermore, three of the AAV1 SIA binding site mutants—S472R, V473D, and N500E—escaped recognition by the anti-AAV1 capsid antibody ADK1a. These observations demonstrate that common key capsid surface residues dictate both virus binding and entry processes, as well as antigenic reactivity. This study identifies an important functional capsid surface “hot spot” dictating receptor attachment, transduction efficiency, and antigenicity which could prove useful for vector engineering. IMPORTANCE The adeno-associated virus (AAV) vector gene delivery system has shown promise in several clinical trials and an AAV1-based vector has been approved as the first gene therapy treatment. However, limitations still exist with respect

  2. Enhanced lubrication on tissue and biomaterial surfaces through peptide-mediated binding of hyaluronic acid

    NASA Astrophysics Data System (ADS)

    Singh, Anirudha; Corvelli, Michael; Unterman, Shimon A.; Wepasnick, Kevin A.; McDonnell, Peter; Elisseeff, Jennifer H.

    2014-10-01

    Lubrication is key for the efficient function of devices and tissues with moving surfaces, such as articulating joints, ocular surfaces and the lungs. Indeed, lubrication dysfunction leads to increased friction and degeneration of these systems. Here, we present a polymer-peptide surface coating platform to non-covalently bind hyaluronic acid (HA), a natural lubricant in the body. Tissue surfaces treated with the HA-binding system exhibited higher lubricity values, and in vivo were able to retain HA in the articular joint and to bind ocular tissue surfaces. Biomaterials-mediated strategies that locally bind and concentrate HA could provide physical and biological benefits when used to treat tissue-lubricating dysfunction and to coat medical devices.

  3. Binding of cyclic carboxylates to octa-acid deep-cavity cavitand

    NASA Astrophysics Data System (ADS)

    Gibb, Corinne L. D.; Gibb, Bruce C.

    2014-04-01

    As part of the fourth statistical assessment of modeling of proteins and ligands (sampl.eyesopen.com) prediction challenge, the strength of association of nine guests ( 1- 9) binding to octa-acid host was determined by a combination of 1H NMR and isothermal titration calorimetry. Association constants in sodium tetraborate buffered (pH 9.2) aqueous solution ranged from 5.39 × 102 M-1 in the case of benzoate 1, up to 3.82 × 105 M-1 for trans-4-methylcyclohexanoate 7. Overall, the free energy difference between the free energies of complexation of these weakest and strongest binding guests was ΔΔG° = 3.88 kcal mol-1. Based on a multitude of previous studies, the anticipated order of strength of binding was close to that which was actually obtained. However, the binding of guest 3 (4-ethylbenzoate) was considerably stronger than initially estimated.

  4. Enhanced lubrication on tissue and biomaterial surfaces through peptide-mediated binding of hyaluronic acid.

    PubMed

    Singh, Anirudha; Corvelli, Michael; Unterman, Shimon A; Wepasnick, Kevin A; McDonnell, Peter; Elisseeff, Jennifer H

    2014-10-01

    Lubrication is key for the efficient function of devices and tissues with moving surfaces, such as articulating joints, ocular surfaces and the lungs. Indeed, lubrication dysfunction leads to increased friction and degeneration of these systems. Here, we present a polymer-peptide surface coating platform to non-covalently bind hyaluronic acid (HA), a natural lubricant in the body. Tissue surfaces treated with the HA-binding system exhibited higher lubricity values, and in vivo were able to retain HA in the articular joint and to bind ocular tissue surfaces. Biomaterials-mediated strategies that locally bind and concentrate HA could provide physical and biological benefits when used to treat tissue-lubricating dysfunction and to coat medical devices.

  5. Towards an understanding of Mesocestoides vogae fatty acid binding proteins' roles.

    PubMed

    Alvite, Gabriela; Garrido, Natalia; Kun, Alejandra; Paulino, Margot; Esteves, Adriana

    2014-01-01

    Two fatty acid binding proteins, MvFABPa and MvFABPb were identified in the parasite Mesocestoides vogae (Platyhelmithes, Cestoda). Fatty acid binding proteins are small intracellular proteins whose members exhibit great diversity. Proteins of this family have been identified in many organisms, of which Platyhelminthes are among the most primitive. These proteins have particular relevance in flatworms since de novo synthesis of fatty acids is absent. Fatty acids should be captured from the media needing an efficient transport system to uptake and distribute these molecules. While HLBPs could be involved in the shuttle of fatty acids to the surrounding host tissues and convey them into the parasite, FABPs could be responsible for the intracellular trafficking. In an effort to understand the role of MvFABPs in fatty acid transport of M. vogae larvae, we analysed the intracellular localization of both MvFABPs and the co-localization with in vivo uptake of fatty acid analogue BODIPY FL C16. Immunohistochemical studies on larvae sections using specific antibodies, showed a diffuse cytoplasmic distribution of each protein with some expression in nuclei and mitochondria. MvFABPs distribution was confirmed by mass spectrometry identification from 2D-electrophoresis of larvae subcellular fractions. This work is the first report showing intracellular distribution of MvFABPs as well as the co-localization of these proteins with the BODIPY FL C16 incorporated from the media. Our results suggest that fatty acid binding proteins could target fatty acids to cellular compartments including nuclei. In this sense, M. vogae FABPs could participate in several cellular processes fulfilling most of the functions attributed to vertebrate's counterparts.

  6. Towards an Understanding of Mesocestoides vogae Fatty Acid Binding Proteins’ Roles

    PubMed Central

    Alvite, Gabriela; Garrido, Natalia; Kun, Alejandra; Paulino, Margot; Esteves, Adriana

    2014-01-01

    Two fatty acid binding proteins, MvFABPa and MvFABPb were identified in the parasite Mesocestoides vogae (Platyhelmithes, Cestoda). Fatty acid binding proteins are small intracellular proteins whose members exhibit great diversity. Proteins of this family have been identified in many organisms, of which Platyhelminthes are among the most primitive. These proteins have particular relevance in flatworms since de novo synthesis of fatty acids is absent. Fatty acids should be captured from the media needing an efficient transport system to uptake and distribute these molecules. While HLBPs could be involved in the shuttle of fatty acids to the surrounding host tissues and convey them into the parasite, FABPs could be responsible for the intracellular trafficking. In an effort to understand the role of MvFABPs in fatty acid transport of M. vogae larvae, we analysed the intracellular localization of both MvFABPs and the co-localization with in vivo uptake of fatty acid analogue BODIPY FL C16. Immunohistochemical studies on larvae sections using specific antibodies, showed a diffuse cytoplasmic distribution of each protein with some expression in nuclei and mitochondria. MvFABPs distribution was confirmed by mass spectrometry identification from 2D-electrophoresis of larvae subcellular fractions. This work is the first report showing intracellular distribution of MvFABPs as well as the co-localization of these proteins with the BODIPY FL C16 incorporated from the media. Our results suggest that fatty acid binding proteins could target fatty acids to cellular compartments including nuclei. In this sense, M. vogae FABPs could participate in several cellular processes fulfilling most of the functions attributed to vertebrate’s counterparts. PMID:25347286

  7. Polysialic acid sustains cancer cell survival and migratory capacity in a hypoxic environment

    PubMed Central

    Elkashef, Sara M.; Allison, Simon J.; Sadiq, Maria; Basheer, Haneen A.; Ribeiro Morais, Goreti; Loadman, Paul M.; Pors, Klaus; Falconer, Robert A.

    2016-01-01

    Polysialic acid (polySia) is a unique carbohydrate polymer expressed on the surface of NCAM (neuronal cell adhesion molecule) in a number of cancers where it modulates cell-cell and cell-matrix adhesion, migration, invasion and metastasis and is strongly associated with poor clinical prognosis. We have carried out the first investigation into the effect of polySia expression on the behaviour of cancer cells in hypoxia, a key source of chemoresistance in tumours. The role of polysialylation and associated tumour cell migration and cell adhesion were studied in hypoxia, along with effects on cell survival and the potential role of HIF-1. Our findings provide the first evidence that polySia expression sustains migratory capacity and is associated with tumour cell survival in hypoxia. Initial mechanistic studies indicate a potential role for HIF-1 in sustaining polySia-mediated migratory capacity, but not cell survival. These data add to the growing body of evidence pointing to a crucial role for the polysialyltransferases (polySTs) in neuroendocrine tumour progression and provide the first evidence to suggest that polySia is associated with an aggressive phenotype in tumour hypoxia. These results have significant potential implications for polyST inhibition as an anti-metastatic therapeutic strategy and for targeting hypoxic cancer cells. PMID:27611649

  8. Isolation and characterization of lactic acid bacteria strains with ornithine producing capacity from natural sea salt.

    PubMed

    Yu, Jin-Ju; Oh, Suk-Heung

    2010-08-01

    Two lactic acid bacteria (LAB) having ornithine-producing capacity were isolated from Korean natural sea salt. They were Gram-positive, short rod-type bacteria, and able to grow anaerobically with CO(2) production. The isolates grew well on MRS broth at 30-37 degrees C and a pH of 6.5-8.0. The optimum temperature and pH for growth are 37 degrees C and pH 7.0. The isolates fermented D-ribose, D-galactose, D-lactose, D-maltose, Dcellobiose, D-tagatose, D-trehalose, sucrose, D-melezitose, gentiobiose, D-glucose but not D-melibiose, inositol, and L-sorbose. The 16S rDNA sequences of the two isolates showed 99.5% and 99.6% homology with the Weissella koreensis S5623 16S rDNA (Access no. AY035891). They were accordingly identified and named as Weissella koreensis MS1-3 and Weissella koreensis MS1-14, and produced intracellular ornithine at levels of 72 mg/100 g cell F.W. and 105 mg/100 g cell F.W. and extracellular ornithine at levels of 4.5 mg/100 ml and 4.6 mg/100 ml medium, respectively, by culturing in MRS broth supplemented with 1% arginine. High cell growth was maintained in MRS broth with a NaCl concentration of 0-6%. These results show for the first time that Korean natural sea salts contain lactic acid bacteria Weissella koreensis strains having ornithine producing capacity.

  9. Structural aspects of catalytic mechanisms of endonucleases and their binding to nucleic acids

    NASA Astrophysics Data System (ADS)

    Zhukhlistova, N. E.; Balaev, V. V.; Lyashenko, A. V.; Lashkov, A. A.

    2012-05-01

    Endonucleases (EC 3.1) are enzymes of the hydrolase class that catalyze the hydrolytic cleavage of deoxyribonucleic and ribonucleic acids at any region of the polynucleotide chain. Endonucleases are widely used both in biotechnological processes and in veterinary medicine as antiviral agents. Medical applications of endonucleases in human cancer therapy hold promise. The results of X-ray diffraction studies of the spatial organization of endonucleases and their complexes and the mechanism of their action are analyzed and generalized. An analysis of the structural studies of this class of enzymes showed that the specific binding of enzymes to nucleic acids is characterized by interactions with nitrogen bases and the nucleotide backbone, whereas the nonspecific binding of enzymes is generally characterized by interactions only with the nucleic-acid backbone. It should be taken into account that the specificity can be modulated by metal ions and certain low-molecular-weight organic compounds. To test the hypotheses about specific and nonspecific nucleic-acid-binding proteins, it is necessary to perform additional studies of atomic-resolution three-dimensional structures of enzyme-nucleic-acid complexes by methods of structural biology.

  10. Structural aspects of catalytic mechanisms of endonucleases and their binding to nucleic acids

    SciTech Connect

    Zhukhlistova, N. E.; Balaev, V. V.; Lyashenko, A. V.; Lashkov, A. A.

    2012-05-15

    Endonucleases (EC 3.1) are enzymes of the hydrolase class that catalyze the hydrolytic cleavage of deoxyribonucleic and ribonucleic acids at any region of the polynucleotide chain. Endonucleases are widely used both in biotechnological processes and in veterinary medicine as antiviral agents. Medical applications of endonucleases in human cancer therapy hold promise. The results of X-ray diffraction studies of the spatial organization of endonucleases and their complexes and the mechanism of their action are analyzed and generalized. An analysis of the structural studies of this class of enzymes showed that the specific binding of enzymes to nucleic acids is characterized by interactions with nitrogen bases and the nucleotide backbone, whereas the nonspecific binding of enzymes is generally characterized by interactions only with the nucleic-acid backbone. It should be taken into account that the specificity can be modulated by metal ions and certain low-molecular-weight organic compounds. To test the hypotheses about specific and nonspecific nucleic-acid-binding proteins, it is necessary to perform additional studies of atomic-resolution three-dimensional structures of enzyme-nucleic-acid complexes by methods of structural biology.

  11. A 25-Amino Acid Sequence of the Arabidopsis TGD2 Protein Is Sufficient for Specific Binding of Phosphatidic Acid*

    PubMed Central

    Lu, Binbin; Benning, Christoph

    2009-01-01

    Genetic analysis suggests that the TGD2 protein of Arabidopsis is required for the biosynthesis of endoplasmic reticulum derived thylakoid lipids. TGD2 is proposed to be the substrate-binding protein of a presumed lipid transporter consisting of the TGD1 (permease) and TGD3 (ATPase) proteins. The TGD1, -2, and -3 proteins are localized in the inner chloroplast envelope membrane. TGD2 appears to be anchored with an N-terminal membrane-spanning domain into the inner envelope membrane, whereas the C-terminal domain faces the intermembrane space. It was previously shown that the C-terminal domain of TGD2 binds phosphatidic acid (PtdOH). To investigate the PtdOH binding site of TGD2 in detail, the C-terminal domain of the TGD2 sequence lacking the transit peptide and transmembrane sequences was fused to the C terminus of the Discosoma sp. red fluorescent protein (DR). This greatly improved the solubility of the resulting DR-TGD2C fusion protein following production in Escherichia coli. The DR-TGD2C protein bound PtdOH with high specificity, as demonstrated by membrane lipid-protein overlay and liposome association assays. Internal deletion and truncation mutagenesis identified a previously undescribed minimal 25-amino acid fragment in the C-terminal domain of TGD2 that is sufficient for PtdOH binding. Binding characteristics of this 25-mer were distinctly different from those of TGD2C, suggesting that additional sequences of TGD2 providing the proper context for this 25-mer are needed for wild type-like PtdOH binding. PMID:19416982

  12. Analysis of the binding interaction in uric acid - Human hemoglobin system by spectroscopic techniques.

    PubMed

    Makarska-Bialokoz, Magdalena

    2017-01-31

    The binding interaction between human hemoglobin and uric acid has been studied for the first time, by UV-vis absorption and steady-state, synchronous and three-dimensional fluorescence techniques. Characteristic effects observed for human hemoglobin intrinsic fluorescence during interaction with uric acid at neutral pH point at the formation of stacking non-covalent and non-fluorescent complexes. All the calculated parameters, the binding, fluorescence quenching and bimolecular quenching rate constants, as well as Förster resonance energy transfer parameters confirm the existence of static quenching. The results of synchronous fluorescence measurements indicate that the fluorescence quenching of human hemoglobin originates both from Trp and Tyr residues and that the addition of uric acid could significantly hinder the physiological functions of human hemoglobin.

  13. The presence of high-affinity, low-capacity estradiol-17β binding in rainbow trout scale indicates a possible endocrine route for the regulation of scale resorption

    USGS Publications Warehouse

    Persson, Petra; Shrimpton, J. Mark; McCormick, Stephen D.; Bjornsson, Bjorn Thrandur

    2000-01-01

    High-affinity, low-capacity estradiol-17β (E2) binding is present in rainbow trout scale. The Kd and Bmax of the scale E2 binding are similar to those of the liver E2 receptor (Kd is 1.6 ± 0.1 and 1.4 ± 0.1 nM, and Bmax is 9.1 ± 1.2 and 23.1 ± 2.2 fmol × mg protein-1, for scale and liver, respectively), but different from those of the high-affinity, low-capacity E2 binding in plasma (Kd is 4.0 ± 0.4 nM and Bmax is 625.4 ± 63.1 fmol × mg protein−1). The E2 binding in scale was displaced by testosterone, but not by diethylstilbestrol. Hence, the ligand binding specificity is different from that of the previously characterized liver E2 receptor, where E2 is displaced by diethylstilbestrol, but not by testosterone. The putative scale E2 receptor thus appears to bind both E2 and testosterone, and it is proposed that the increased scale resorption observed during sexual maturation in both sexes of several salmonid species may be mediated by this receptor. No high-affinity, low-capacity E2 binding could be detected in rainbow trout gill or skin.

  14. Acid-base and copper-binding properties of three organic matter fractions isolated from a forest floor soil solution

    NASA Astrophysics Data System (ADS)

    van Schaik, Joris W. J.; Kleja, Dan B.; Gustafsson, Jon Petter

    2010-02-01

    Vast amounts of knowledge about the proton- and metal-binding properties of dissolved organic matter (DOM) in natural waters have been obtained in studies on isolated humic and fulvic (hydrophobic) acids. Although macromolecular hydrophilic acids normally make up about one-third of DOM, their proton- and metal-binding properties are poorly known. Here, we investigated the acid-base and Cu-binding properties of the hydrophobic (fulvic) acid fraction and two hydrophilic fractions isolated from a soil solution. Proton titrations revealed a higher total charge for the hydrophilic acid fractions than for the hydrophobic acid fraction. The most hydrophilic fraction appeared to be dominated by weak acid sites, as evidenced by increased slope of the curve of surface charge versus pH at pH values above 6. The titration curves were poorly predicted by both Stockholm Humic Model (SHM) and NICA-Donnan model calculations using generic parameter values, but could be modelled accurately after optimisation of the proton-binding parameters (pH ⩽ 9). Cu-binding isotherms for the three fractions were determined at pH values of 4, 6 and 9. With the optimised proton-binding parameters, the SHM model predictions for Cu binding improved, whereas the NICA-Donnan predictions deteriorated. After optimisation of Cu-binding parameters, both models described the experimental data satisfactorily. Iron(III) and aluminium competed strongly with Cu for binding sites at both pH 4 and pH 6. The SHM model predicted this competition reasonably well, but the NICA-Donnan model underestimated the effects significantly at pH 6. Overall, the Cu-binding behaviour of the two hydrophilic acid fractions was very similar to that of the hydrophobic acid fraction, despite the differences observed in proton-binding characteristics. These results show that for modelling purposes, it is essential to include the hydrophilic acid fraction in the pool of 'active' humic substances.

  15. In Vitro bile acid binding of kale, mustard greens, broccoli, cabbage and green bell pepper improves with microwave cooking

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bile acid binding potential of foods and food fractions has been related to lowering the risk of heart disease and that of cancer. Sautéing or steam cooking has been observed to significantly improve bile acid binding of green/leafy vegetables. It was hypothesized that microwave cooking could impr...

  16. Tolerance to acetic acid is improved by mutations of the TATA-binding protein gene.

    PubMed

    An, Jieun; Kwon, Hyeji; Kim, Eunjung; Lee, Young Mi; Ko, Hyeok Jin; Park, Hongjae; Choi, In-Geol; Kim, Sooah; Kim, Kyoung Heon; Kim, Wankee; Choi, Wonja

    2015-03-01

    Screening a library of overexpressing mutant alleles of the TATA-binding gene SPT15 yielded two Saccharomyces cerevisiae strains (MRRC 3252 and 3253) with enhanced tolerance to acetic acid. They were also tolerant to propionic acid and hydrogen peroxide. Transcriptome profile analysis identified 58 upregulated genes and 106 downregulated genes in MRRC 3252. Stress- and protein synthesis-related transcription factors were predominantly enriched in the upregulated and downregulated genes respectively. Eight deletion mutants for some of the highly downregulated genes were acetic acid-tolerant. The level of intracellular reactive oxygen species was considerably lessened in MRRC 3252 and 3253 upon exposure to acetic acid. Metabolome profile analysis revealed that intracellular concentrations of 5 and 102 metabolites were increased and decreased, respectively, in MRRC 3252, featuring a large increase of urea and a significant decrease of amino acids. The dur1/2Δmutant, in which the urea degradation gene DUR1/2 is deleted, displayed enhanced tolerance to acetic acid. Enhanced tolerance to acetic acid was also observed on the medium containing a low concentration of amino acids. Taken together, this study identified two SPT15 alleles, nine gene deletions and low concentration of amino acids in the medium that confer enhanced tolerance to acetic acid.

  17. Variation of the Detergent-Binding Capacity and Phospholipid Content of Membrane Proteins When Purified in Different Detergents

    PubMed Central

    Ilgü, Hüseyin; Jeckelmann, Jean-Marc; Gachet, María Salomé; Boggavarapu, Rajendra; Ucurum, Zöhre; Gertsch, Jürg; Fotiadis, Dimitrios

    2014-01-01

    Purified membrane proteins are ternary complexes consisting of protein, lipid, and detergent. Information about the amounts of detergent and endogenous phospholipid molecules bound to purified membrane proteins is largely lacking. In this systematic study, three model membrane proteins of different oligomeric states were purified in nine different detergents at commonly used concentrations and characterized biochemically and biophysically. Detergent-binding capacities and phospholipid contents of the model proteins were determined and compared. The insights on ternary complexes obtained from the experimental results, when put into a general context, are summarized as follows. 1), The amount of detergent and 2) the amount of endogenous phospholipids bound to purified membrane proteins are dependent on the size of the hydrophobic lipid-accessible protein surface areas and the physicochemical properties of the detergents used. 3), The size of the detergent and lipid belt surrounding the hydrophobic lipid-accessible surface of purified membrane proteins can be tuned by the appropriate choice of detergent. 4), The detergents n-nonyl-β-D-glucopyranoside and Cymal-5 have exceptional delipidating effects on ternary complexes. 5), The types of endogenous phospholipids bound to membrane proteins can vary depending on the detergent used for solubilization and purification. 6), Furthermore, we demonstrate that size-exclusion chromatography can be a suitable method for estimating the molecular mass of ternary complexes. The findings presented suggest a strategy to control and tune the numbers of detergent and endogenous phospholipid molecules bound to membrane proteins. These two parameters are potentially important for the successul crystallization of membrane proteins for structure determination by crystallographic approaches. PMID:24739165

  18. Acrosin-binding protein (ACRBP) and triosephosphate isomerase (TPI) are good markers to predict boar sperm freezing capacity.

    PubMed

    Vilagran, Ingrid; Castillo, Judit; Bonet, Sergi; Sancho, Sílvia; Yeste, Marc; Estanyol, Josep M; Oliva, Rafael

    2013-09-15

    Sperm cryopreservation is the most efficient method for storing boar sperm samples for a long time. However, one of the inconveniences of this method is the large variation between and within boars in the cryopreservation success of their sperm. The aim of the present work was thus to find reliable and useful predictive biomarkers of the good and poor capacity to withstand the freeze-thawing process in boar ejaculates. To find these biomarkers, the amount of proteins present in the total proteome in sperm cells were compared between good freezability ejaculates (GFE) and poor freezability ejaculates (PFE) using the two-dimensional difference gel electrophoresis technique. Samples were classified as GFE and PFE using progressive motility and viability of the sperm at 30 and 240 minutes after thawing, and the proteomes from each group, before starting cryopreservation protocols, were compared. Because two proteins, acrosin binding protein (ACRBP) and triosephosphate isomerase (TPI), presented the highest significant differences between GFE and PFE groups in two-dimensional difference gel electrophoresis assessment, Western blot analyses for ACRBP and TPI were also performed for validation. ACRBP normalized content was significantly lower in PFE than in GFE (P < 0.05), whereas the TPI amounts were significantly lower in GFE (P < 0.05) than in PFE. The association of ACRBP and TPI with postthaw sperm viability and motility was confirmed using Pearson's linear correlation. In conclusion, ACRBP and TPI can be used as markers of boar sperm freezability before starting the cryopreservation procedure, thereby avoiding unnecessary costs involved in this practice.

  19. Variation of the detergent-binding capacity and phospholipid content of membrane proteins when purified in different detergents.

    PubMed

    Ilgü, Hüseyin; Jeckelmann, Jean-Marc; Gachet, María Salomé; Boggavarapu, Rajendra; Ucurum, Zöhre; Gertsch, Jürg; Fotiadis, Dimitrios

    2014-04-15

    Purified membrane proteins are ternary complexes consisting of protein, lipid, and detergent. Information about the amounts of detergent and endogenous phospholipid molecules bound to purified membrane proteins is largely lacking. In this systematic study, three model membrane proteins of different oligomeric states were purified in nine different detergents at commonly used concentrations and characterized biochemically and biophysically. Detergent-binding capacities and phospholipid contents of the model proteins were determined and compared. The insights on ternary complexes obtained from the experimental results, when put into a general context, are summarized as follows. 1), The amount of detergent and 2) the amount of endogenous phospholipids bound to purified membrane proteins are dependent on the size of the hydrophobic lipid-accessible protein surface areas and the physicochemical properties of the detergents used. 3), The size of the detergent and lipid belt surrounding the hydrophobic lipid-accessible surface of purified membrane proteins can be tuned by the appropriate choice of detergent. 4), The detergents n-nonyl-β-D-glucopyranoside and Cymal-5 have exceptional delipidating effects on ternary complexes. 5), The types of endogenous phospholipids bound to membrane proteins can vary depending on the detergent used for solubilization and purification. 6), Furthermore, we demonstrate that size-exclusion chromatography can be a suitable method for estimating the molecular mass of ternary complexes. The findings presented suggest a strategy to control and tune the numbers of detergent and endogenous phospholipid molecules bound to membrane proteins. These two parameters are potentially important for the successul crystallization of membrane proteins for structure determination by crystallographic approaches.

  20. Fatty acid binding sites of human and bovine albumins: Differences observed by spin probe ESR

    NASA Astrophysics Data System (ADS)

    Muravsky, Vladimir; Gurachevskaya, Tatjana; Berezenko, Stephen; Schnurr, Kerstin; Gurachevsky, Andrey

    2009-09-01

    Bovine and human serum albumins and recombinant human albumin, all non-covalently complexed with 5- and 16-doxyl stearic acids, were investigated by ESR spectroscopy in solution over a range of pH values (5.5-8.0) and temperatures (25-50 °C), with respect to the allocation and mobility of fatty acid (FA) molecules bound to the proteins and conformation of the binding sites. In all proteins bound FA undergo a permanent intra-albumin migration between the binding sites and inter-domain residence. Nature identity of the recombinant human albumin to its serum-derived analog was observed. However, the binding sites of bovine albumin appeared shorter in length and wider in diameter than those of human albumin. Presumably, less tightly folded domains in bovine albumin allow better penetration of water molecules in the interior of the globule that resulted in higher activation energy of FA dissociation from the binding site. Thus, the sensitive technique based on ESR non-covalent spin labeling allowed quantitative analysis and reliable comparison of the fine features of binding proteins.

  1. SAAMBE: Webserver to Predict the Charge of Binding Free Energy Caused by Amino Acids Mutations

    PubMed Central

    Petukh, Marharyta; Dai, Luogeng; Alexov, Emil

    2016-01-01

    Predicting the effect of amino acid substitutions on protein–protein affinity (typically evaluated via the change of protein binding free energy) is important for both understanding the disease-causing mechanism of missense mutations and guiding protein engineering. In addition, researchers are also interested in understanding which energy components are mostly affected by the mutation and how the mutation affects the overall structure of the corresponding protein. Here we report a webserver, the Single Amino Acid Mutation based change in Binding free Energy (SAAMBE) webserver, which addresses the demand for tools for predicting the change of protein binding free energy. SAAMBE is an easy to use webserver, which only requires that a coordinate file be inputted and the user is provided with various, but easy to navigate, options. The user specifies the mutation position, wild type residue and type of mutation to be made. The server predicts the binding free energy change, the changes of the corresponding energy components and provides the energy minimized 3D structure of the wild type and mutant proteins for download. The SAAMBE protocol performance was tested by benchmarking the predictions against over 1300 experimentally determined changes of binding free energy and a Pearson correlation coefficient of 0.62 was obtained. How the predictions can be used for discriminating disease-causing from harmless mutations is discussed. The webserver can be accessed via http://compbio.clemson.edu/saambe_webserver/. PMID:27077847

  2. SAAMBE: Webserver to Predict the Charge of Binding Free Energy Caused by Amino Acids Mutations.

    PubMed

    Petukh, Marharyta; Dai, Luogeng; Alexov, Emil

    2016-04-12

    Predicting the effect of amino acid substitutions on protein-protein affinity (typically evaluated via the change of protein binding free energy) is important for both understanding the disease-causing mechanism of missense mutations and guiding protein engineering. In addition, researchers are also interested in understanding which energy components are mostly affected by the mutation and how the mutation affects the overall structure of the corresponding protein. Here we report a webserver, the Single Amino Acid Mutation based change in Binding free Energy (SAAMBE) webserver, which addresses the demand for tools for predicting the change of protein binding free energy. SAAMBE is an easy to use webserver, which only requires that a coordinate file be inputted and the user is provided with various, but easy to navigate, options. The user specifies the mutation position, wild type residue and type of mutation to be made. The server predicts the binding free energy change, the changes of the corresponding energy components and provides the energy minimized 3D structure of the wild type and mutant proteins for download. The SAAMBE protocol performance was tested by benchmarking the predictions against over 1300 experimentally determined changes of binding free energy and a Pearson correlation coefficient of 0.62 was obtained. How the predictions can be used for discriminating disease-causing from harmless mutations is discussed. The webserver can be accessed via http://compbio.clemson.edu/saambe_webserver/.

  3. Recent insights into the biological functions of liver fatty acid binding protein 1

    PubMed Central

    Wang, GuQi; Bonkovsky, Herbert L.; de Lemos, Andrew; Burczynski, Frank J.

    2015-01-01

    Over four decades have passed since liver fatty acid binding protein (FABP)1 was first isolated. There are few protein families for which most of the complete tertiary structures, binding properties, and tissue occurrences are described in such detail and yet new functions are being uncovered for this protein. FABP1 is known to be critical for fatty acid uptake and intracellular transport and also has an important role in regulating lipid metabolism and cellular signaling pathways. FABP1 is an important endogenous cytoprotectant, minimizing hepatocyte oxidative damage and interfering with ischemia-reperfusion and other hepatic injuries. The protein may be targeted for metabolic activation through the cross-talk among many transcriptional factors and their activating ligands. Deficiency or malfunction of FABP1 has been reported in several diseases. FABP1 also influences cell proliferation during liver regeneration and may be considered as a prognostic factor for hepatic surgery. FABP1 binds and modulates the action of many molecules such as fatty acids, heme, and other metalloporphyrins. The ability to bind heme is another cytoprotective property and one that deserves closer investigation. The role of FABP1 in substrate availability and in protection from oxidative stress suggests that FABP1 plays a pivotal role during intracellular bacterial/viral infections by reducing inflammation and the adverse effects of starvation (energy deficiency). PMID:26443794

  4. Improved binding of acidic bone matrix proteins to cationized filters during solid phase assays.

    PubMed

    Farach-Carson, M C; Wright, G C; Butler, W T

    1992-01-01

    A number of commercially available matrix filter supports have been designed for the immobilization of proteins following either electrotransfer from sodium dodecyl sulfate (SDS) polyacrylamide gels or direct application during dot blotting assays. These matrices differ with respect to chemical composition, charge, pore size, and degree of hydrophobicity. It follows that the properties of the protein(s) of interest will greatly influence the degree to which they interact with and ultimately bind to various filters. Acidic bone proteins contain diverse post-translational modifications that influence their interactions with solid phase matrices such as those used in immunoblotting (Western or dot blotting) or ion binding (overlay) procedures. This communication describes the results of a study comparing binding of various mixtures of non-collagenous acidic bone matrix phosphoproteins as well as purified osteopontin and osteocalcin to various filters including nitrocellulose and cationized paper or nylon. Based on our findings, we recommend the use of cationized filters for solid phase assays requiring the binding of these acidic macromolecules to background supports.

  5. IgE binding to peanut allergens is inhibited by combined D-aspartic and D-glutamic acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    D-amino acids (D-aas) are reported to bind to IgE antibodies from people with allergy and asthma. The objectives of this study were to determine if D-aas bind or inhibit IgE binding to peanut allergens, and if they are more effective than L-amino acids (L-aas) in this respect. Several D-aa cocktails...

  6. Kinematic variables and blood Acid-base status in the analysis of collegiate swimmers' anaerobic capacity.

    PubMed

    Bielec, G; Makar, P; Laskowski, R; Olek, R A

    2013-09-01

    Short duration repeated maximal efforts are often used in swimming training to improve lactate tolerance, which gives swimmers the ability to maintain a high work rate for a longer period of time. The aim of the study was to examine the kinematics of swimming and its relation to the changes in blood acid-base status and potassium level. Seven collegiate swimmers, with at least 6 years of training experience, volunteered to participate in the study. The test consisted of 8 x 25 m front crawl performed with maximum effort. The rest period between repetitions was set to five seconds. Blood samples were taken from the fingertip at rest, after warm-up and in the 3rd minute after completion of the test. The swimming was recorded with a video recorder, for later analysis of time, velocity and technique (stroke index). Based on the swimming velocity results, the obtained curve can be divided into rapid decrease of velocity and relatively stable velocities. The breaking point of repetition in swimming velocity was assumed as the swimming velocity threshold and it was highly correlated with the decrease of the blood acid-base status (pH r=0.82, BE r=0.87, HCO3 (-) r=0.76; p<0.05 in all cases). There was no correlation between stroke index or fatigue index and blood acid-base status. Analysis of the swimming speed in the 8 x 25 m test seems to be helpful in evaluation of lactate tolerance (anaerobic capacity) in collegiate swimmers.

  7. Genetic effects of sterol regulatory element binding proteins and fatty acid-binding protein4 on the fatty acid composition of Korean cattle (Hanwoo)

    PubMed Central

    Oh, Dong-Yep; Lee, Jea-Young; Jang, Ji-Eun; Lee, Seung-Uk

    2017-01-01

    Objective This study identifies single-nucleotide polymorphisms (SNP) or gene combinations that affect the flavor and quality of Korean cattle (Hanwoo) by using the SNP Harvester method. Methods Four economic traits (oleic acid [C18:1], saturated fatty acids), monounsaturated fatty acids, and marbling score) were adjusted for environmental factors in order to focus solely on genetic effects. The SNP Harvester method was used to investigate gene combinations (two-way gene interactions) associated with these economic traits. Further, a multifactor dimensionality reduction method was used to identify superior genotypes in gene combinations. Results Table 3 to 4 show the analysis results for differences between superior genotypes and others for selected major gene combinations using the multifactor dimensionality reduction method. Environmental factors were adjusted for in order to evaluate only the genetic effect. Table 5 shows the adjustment effect by comparing the accuracy before and after correction in two-way gene interactions. Conclusion The g.3977-325 T>C and (g.2988 A>G, g.3977-325 T>C) combinations of fatty acid-binding protein4 were the superior gene, and the superior genotype combinations across all economic traits were the CC genotype at g.3977-325 T>C and the AACC, GACC, GGCC genotypes of (g.2988 A>G, g.3977-325 T>C). PMID:27492349

  8. Biochemical Roles for Conserved Residues in the Bacterial Fatty Acid-binding Protein Family*

    PubMed Central

    Broussard, Tyler C.; Miller, Darcie J.; Jackson, Pamela; Nourse, Amanda; White, Stephen W.; Rock, Charles O.

    2016-01-01

    Fatty acid kinase (Fak) is a ubiquitous Gram-positive bacterial enzyme consisting of an ATP-binding protein (FakA) that phosphorylates the fatty acid bound to FakB. In Staphylococcus aureus, Fak is a global regulator of virulence factor transcription and is essential for the activation of exogenous fatty acids for incorporation into phospholipids. The 1.2-Å x-ray structure of S. aureus FakB2, activity assays, solution studies, site-directed mutagenesis, and in vivo complementation were used to define the functions of the five conserved residues that define the FakB protein family (Pfam02645). The fatty acid tail is buried within the protein, and the exposed carboxyl group is bound by a Ser-93-fatty acid carboxyl-Thr-61-His-266 hydrogen bond network. The guanidinium of the invariant Arg-170 is positioned to potentially interact with a bound acylphosphate. The reduced thermal denaturation temperatures of the T61A, S93A, and H266A FakB2 mutants illustrate the importance of the hydrogen bond network in protein stability. The FakB2 T61A, S93A, and H266A mutants are 1000-fold less active in the Fak assay, and the R170A mutant is completely inactive. All FakB2 mutants form FakA(FakB2)2 complexes except FakB2(R202A), which is deficient in FakA binding. Allelic replacement shows that strains expressing FakB2 mutants are defective in fatty acid incorporation into phospholipids and virulence gene transcription. These conserved residues are likely to perform the same critical functions in all bacterial fatty acid-binding proteins. PMID:26774272

  9. Polymerization and nucleic acid-binding properties of human L1 ORF1 protein

    PubMed Central

    Callahan, Kathryn E.; Hickman, Alison B.; Jones, Charles E.; Ghirlando, Rodolfo; Furano, Anthony V.

    2012-01-01

    The L1 (LINE 1) retrotransposable element encodes two proteins, ORF1p and ORF2p. ORF2p is the L1 replicase, but the role of ORF1p is unknown. Mouse ORF1p, a coiled-coil-mediated trimer of ∼42-kDa monomers, binds nucleic acids and has nucleic acid chaperone activity. We purified human L1 ORF1p expressed in insect cells and made two findings that significantly advance our knowledge of the protein. First, in the absence of nucleic acids, the protein polymerizes under the very conditions (0.05 M NaCl) that are optimal for high (∼1 nM)-affinity nucleic acid binding. The non-coiled-coil C-terminal half mediates formation of the polymer, an active conformer that is instantly resolved to trimers, or multimers thereof, by nucleic acid. Second, the protein has a biphasic effect on mismatched double-stranded DNA, a proxy chaperone substrate. It protects the duplex from dissociation at 37°C before eventually melting it when largely polymeric. Therefore, polymerization of ORF1p seemingly affects its interaction with nucleic acids. Additionally, polymerization of ORF1p at its translation site could explain the heretofore-inexplicable phenomenon of cis preference—the favored retrotransposition of the actively translated L1 transcript, which is essential for L1 survival. PMID:21937507

  10. The endothelial cell binding determinant of human factor IX resides in the. gamma. -carboxyglutamic acid domain

    SciTech Connect

    Toomey, J.R.; Roberts, H.R.; Stafford, D.W. ); Smith, K.J. United Blood Services, Albuquerque, NM )

    1992-02-18

    The blood coagulation factor IX(a) binds specifically to a site on endothelial cells with a K{sub d} of 2.0-3.0 nM. A number of previous studies have attempted to define the region(s) of factor IX(a) that mediate this interaction. These studies suggested that there are two regions of factor IX(a), the {gamma}-carboxyglutamic acid (Gla) domain and the epidermal growth factor like (EGF-like) domains, that mediate high-affinity binding to endothelial cells. Recently, however, the participation of the EGF1 domain has been excluded from the interaction. This indicated that if there was an EGF component of factor IX contributing to the binding affinity, then it must be in the second EGF-like domain. In order to further evaluate this relationship, the authors performed competitive binding experiments between {sup 125}I plasma factor IX and a set of six chimeric proteins composed of portions of factor VII and factor IX. The data suggest that the high-affinity interaction between factor IX and the endothelial cell binding site is mediated by the factor IX Gla domain and that the factor IX EGF domains are not involved in binding specificity.

  11. Three Dimensional Structure Prediction of Fatty Acid Binding Site on Human Transmembrane Receptor CD36.

    PubMed

    Tarhda, Zineb; Semlali, Oussama; Kettani, Anas; Moussa, Ahmed; Abumrad, Nada A; Ibrahimi, Azeddine

    2013-01-01

    CD36 is an integral membrane protein which is thought to have a hairpin-like structure with alpha-helices at the C and N terminals projecting through the membrane as well as a larger extracellular loop. This receptor interacts with a number of ligands including oxidized low density lipoprotein and long chain fatty acids (LCFAs). It is also implicated in lipid metabolism and heart diseases. It is therefore important to determine the 3D structure of the CD36 site involved in lipid binding. In this study, we predict the 3D structure of the fatty acid (FA) binding site [127-279 aa] of the CD36 receptor based on homology modeling with X-ray structure of Human Muscle Fatty Acid Binding Protein (PDB code: 1HMT). Qualitative and quantitative analysis of the resulting model suggests that this model was reliable and stable, taking in consideration over 97.8% of the residues in the most favored regions as well as the significant overall quality factor. Protein analysis, which relied on the secondary structure prediction of the target sequence and the comparison of 1HMT and CD36 [127-279 aa] secondary structures, led to the determination of the amino acid sequence consensus. These results also led to the identification of the functional sites on CD36 and revealed the presence of residues which may play a major role during ligand-protein interactions.

  12. Three Dimensional Structure Prediction of Fatty Acid Binding Site on Human Transmembrane Receptor CD36

    PubMed Central

    Tarhda, Zineb; Semlali, Oussama; Kettani, Anas; Moussa, Ahmed; Abumrad, Nada A.; Ibrahimi, Azeddine

    2013-01-01

    CD36 is an integral membrane protein which is thought to have a hairpin-like structure with alpha-helices at the C and N terminals projecting through the membrane as well as a larger extracellular loop. This receptor interacts with a number of ligands including oxidized low density lipoprotein and long chain fatty acids (LCFAs). It is also implicated in lipid metabolism and heart diseases. It is therefore important to determine the 3D structure of the CD36 site involved in lipid binding. In this study, we predict the 3D structure of the fatty acid (FA) binding site [127–279 aa] of the CD36 receptor based on homology modeling with X-ray structure of Human Muscle Fatty Acid Binding Protein (PDB code: 1HMT). Qualitative and quantitative analysis of the resulting model suggests that this model was reliable and stable, taking in consideration over 97.8% of the residues in the most favored regions as well as the significant overall quality factor. Protein analysis, which relied on the secondary structure prediction of the target sequence and the comparison of 1HMT and CD36 [127–279 aa] secondary structures, led to the determination of the amino acid sequence consensus. These results also led to the identification of the functional sites on CD36 and revealed the presence of residues which may play a major role during ligand-protein interactions. PMID:24348024

  13. Recent Advances in Nucleic Acid Binding Aspects of Berberine Analogs and Implications for Drug Design.

    PubMed

    Bhowmik, Debipreeta; Kumar, Gopinatha Suresh

    2016-01-01

    Berberine is one of the most widely known alkaloids belonging to the protoberberine group exhibiting myriad therapeutic properties. The anticancer potency of berberine appears to derive from its multiple actions including strong interaction with nucleic acids exhibiting adenine-thymine base pair specificity, inhibition of the enzymes topoisomerases and telomerases, and stabilizing the quadruplex structures. It was realized that the development of berberine as a potential anticancer agent necessitates enhancing its nucleic acid binding efficacy through appropriate structural modifications. More recently a number of such approaches have been attempted in various laboratories with great success. Several derivatives have been synthesized mostly with substitutions at the 8, 9 and 13 positions of the isoquinoline chromophore, and studied for enhanced nucleic acid binding activity. In this article, we present an up to date review of the details of the interaction of berberine and several of its important synthetic 8, 9 and 13 substituted derivatives with various nucleic acid structures reported recently. These studies provide interesting knowledge on the mode, mechanism, sequence and structural specificity of the binding of berberine derivatives and correlate structural and energetic aspects of the interaction providing better understanding of the structure- activity relations for designing and development of berberine based therapeutic agents with higher efficacy and therapeutic potential.

  14. Cyclic Phosphopeptides to Rationalize the Role of Phosphoamino Acids in Uranyl Binding to Biological Targets.

    PubMed

    Starck, Matthieu; Laporte, Fanny A; Oros, Stephane; Sisommay, Nathalie; Gathu, Vicky; Solari, Pier Lorenzo; Creff, Gaëlle; Roques, Jérôme; Den Auwer, Christophe; Lebrun, Colette; Delangle, Pascale

    2017-02-05

    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.

  15. Molecular Dynamics Simulation of Ligand Dissociation from Liver Fatty Acid Binding Protein

    PubMed Central

    Long, Dong; Mu, Yuguang; Yang, Daiwen

    2009-01-01

    The mechanisms of how ligands enter and leave the binding cavity of fatty acid binding proteins (FABPs) have been a puzzling question over decades. Liver fatty acid binding protein (LFABP) is a unique family member which accommodates two molecules of fatty acids in its cavity and exhibits the capability of interacting with a variety of ligands with different chemical structures and properties. Investigating the ligand dissociation processes of LFABP is thus a quite interesting topic, which however is rather difficult for both experimental approaches and ordinary simulation strategies. In the current study, random expulsion molecular dynamics simulation, which accelerates ligand motions for rapid dissociation, was used to explore the potential egress routes of ligands from LFABP. The results showed that the previously hypothesized “portal region” could be readily used for the dissociation of ligands at both the low affinity site and the high affinity site. Besides, one alternative portal was shown to be highly favorable for ligand egress from the high affinity site and be related to the unique structural feature of LFABP. This result lends strong support to the hypothesis from the previous NMR exchange studies, which in turn indicates an important role for this alternative portal. Another less favored potential portal located near the N-terminal end was also identified. Identification of the dissociation pathways will allow further mechanistic understanding of fatty acid uptake and release by computational and/or experimental techniques. PMID:19564911

  16. Regulation of GABA-modulin phosphorylation and GABA receptor binding by excitatory amino acids

    SciTech Connect

    Vaccarino, F.; Guidotti, A.

    1987-05-01

    Primary cultures of cerebellar granule cells phosphorylate numerous proteins including GABA-modulin (GM), which is a putative allosteric modulator of GABA receptors. Cell depolarization and treatment with dicarboxylic excitatory amino acids, which activate PI turnover, Ca/sup 2 +/ influx and guanylate cyclase in granule cells increase the phosphorylation of specific proteins. To determine GM phosphorylation by endogenous protein kinases in living granule cell cultures, GM was isolated by immunoprecipitation and reverse-phase HPLC. High K/sup +/, veratridine, glutamate and NMDA treatment stimulated GM phosphorylation over 2-fold. This increase was abolished by the absence of extracellular Ca/sup 2 +/ and was antagonized by Mg/sup 2 +/ ions and by AVP. The excitatory amino acid action was mimicked by phorbol esters but not by forskolin or by cGMP, and thus may be mediated by an activation of protein kinase C (PKC). Moreover, excitatory amino acids increase /sup 3/H-labelled phorbol ester binding sites in granule cell membrane. The same cultures, treated with glutamate or kainate, showed a 50-fold greater efficacy of muscimol for the stimulation of benzodiazepine (BZ) binding. These data-suggest that excitatory amino acid stimulation of neurons triggers PKC translocation and the activated enzyme phosphorylates GM. The extent of GM phosphorylation may regulate the coupling between GABA and BZ binding sites.

  17. Short-duration intermittent hypoxia enhances endurance capacity by improving muscle fatty acid metabolism in mice.

    PubMed

    Suzuki, Junichi

    2016-04-01

    This study was designed to (1) investigate the effects of acute short-duration intermittent hypoxia on musclemRNAand microRNAexpression levels; and (2) clarify the mechanisms by which short-duration intermittent hypoxia improves endurance capacity. Experiment-1: Male mice were subjected to either acute 1-h hypoxia (12% O2), acute short-duration intermittent hypoxia (12% O2for 15 min, room air for 10 min, 4 times, Int-Hypo), or acute endurance exercise (Ex). The expression of vascular endothelial growth factor-AmRNAwas significantly greater than the control at 0 h post Ex and 6 h post Int-Hypo in the deep red region of the gastrocnemius muscle. miR-16 expression levels were significantly lower at 6 and 10 h post Int-Hypo. Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α)mRNAlevels were significantly greater than the control at 3 h post Ex and 6 h post Int-Hypo. miR-23a expression levels were lower than the control at 6-24 h post Int-Hypo. Experiment-2: Mice were subjected to normoxic exercise training with or without intermittent hypoxia for 3 weeks. Increases in maximal exercise capacity were significantly greater by training with short-duration intermittent hypoxia (IntTr) than without hypoxia. Both 3-Hydroxyacyl-CoA-dehydrogenase and total carnitine palmitoyl transferase activities were significantly enhanced in IntTr. Peroxisome proliferator-activated receptor delta andPGC-1α mRNAlevels were both significantly greater in IntTr than in the sedentary controls. These results suggest that exercise training under normoxic conditions with exposure to short-duration intermittent hypoxia represents a beneficial strategy for increasing endurance performance by enhancing fatty acid metabolism in skeletal muscle.

  18. Combined spectroscopy and molecular modeling studies on the binding of galbanic acid and MMP9.

    PubMed

    Kiani, Amir; Almasi, Khadijeh; Shokoohinia, Yalda; Sadrjavadi, Komail; Nowroozi, Amin; Shahlaei, Mohsen

    2015-11-01

    The molecular mechanism of galbanic acid (GBA) binding to matrix metalloproteinase 9 (MMP9) was investigated by fluorescence quenching, absorption spectroscopy, FT-IR, molecular docking and molecular dynamics (MD) simulation procedures. The fluorescence emission of MMP9 was quenched by GBA. The titration of MMP9 by various amount of GBA was also followed by UV-Vis absorption spectroscopy. The results revealed that GBA, as a biologically active sesquiterpene coumarin derivative, has an ability to bind strongly to MMP9. Molecular docking results indicated that the main active binding site for GBA has been located in a hydrophobic cavity in the vicinity of Zn atom. Moreover, MD simulation results suggested that GBA as a coumarin derivative can interact with MMP9, without affecting the secondary structure of MMP9. MD simulations, molecular docking as computational methods from one hand and experimental data from other hand reciprocally supported each other.

  19. BEDAM Binding Free Energy Predictions for the SAMPL4 Octa-Acid Host Challenge

    PubMed Central

    Gallicchio, Emilio; Chen, Haoyuan; Chen, He; Fitzgerald, Michael; Gao, Yang; He, Peng; Kalyanikar, Malathi; Kao, Chuan; Lu, Beidi; Niu, Yijie; Pethe, Manasi; Zhu, Jie; Levy, Ronald M.

    2015-01-01

    The Binding Energy Distribution Analysis Method (BEDAM) protocol has been employed as part of the SAMPL4 blind challenge to predict the binding free energies of a set of octa-acid host-guest complexes. The resulting predictions were consistently judged as some of the most accurate predictions in this category of the SAMPL4 challenge in terms of quantitative accuracy and statistical correlation relative to the experimental values, which were not known at the time the predictions were made. The work has been conducted as part of a hands-on graduate class laboratory session. Collectively the students, aided by automated setup and analysis tools, performed the bulk of the calculations and the numerical and structural analysis. The success of the experiment confirms the reliability of the BEDAM methodology and it shows that physics-based atomistic binding free energy estimation models, when properly streamlined and automated, can be successfully employed by non-specialists. PMID:25726024

  20. A report on emergent uranyl binding phenomena by an amidoxime phosphonic acid co-polymer

    SciTech Connect

    Abney, C. W.; Das, S.; Mayes, R. T.; Kuo, L. -J.; Wood, J.; Gill, G.; Piechowicz, M.; Lin, Z.; Lin, W.; Dai, S.

    2016-01-01

    The development of technology to harvest the uranium dissolved in seawater would enable access to vast quantities of this critical metal for nuclear power generation. Amidoxime polymers are the most promising platforms for achieving this separation, yet the design of advanced adsorbents is hindered by uncertainty regarding the uranium binding mode. In this work we use XAFS to investigate the uranium coordination environment in an amidoxime–phosphonic acid copolymer adsorbent. In contrast to the binding mode predicted computationally and from small molecule studies, a cooperative chelating model is favoured, attributable to emergent behavior resulting from inclusion of amidoxime in the polymer. Samples exposed to seawater also display a feature consistent with a μ2-oxo-bridged transition metal, suggesting the formation of an in situ specific binding site. These findings challenge long held assumptions and provide new opportunities for the design of advanced adsorbent materials.

  1. A report on emergent uranyl binding phenomena by an amidoxime phosphonic acid co-polymer

    DOE PAGES

    Abney, C. W.; Das, S.; Mayes, R. T.; ...

    2016-08-01

    Development of technology to harvest the uranium dissolved in seawater would enable access to vast quantities of this critical metal for nuclear power generation. Amidoxime polymers are the most promising platform for achieving this separation, yet design of advanced adsorbents is hindered by uncertainty regarding the uranium binding mode. In this work we use XAFS to investigate the uranium coordination environment in an amidoxime-phosphonic acid copolymer adsorbent. In contrast to the binding mode predicted computationally and from small molecule studies, a cooperative chelating model is favoured, attributable to emergent behavior resulting from inclusion of amidoxime in a polymer. Samples exposedmore » to seawater also display a feature consistent with a 2-oxo-bridged transition metal, suggesting formation of an in situ specific binding site. As a result, these findings challenge long held assumptions and provide new opportunities for the design of advanced adsorbent materials.« less

  2. A report on emergent uranyl binding phenomena by an amidoxime phosphonic acid co-polymer

    SciTech Connect

    Abney, C. W.; Das, S.; Mayes, R. T.; Kuo, L. -J.; Wood, J.; Gill, G.; Piechowicz, M.; Lin, Z.; Lin, W.; Dai, S.

    2016-08-01

    Development of technology to harvest the uranium dissolved in seawater would enable access to vast quantities of this critical metal for nuclear power generation. Amidoxime polymers are the most promising platform for achieving this separation, yet design of advanced adsorbents is hindered by uncertainty regarding the uranium binding mode. In this work we use XAFS to investigate the uranium coordination environment in an amidoxime-phosphonic acid copolymer adsorbent. In contrast to the binding mode predicted computationally and from small molecule studies, a cooperative chelating model is favoured, attributable to emergent behavior resulting from inclusion of amidoxime in a polymer. Samples exposed to seawater also display a feature consistent with a 2-oxo-bridged transition metal, suggesting formation of an in situ specific binding site. As a result, these findings challenge long held assumptions and provide new opportunities for the design of advanced adsorbent materials.

  3. Amino Acid Derivatives as New Zinc Binding Groups for the Design of Selective Matrix Metalloproteinase Inhibitors

    PubMed Central

    Giustiniano, Mariateresa; Agamennone, Mariangela; Rossello, Armando; Gomez-Monterrey, Isabel; Novellino, Ettore; Campiglia, Pietro; Vernieri, Ermelinda; Bertamino, Alessia; Carotenuto, Alfonso

    2013-01-01

    A number of matrix metalloproteinases (MMPs) are important medicinal targets for conditions ranging from rheumatoid arthritis to cardiomyopathy, periodontal disease, liver cirrhosis, multiple sclerosis, and cancer invasion and metastasis, where they showed to have a dual role, inhibiting or promoting important processes involved in the pathology. MMPs contain a zinc (II) ion in the protein active site. Small-molecule inhibitors of these metalloproteins are designed to bind directly to the active site metal ions. In an effort to devise new approaches to selective inhibitors, in this paper, we describe the synthesis and preliminary biological evaluation of amino acid derivatives as new zinc binding groups (ZBGs). The incorporation of selected metal-binding functions in more complex biphenyl sulfonamide moieties allowed the identification of one compound able to interact selectively with different MMP enzymatic isoforms. PMID:23555050

  4. Identification of cytosolic and microsomal bile acid-binding proteins in rat ileal enterocytes

    SciTech Connect

    Lin, M.C.; Kramer, W.; Wilson, F.A. )

    1990-09-05

    Studies were performed to determine the subcellular fractions and proteins involved in the intracellular transport of bile acids in rat ileal cells. The photolabile derivative 7,7-azo-taurocholate inhibited the Na(+)-dependent uptake of taurocholate into rat ileal enterocytes reversibly in the dark and irreversibly following photolysis. When photolabeled cells were submitted to subcellular fractionation, greatest radioactivity was found in the soluble protein (SP) fraction with decreasing radioactivity in the brush-border-(BBM), basolateral-(BLM), mitochondria-(MT), microsome-(MC), and Golgi-(GO) enriched fractions. Following trichloroacetic acid precipitation, delipidation, and correction for loss of marker enzyme activity, protein bound radioactivity was in SP greater than BBM greater than MC greater than BLM greater than GO greater than MT. When photolabeled cells were first fractionated and then submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a 99-kDa polypeptide was associated with BBM, 54- and 59-kDa polypeptides with BLM, 14-, 35-, 43-, 59-, and 68-kDa polypeptides with SP and a 20-kDa polypeptide with MC fractions. Immunoprecipitation with known antisera identified the 68-kDa polypeptide as albumin and the 43-kDa polypeptide as actin. No precipitation on the 14-kDa polypeptide was noted with anti-hepatic and anti-intestinal fatty acid-binding proteins. No precipitation of the 35-kDa polypeptide occurred with antibody to the hepatic cytosolic bile acid-binding protein. These studies reveal a previously unrecognized 20-kDa microsomal, and 14- and 35-kDa cytosolic bile acid-binding polypeptides which may be involved in the transcellular movement of bile acids.

  5. Absolute binding free energies for octa-acids and guests in SAMPL5

    NASA Astrophysics Data System (ADS)

    Tofoleanu, Florentina; Lee, Juyong; Pickard, Frank C., IV; König, Gerhard; Huang, Jing; Baek, Minkyung; Seok, Chaok; Brooks, Bernard R.

    2017-01-01

    As part of the SAMPL5 blind prediction challenge, we calculate the absolute binding free energies of six guest molecules to an octa-acid (OAH) and to a methylated octa-acid (OAMe). We use the double decoupling method via thermodynamic integration (TI) or Hamiltonian replica exchange in connection with the Bennett acceptance ratio (HREM-BAR). We produce the binding poses either through manual docking or by using GalaxyDock-HG, a docking software developed specifically for this study. The root mean square deviations for our most accurate predictions are 1.4 kcal mol-1 for OAH with TI and 1.9 kcal mol-1 for OAMe with HREM-BAR. Our best results for OAMe were obtained for systems with ionic concentrations corresponding to the ionic strength of the experimental solution. The most problematic system contains a halogenated guest. Our attempt to model the σ-hole of the bromine using a constrained off-site point charge, does not improve results. We use results from molecular dynamics simulations to argue that the distinct binding affinities of this guest to OAH and OAMe are due to a difference in the flexibility of the host. We believe that the results of this extensive analysis of host-guest complexes will help improve the protocol used in predicting binding affinities for larger systems, such as protein-substrate compounds.

  6. Zinc complexes of the antibacterial drug oxolinic acid: structure and DNA-binding properties.

    PubMed

    Tarushi, Alketa; Psomas, George; Raptopoulou, Catherine P; Kessissoglou, Dimitris P

    2009-06-01

    The neutral mononuclear zinc complexes with the quinolone antibacterial drug oxolinic acid in the absence or presence of a nitrogen donor heterocyclic ligand 2,2'-bipyridine or 1,10-phenanthroline have been synthesized and characterized. The experimental data suggest that oxolinic acid is on deprotonated mode acting as a bidentate ligand coordinated to the metal ion through the ketone and one carboxylato oxygen atoms. The crystal structures of (chloro)(oxolinato)(2,2'-bipyridine)zinc(II), 2, and bis(oxolinato)(1,10-phenanthroline)zinc(II), 3, have been determined with X-ray crystallography. The biological activity of the complexes has been evaluated by examining their ability to bind to calf-thymus DNA (CT DNA) with UV and fluorescence spectroscopies. UV studies of the interaction of the complexes with DNA have shown that they can bind to CT DNA and the DNA-binding constants have been calculated. Competitive studies with ethidium bromide (EB) have shown that complex 3 exhibits the ability to displace the DNA-bound EB indicating that it binds to DNA in strong competition with EB.

  7. Diverse roles of the nucleic acid binding protein KHSRP in cell differentiation and disease

    PubMed Central

    Briata, Paola; Bordo, Domenico; Puppo, Margherita; Gorlero, Franco; Rossi, Martina; Bizzozzero, Nora Perrone; Gherzi, Roberto

    2015-01-01

    The single-stranded nucleic acid binding protein KHSRP (KH-Type Splicing Regulatory Protein) modulates RNA life and gene expression at various levels. KHSRP controls important cellular functions as different as proliferation, differentiation, metabolism and response to infectious agents. We summarize and discuss experimental evidence providing a potential link between changes in KHSRP expression/function and human diseases including neuromuscular disorders, obesity, type II diabetes, and cancer. PMID:26708421

  8. Diverse roles of the nucleic acid-binding protein KHSRP in cell differentiation and disease.

    PubMed

    Briata, Paola; Bordo, Domenico; Puppo, Margherita; Gorlero, Franco; Rossi, Martina; Perrone-Bizzozero, Nora; Gherzi, Roberto

    2016-01-01

    The single-stranded nucleic acid-binding protein KHSRP (KH-type splicing regulatory protein) modulates RNA life and gene expression at various levels. KHSRP controls important cellular functions as different as proliferation, differentiation, metabolism, and response to infectious agents. We summarize and discuss experimental evidence providing a potential link between changes in KHSRP expression/function and human diseases including neuromuscular disorders, obesity, type II diabetes, and cancer.

  9. Urinary liver-type fatty acid-binding protein change in gestational diabetes mellitus.

    PubMed

    Fu, Wen-Jin; Wang, Du-Juan; Deng, Ren-Tang; Huang, Zhi-Hong; Chen, Mei-Lian; Jang, You-Ming; Wen, Shu; Yang, Hong-Ling; Huang, Xian-zhang

    2015-09-01

    We compared urinary liver-type fatty acid-binding protein (L-FABP) among non-pregnant and pregnant women with and without gestational diabetes mellitus (GDM). Higher urinary L-FABP was found in pregnant with and without GDM, and considerably higher urinary L-FABP was found in the GDM group compared with the non-GDM group. Hyperglycemia and anemia were related with high urinary L-FABP expression.

  10. Correlation between carbohydrate-binding specificity and amino acid sequence of carbohydrate-binding regions of Cytisus-type anti-H(O) lectins.

    PubMed

    Konami, Y; Yamamoto, K; Osawa, T; Irimura, T

    1992-06-15

    A carbohydrate-binding peptide of the di-N-acetylchitobiose-binding Cytisus sessilifolius anti-H(O) lectin I (CSA-I) was isolated from the endoproteinase Asp-N digest of CSA-I by affinity chromatography on a column of N-acetyl-D-glucosamine oligomer-Sepharose (GlcNAc oligomer-Sepharose). The amino acid sequence of the carbohydrate-binding peptide of CSA-I was determined to be DTYFGKTYNPW using a gas-phase protein sequencer. This sequence corresponds to the sequence from Asp-129 to Trp-139 based on the primary structure of CSA-I, and shows a high degree of homology to those of the putative carbohydrate-binding peptide of the Laburnum alpinum lectin I (LAA-I) (DTYFGKAYNPW) and of the Ulex europaeus lectin II (UEA-II) (DSYFGKTYNPW). The binding of these three anti-H(O) lectins is known to be inhibited by di-N-acetylchitobiose but not by L-fucose. These results strongly suggest that there is a good correlation between the carbohydrate-binding specificity and the amino acid sequence of the carbohydrate-binding regions of di-N-acetylchitobiose-binding lectins.

  11. Effects of oleic acid-induced lung injury on oxygen transport and aerobic capacity.

    PubMed

    Crocker, George H; Jones, James H

    2014-06-01

    We tested the hypothesis that oleic-acid (OA) infusion impairs gas exchange, decreases total cardiopulmonary O2 delivery and lowers maximal aerobic capacity ( [Formula: see text] ). We infused 0.05ml OAkg(-1) (∼3ml) and ∼563ml saline into the right atria of four goats [59.1±14.0 (SD) kg] prior to running them on a treadmill at [Formula: see text] 2-h and 1-d following OA-induced acute lung injury, and with no lung injury. Acute lung injury decreased [Formula: see text] , O2 delivery, arterial O2 concentration and arterial O2 partial pressure compared to no lung injury. The [Formula: see text] positively correlated with O2 delivery and inversely correlated with alveolar-arterial O2 partial pressure difference, suggesting that impaired pulmonary gas exchange decreased O2 delivery and uptake. Results indicate OA infusion may be a useful model for acutely impairing pulmonary gas exchange for exercise studies. Seven OA infusions induced smaller chronic gas exchange and arterial O2 partial pressure changes than acute infusion.

  12. Binding of [alpha, alpha]-Disubstituted Amino Acids to Arginase Suggests New Avenues for Inhibitor Design

    SciTech Connect

    Ilies, Monica; Di Costanzo, Luigi; Dowling, Daniel P.; Thorn, Katherine J.; Christianson, David W.

    2011-10-21

    Arginase is a binuclear manganese metalloenzyme that hydrolyzes L-arginine to form L-ornithine and urea, and aberrant arginase activity is implicated in various diseases such as erectile dysfunction, asthma, atherosclerosis, and cerebral malaria. Accordingly, arginase inhibitors may be therapeutically useful. Continuing our efforts to expand the chemical space of arginase inhibitor design and inspired by the binding of 2-(difluoromethyl)-L-ornithine to human arginase I, we now report the first study of the binding of {alpha},{alpha}-disubstituted amino acids to arginase. Specifically, we report the design, synthesis, and assay of racemic 2-amino-6-borono-2-methylhexanoic acid and racemic 2-amino-6-borono-2-(difluoromethyl)hexanoic acid. X-ray crystal structures of human arginase I and Plasmodium falciparum arginase complexed with these inhibitors reveal the exclusive binding of the L-stereoisomer; the additional {alpha}-substituent of each inhibitor is readily accommodated and makes new intermolecular interactions in the outer active site of each enzyme. Therefore, this work highlights a new region of the protein surface that can be targeted for additional affinity interactions, as well as the first comparative structural insights on inhibitor discrimination between a human and a parasitic arginase.

  13. Binding of α,α-disubstituted amino acids to arginase suggests new avenues for inhibitor design.

    PubMed

    Ilies, Monica; Di Costanzo, Luigi; Dowling, Daniel P; Thorn, Katherine J; Christianson, David W

    2011-08-11

    Arginase is a binuclear manganese metalloenzyme that hydrolyzes L-arginine to form L-ornithine and urea, and aberrant arginase activity is implicated in various diseases such as erectile dysfunction, asthma, atherosclerosis, and cerebral malaria. Accordingly, arginase inhibitors may be therapeutically useful. Continuing our efforts to expand the chemical space of arginase inhibitor design and inspired by the binding of 2-(difluoromethyl)-L-ornithine to human arginase I, we now report the first study of the binding of α,α-disubstituted amino acids to arginase. Specifically, we report the design, synthesis, and assay of racemic 2-amino-6-borono-2-methylhexanoic acid and racemic 2-amino-6-borono-2-(difluoromethyl)hexanoic acid. X-ray crystal structures of human arginase I and Plasmodium falciparum arginase complexed with these inhibitors reveal the exclusive binding of the L-stereoisomer; the additional α-substituent of each inhibitor is readily accommodated and makes new intermolecular interactions in the outer active site of each enzyme. Therefore, this work highlights a new region of the protein surface that can be targeted for additional affinity interactions, as well as the first comparative structural insights on inhibitor discrimination between a human and a parasitic arginase.

  14. Microbial alginate dressings show improved binding capacity for pathophysiological factors in chronic wounds compared to commercial alginate dressings of marine origin.

    PubMed

    Fischer, Melissa; Gebhard, Florian; Hammer, Timo; Zurek, Christian; Meurer, Guido; Marquardt, Christoph; Hoefer, Dirk

    2017-01-01

    Marine alginates are well established in wound management. Compared with different modern wound dressings, marine alginates cannot prove superior effects on wound healing. Alginates from bacteria have never been studied for medical applications so far, although the microbial polymer raises expectations for improved binding of wound factors because of its unique O-acetylation. Due to its possible positive effects on wound healing, alginates from bacteria might be a superior future medical product for clinical use. To prove the binding capacity of microbial alginates to pathophysiological factors in chronic wounds, we processed microbial alginate fibres, produced from fermentation of the soil bacterium Azotobacter vinelandii ATCC 9046, into needle web dressings and compared them with commercial dressings made of marine alginate. Four dressings were assessed: Marine alginate dressings containing either ionic silver or zinc/manganese/calcium, and microbial alginate dressings with and without nanosilver. All dressings were tested in an in vitro approach for influence on chronic wound parameters such as elastase, matrix metalloproteases-2, tumour necrosis factor-α, interleukin-8, and free radical formation. Despite the alginate origin or addition of antimicrobials, all dressings were able to reduce the concentration of the proinflammatory cytokines TNF-α and IL-8. However, microbial alginate was found to bind considerable larger amounts of elastase and matrix metalloproteases-2 in contrast to the marine alginate dressings. The incorporation of zinc, silver or nanosilver into alginate fibres did not improve their binding capacity for proteases or cytokines. The addition of nanosilver slightly enhanced the antioxidant capacity of microbial alginate dressings, whereas the marine alginate dressing containing zinc/manganese/calcium was unable to inhibit the formation of free radicals. The enhanced binding affinity by microbial alginate of Azotobacter vinelandii to

  15. Hyaluronic acid binding, endocytosis and degradation by sinusoidal liver endothelial cells

    SciTech Connect

    McGary, C.T.

    1988-01-01

    The binding, endocytosis, and degradation of {sup 125}I-hyaluronic acid ({sup 125}I-HA) by liver endothelial cells (LEC) was studied under several conditions. The dissociation of receptor-bound {sup 125}I-HA was rapid, with a half time of {approx}31 min and a K{sub off} of 6.3 {times} 10{sup {minus}4}/sec. A large reversible increase in {sup 125}I-HA binding to LEC at pH 5.0 was due to an increase in the observed affinity of the binding interaction. Pronase digestion suggested the protein nature of the receptor and the intracellular location of the digitonin exposed binding activity. Binding and endocytosis occur in the presence of 10 mM EGTA indicating that divalent cations are not required for receptor function. To study the degradation of {sup 125}I-HA by LEC, a cetylpyridinium chloride (CPC) precipitation assay was characterized. The minimum HA length required for precipitation was elucidated. The fate of the LEC HA receptor after endocytosis was examined.

  16. Binding modes of zaragozic acid A to human squalene synthase and staphylococcal dehydrosqualene synthase.

    PubMed

    Liu, Chia-I; Jeng, Wen-Yih; Chang, Wei-Jung; Ko, Tzu-Ping; Wang, Andrew H-J

    2012-05-25

    Zaragozic acids (ZAs) belong to a family of fungal metabolites with nanomolar inhibitory activity toward squalene synthase (SQS). The enzyme catalyzes the committed step of sterol synthesis and has attracted attention as a potential target for antilipogenic and antiinfective therapies. Here, we have determined the structure of ZA-A complexed with human SQS. ZA-A binding induces a local conformational change in the substrate binding site, and its C-6 acyl group also extends over to the cofactor binding cavity. In addition, ZA-A effectively inhibits a homologous bacterial enzyme, dehydrosqualene synthase (CrtM), which synthesizes the precursor of staphyloxanthin in Staphylococcus aureus to cope with oxidative stress. Size reduction at Tyr(248) in CrtM further increases the ZA-A binding affinity, and it reveals a similar overall inhibitor binding mode to that of human SQS/ZA-A except for the C-6 acyl group. These structures pave the way for further improving selectivity and development of a new generation of anticholesterolemic and antimicrobial inhibitors.

  17. Phosphatidic Acid Binds to Cytosolic Glyceraldehyde-3-phosphate Dehydrogenase and Promotes Its Cleavage in Arabidopsis *

    PubMed Central

    Kim, Sang-Chul; Guo, Liang; Wang, Xuemin

    2013-01-01

    Phosphatidic acid (PA) is a class of lipid messengers involved in a variety of physiological processes. To understand how PA mediates cell functions in plants, we used a PA affinity membrane assay to isolate PA-binding proteins from Camelina sativa followed by mass spectrometric sequencing. A cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC) was identified to bind to PA, and detailed analysis was carried out subsequently using GAPC1 and GAPC1 from Arabidopsis. The PA and GAPC binding was abolished by the cation zinc whereas oxidation of GAPCs promoted the PA binding. PA had little impact on the GAPC catalytic activity in vitro, but the PA treatment of Arabidopsis seedlings induced proteolytic cleavage of GAPC2 and inhibited Arabidopsis seedling growth. The extent of PA inhibition was greater in GAPC-overexpressing than wild-type seedlings, but the greater PA inhibition was abolished by application of zinc to the seedling. The PA treatment also reduced the expression of genes involved in PA synthesis and utilization, and the PA-reduced gene expression was partially recovered by zinc treatment. These data suggest that PA binds to oxidized GAPDH and promotes its cleavage and that the PA and GAPC interaction may provide a signaling link coordinating carbohydrate and lipid metabolism. PMID:23504314

  18. Study on the binding of chlorogenic acid to pepsin by spectral and molecular docking.

    PubMed

    Zeng, Hua-jin; Liang, Hui-li; You, Jing; Qu, Ling-bo

    2014-11-01

    The interaction of pepsin with chlorogenic acid (CHA) was investigated using fluorescence, UV/vis spectroscopy and molecular modeling methods. Stern-Volmer analysis indicated that the fluorescence quenching of pepsin by CHA resulted from a static mechanism, and the binding constant was 1.1846 × 10(5) and 1.1587 × 10(5) L/mol at 288 and 310 K, respectively. The distance between donor (pepsin) and acceptor (CHA) was calculated to be 2.39 nm and the number of binding sites for CHA binding on pepsin was ~ 1. The results of synchronous fluorescence and three-dimensional fluorescence showed that binding of CHA to pepsin could induce conformational changes in pepsin. Molecular docking experiments found that CHA bonded with pepsin in the area of the hydrophobic cavity with Van der Waals' forces or hydrogen bonding interaction, which were consistent with the results obtained from the thermodynamic parameter analysis. Furthermore, the binding of CHA can inhibit pepsin activity in vitro.

  19. Composition and copper binding properties of aquatic fulvic acids in eutrophic Taihu Lake, China.

    PubMed

    Li, Weiwei; Zhang, Fenfen; Ye, Qi; Wu, Dan; Wang, Liying; Yu, Yihua; Deng, Bing; Du, Jinzhou

    2017-04-01

    Fulvic acid (FA) plays a significant role in biogenic-elemental cycling in aquatic ecosystems which is highly dependent on their organic composition. In this study, the aquatic FA contents and binding properties during bloom and non-bloom periods in Taihu Lake were investigated by two-dimensional correlation spectroscopy Fourier transform infrared spectroscopy (2D-COS-FTIR), nuclear magnetic resonance (NMR) and elemental analysis. Compared with non-bloom FA, bloom FA was of lower nitrogen content and higher C/N ratio. It contained more carboxylic and aliphatic groups while less amide groups. 2D-COS-FTIR spectra evidenced the carboxyl groups in bloom FA had the fastest response to Cu(II) binding. Also, polysaccharide in bloom FA was more susceptive to Cu(II) concentrations than that in non-bloom FA. While comparing with bloom FA, the N-rich organic compounds in non-bloom FA exhibited faster binding sequence with Cu(II). A comprehensive scheme about the interaction process of FA-Cu(II) showed that both nitrogenous and oxygenic groups in FAs were active in binding to Cu(II). The alteration in binding behaviors of organic groups in FAs to Cu(II) may have been driven by algal products and microbial community variety in Taihu Lake. Our results here have the potential to contribute significantly to future studies of dissolved organic matter dynamic biogeochemistry processes and trace metal cycling processes in eutrophic lakes.

  20. Iron binding efficiency of polyphenols: Comparison of effect of ascorbic acid and ethylenediaminetetraacetic acid on catechol and galloyl groups.

    PubMed

    Tamilmani, Poonkodi; Pandey, Mohan Chandra

    2016-04-15

    Dietary polyphenols are markedly studied for their antioxidant activity. They also have a negative impact on nutrition whereby they interfere with iron absorption. Common dietary polyphenols include: catechins, flavonols, flavanols, flavones, anthocyanins, proanthocyanidins and phenolic acids. Ascorbic acid (AA) and Ethylenediaminetetraacetic acid (EDTA) are commonly used to counter act this reaction and increase iron bioavailability. This study was aimed at determining the effect of AA and EDTA on the catechol or galloyl iron binding ability of pure phenolics, coffee and tea. Phenolic concentrations of 40, 80, 610, 240, 320, 400, 520 and 900 μg/ml were tested against six levels of AA and EDTA. These effects were studied in detail using Multivariate Analysis of Variance (MANOVA) with the hypothesis that there would be one or more mean differences between the ratio of enhancer and the different concentrations of samples tested. AA was found to be more efficient than EDTA in a way that lesser quantity is required for completely overcoming negative iron binding effects of polyphenols and similar samples.

  1. Synthesis, saccharide-binding and anti-cancer cell proliferation properties of arylboronic acid derivatives of indoquinolines.

    PubMed

    Meng, Junxiu; Yu, Shaoqing; Wan, Shengbiao; Ren, Sumei; Jiang, Tao

    2011-11-01

    A facile synthesis of a series of saccharide-binding arylboronic acid derivatives of indoloquinoline was described. The key synthetic steps were polyphosphoric acid-mediated cyclization, chlorinative aromatization, and amidation. Mass spectrometry experiments revealed these synthetic arylboronic acid derivatives of indoquinolines could bind to biologically important carbohydrates (sialic acid, fucose, glucose, and galactose) by forming boronate di-esters in alkaline aqueous solution. Most of the arylboronic acid derivatives of indoquinolines inhibited human breast cancer cell (MDA-231) proliferation at a concentration of 5 μm, whereas the compound 17 exhibited highest percentages (76.74%) of the cancer cell proliferation inhibition.

  2. Mutational Insights into the Roles of Amino Acid Residues in Ligand Binding for Two Closely Related Family 16 Carbohydrate Binding Modules

    SciTech Connect

    Su, Xiaoyun; Agarwal, Vinayak; Dodd, Dylan; Bae, Brian; Mackie, Roderick I.; Nair, Satish K.; Cann, Isaac K.O.

    2010-11-22

    Carbohydrate binding modules (CBMs) are specialized proteins that bind to polysaccharides and oligosaccharides. Caldanaerobius polysaccharolyticus Man5ACBM16-1/CBM16-2 bind to glucose-, mannose-, and glucose/mannose-configured substrates. The crystal structures of the two proteins represent the only examples in CBM family 16, and studies that evaluate the roles of amino acid residues in ligand binding in this family are lacking. In this study, we probed the roles of amino acids (selected based on CBM16-1/ligand co-crystal structures) on substrate binding. Two tryptophan (Trp-20 and Trp-125) and two glutamine (Gln-81 and Gln-93) residues are shown to be critical in ligand binding. Additionally, several polar residues that flank the critical residues also contribute to ligand binding. The CBM16-1 Q121E mutation increased affinity for all substrates tested, whereas the Q21G and N97R mutants exhibited decreased substrate affinity. We solved CBM/substrate co-crystal structures to elucidate the molecular basis of the increased substrate binding by CBM16-1 Q121E. The Gln-121, Gln-21, and Asn-97 residues can be manipulated to fine-tune ligand binding by the Man5A CBMs. Surprisingly, none of the eight residues investigated was absolutely conserved in CBM family 16. Thus, the critical residues in the Man5A CBMs are either not essential for substrate binding in the other members of this family or the two CBMs are evolutionarily distinct from the members available in the current protein database. Man5A is dependent on its CBMs for robust activity, and insights from this study should serve to enhance our understanding of the interdependence of its catalytic and substrate binding modules.

  3. Interactions of chrysotile and crocidolite asbestos with red blood cell membranes. Chrysotile binds to sialic acid.

    PubMed

    Brody, A R; George, G; Hill, L H

    1983-10-01

    Chrysotile and crocidolite are commonly used forms of asbestos. Hemolysis has been widely used as a test of membrane injury, and it has been shown previously that chrysotile causes rapid breakdown of red blood cells (RBCs), whereas crocidolite is only weakly hemolytic. A reasonable hypothesis set forth to explain the cytotoxic effects of chrysotile maintains that positively charged chrysotile fibers bind to negatively charged sialic acid residues on RBC membranes causing clustering of membrane proteins and increased cell permeability to Na and K ions. Our studies presented here provide two lines of evidence in direct support of this hypothesis. (a) Morphologic--Ultrastructural techniques showed that both chrysotile and crocidolite asbestos bind to and distort more than 85% of RBCs treated for 15 minutes. The distorting effects of chrysotile, but not crocidolite, were almost totally ablated by pretreating the cells with neuraminidase. In addition, gold-conjugated wheat germ agglutinin was used to label the distribution of sialic acid groups on RBC membranes. Pretreatment of the RBCs with chrysotile, but not crocidolite, reduced the number of gold-conjugated wheat germ agglutinin-labeled sites to less than 30% of the control level. (b) Biochemical--The thiobarbituric acid assay was used to determine the percentage of sialic acid that remained with the cell pellet after neuraminidase and/or asbestos treatment. Asbestos treatment alone caused no release of sialic acid from the cells. Neuraminidase treatment for 3.5 hours removed more than 80% of the sialic acid from cell surfaces. Chrysotile, but not crocidolite, asbestos prevented neuraminidase-mediated removal of sialic acid from RBCs. In addition, x-ray energy spectrometry of freeze-dried cells showed that RBCs distorted by chrysotile, but not by crocidolite, exhibited significant alterations in intracellular Na:K ratios. The morphologic and biochemical data strongly support the hypothesis that chrysotile asbestos

  4. High fatty acid oxidation capacity and phosphorylation control despite elevated leak and reduced respiratory capacity in northern elephant seal muscle mitochondria.

    PubMed

    Chicco, Adam J; Le, Catherine H; Schlater, Amber; Nguyen, Alex; Kaye, Spencer; Beals, Joseph W; Scalzo, Rebecca L; Bell, Christopher; Gnaiger, Erich; Costa, Daniel P; Crocker, Daniel E; Kanatous, Shane B

    2014-08-15

    Northern elephant seals (Mirounga angustirostris) are extreme, hypoxia-adapted endotherms that rely largely on aerobic metabolism during extended breath-hold dives in near-freezing water temperatures. While many aspects of their physiology have been characterized to account for these remarkable feats, the contribution of adaptations in the aerobic powerhouses of muscle cells, the mitochondria, are unknown. In the present study, the ontogeny and comparative physiology of elephant seal muscle mitochondrial respiratory function was investigated under a variety of substrate conditions and respiratory states. Intact mitochondrial networks were studied by high-resolution respirometry in saponin-permeabilized fiber bundles obtained from primary swimming muscles of pup, juvenile and adult seals, and compared with fibers from adult human vastus lateralis. Results indicate that seal muscle maintains a high capacity for fatty acid oxidation despite a progressive decrease in total respiratory capacity as animals mature from pups to adults. This is explained by a progressive increase in phosphorylation control and fatty acid utilization over pyruvate in adult seals compared with humans and seal pups. Interestingly, despite higher indices of oxidative phosphorylation efficiency, juvenile and adult seals also exhibit a ~50% greater capacity for respiratory 'leak' compared with humans and seal pups. The ontogeny of this phenotype suggests it is an adaptation of muscle to the prolonged breath-hold exercise and highly variable ambient temperatures experienced by mature elephant seals. These studies highlight the remarkable plasticity of mammalian mitochondria to meet the demands for both efficient ATP production and endothermy in a cold, oxygen-limited environment.

  5. Binding of diazepam, salicylic acid and digitoxin to albumin isolated from fetal and adult serum.

    PubMed

    Viani, A; Cappiello, M; Pacifici, G M

    1991-01-01

    Albumin was isolated from pooled fetal serum obtained at normal delivery at term and from pooled adult plasma. Albumin isolation was carried out by means of PEG precipitation followed by ion exchange chromatography on DEAE-Sephadex A 50 and then on SP-Sephadex C 50. The binding of diazepam (1 microM), salicylic acid (2 mM) and digitoxin (6 nM) to albumin (40 g/l) was measured by equilibrium dialysis at 37 degrees C. The unbound fraction (mean +/- SD) for fetal and adult albumin of diazepam was 1.86 +/- 0.24 and 1.82 +/- 0.15% (NS), that of digitoxin was 3.18 +/- 0.27 and 3.36 +/- 0.04% (NS) and that of salicylic acid was 11.65 +/- 0.99 and 9.47 +/- 0.75% (p less than 0.05), respectively. With both fetal and adult albumin, a single class of binding sites was observed for diazepam and digitoxin, whereas two classes of binding sites were observed for salicylic acid. The number of binding sites (n, moles of drug per mole of albumin) for fetal and adult albumin was 0.83 and 1.02 for diazepam and 0.014 and 0.018 for digitoxin, respectively. For salicylic acid, n was 1.45 (fetal albumin) and 1.55 (adult albumin) for the higher affinity site, and 3.06 (fetal albumin) and 3.27 (adult albumin) for the lower affinity site. The association constant (Ka, M-1) for diazepam was 1.36 x 10(5) (fetal albumin) and 1.00 x 10(5) (adult albumin) and that for digitoxin was 4.12 x 10(6) (fetal albumin) and 2.7 x 10(6) (adult albumin). For salicylic acid, Ka was 38.4 x 10(3) (fetal albumin) and 35.8 x 10(3) (adult albumin) for the higher affinity site, and 2.7 x 10(3) (fetal albumin) and 4.3 x 10(3) (adult albumin) for the lower affinity site. This work shows that fetal and adult albumin have similar binding properties and corroborates our previous findings with furosemide.

  6. Electrostatic binding and hydrophobic collapse of peptide-nucleic acid aggregates quantified using force spectroscopy.

    PubMed

    Camunas-Soler, Joan; Frutos, Silvia; Bizarro, Cristiano V; de Lorenzo, Sara; Fuentes-Perez, Maria Eugenia; Ramsch, Roland; Vilchez, Susana; Solans, Conxita; Moreno-Herrero, Fernando; Albericio, Fernando; Eritja, Ramón; Giralt, Ernest; Dev, Sukhendu B; Ritort, Felix

    2013-06-25

    Knowledge of the mechanisms of interaction between self-aggregating peptides and nucleic acids or other polyanions is key to the understanding of many aggregation processes underlying several human diseases (e.g., Alzheimer's and Parkinson's diseases). Determining the affinity and kinetic steps of such interactions is challenging due to the competition between hydrophobic self-aggregating forces and electrostatic binding forces. Kahalalide F (KF) is an anticancer hydrophobic peptide that contains a single positive charge that confers strong aggregative properties with polyanions. This makes KF an ideal model to elucidate the mechanisms by which self-aggregation competes with binding to a strongly charged polyelectrolyte such as DNA. We use optical tweezers to apply mechanical forces to single DNA molecules and show that KF and DNA interact in a two-step kinetic process promoted by the electrostatic binding of DNA to the aggregate surface followed by the stabilization of the complex due to hydrophobic interactions. From the measured pulling curves we determine the spectrum of binding affinities, kinetic barriers, and lengths of DNA segments sequestered within the KF-DNA complex. We find there is a capture distance beyond which the complex collapses into compact aggregates stabilized by strong hydrophobic forces and discuss how the bending rigidity of the nucleic acid affects this process. We hypothesize that within an in vivo context, the enhanced electrostatic interaction of KF due to its aggregation might mediate the binding to other polyanions. The proposed methodology should be useful to quantitatively characterize other compounds or proteins in which the formation of aggregates is relevant.

  7. A novel method for the determination of ascorbic acid and antioxidant capacity in Opuntia ficus indica using in vivo microdialysis.

    PubMed

    Pretti, L; Bazzu, G; Serra, P A; Nieddu, G

    2014-03-15

    A simple and rapid method was developed for in vivo simultaneous determination of ascorbic-acid and antioxidant capacity in microdialysates from cladodes of Opuntia ficus-indica (L.) Miller. The method is verified in water-stressed plants, as compared with a well-watered test controls. The microdialysis probe construction and insertion procedure was specifically developed to minimise the tissue trauma of the plant and to obtain optimal dialysis performance. Microdialysis was performed using a flow rate of 3 μL/min and the samples were analysed by HPLC coupled to electrochemical detection of ascorbic-acid and DPPH-determined antioxidant capacity. Our data indicate exponential decay of the concentrations of the analysed compounds as a function of microdialysis sampling time. Water-stressed Opuntia show decreased ascorbic acid levels and increased the others antioxidants.

  8. Antioxidant capacity and stability of liposomes containing a triglyceride derivative of lipoic acid

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The multi-functional nutritional agent lipoic acid offers numerous beneficial effects to oxidatively stressed tissues. Lipoic acid was enzymatically incorporated into a triglyceride in conjunction with oleic acid, creating lipoyl dioleoylglycerol, and then chemically reduced to form dihydrolipoyl d...

  9. Dietary intake, neutrophil fatty acid profile, serum antioxidant vitamins and oxygen radical absorbance capacity in patients with ulcerative colitis.

    PubMed

    Kawakami, Yuko; Okada, Hiroyuki; Murakami, Yasuko; Kawakami, Takayo; Ueda, Yukiko; Kunii, Daisuke; Sakamoto, Yachiyo; Shiratori, Yasushi; Okita, Misako

    2007-04-01

    Nutrition may play an important role in the pathogenesis and treatment of ulcerative colitis. Several studies suggest an association between dietary factors and the onset of ulcerative colitis; however, only few studies have examined the relationship between dietary intake and relapse of ulcerative colitis. The aim of this study was to assess the dietary intake and antioxidative capacity of ulcerative colitis patients and to elucidate the efficacy of dietary therapy for ulcerative colitis. Dietary intake, fatty acid composition of phospholipids in plasma and neutrophils, serum fat-soluble vitamin levels, and oxygen radical absorbance capacity were analyzed in 29 ulcerative colitis patients (7 males and 22 females), who were treated at the Department of Gastroenterology, Okayama University Hospital. Total fat intake, fat energy ratio and linoleic acid intake were significantly lower, while protein and carbohydrate intakes were significantly higher, in the patients than age- and sex-matched controls. In the neutrophil phospholipids of ulcerative colitis patients, significantly higher levels of linoleic aicd and arachidonic acid and a lower level of eicosapentaenoic acid were observed. The concentrations of serum retinol and beta-carotene but not alpha-tocopherol were significantly lower and serum oxygen radical absorbance capacity was also lower than in the controls. Significant correlations between serum oxygen radical absorbance capacity and retinol (r = 0.567, p = 0.0031), alpha-tocopherol (r = 0.560, p = 0.0036) and beta-carotene (r = 0.440, p = 0.0279) concentrations were observed in the ulcerative colitis patients. A diet restricting the intake of linoleic acid and supplemented with eicosapentaenoic acid and antioxidative vitamins may be recommendable for the nutritional management of ulcerative colitis patients.

  10. From keys to bulldozers: expanding roles for winged helix domains in nucleic-acid-binding proteins.

    PubMed

    Harami, Gábor M; Gyimesi, Máté; Kovács, Mihály

    2013-07-01

    The winged helix domain (WHD) is a widespread nucleic-acid-binding protein structural element found in all kingdoms of life. Although the overall structure of the WHD is conserved, its functional properties and interaction profiles are extremely versatile. WHD-containing proteins can exploit nearly the full spectrum of nucleic acid structural features for recognition and even covalent modification or noncovalent rearrangement of target molecules. WHD functions range from sequence-recognizing keys in transcription factors and bulldozer-like strand-separating wedges in helicases to mediators of protein-protein interactions (PPIs). Further investigations are needed to understand the contribution of WHD structural dynamics to nucleic-acid-modifying enzymatic functions.

  11. Sex Differences in Long Chain Fatty Acid Utilization and Fatty Acid Binding Protein Concentration in Rat Liver

    PubMed Central

    Ockner, Robert K.; Burnett, David A.; Lysenko, Nina; Manning, Joan A.

    1979-01-01

    Female sex and estrogen administration are associated with increased hepatic production of triglyceride-rich lipoproteins; the basis for this has not been fully elucidated. Inasmuch as hepatic lipoprotein production is also influenced by FFA availability and triglyceride biosynthesis, we investigated sex differences in FFA utilization in rat hepatocyte suspensions and in the components of the triglyceride biosynthetic pathway. Isolated adult rat hepatocyte suspensions were incubated with albumin-bound [14C]oleate for up to 15 min. At physiological and low oleate concentrations, cells from females incorporated significantly more 14C into glycerolipids, especially triglycerides, and into oxidation products than did male cells, per milligram cell protein. At 0.44 mM oleate, incorporation into triglycerides in female cells was approximately twice that in male cells. Comparable sex differences were observed in cells from fasted animals and when [14C]-glycerol incorporation was measured. At higher oleate concentrations, i.e., fatty acid:albumin mole ratios in excess of 2:1, these sex differences were no longer demonstrable, suggesting that maximal rates of fatty acid esterification and oxidation were similar in female and male cells. In female and male hepatic microsomes, specific activities of long chain acyl coenzyme A synthetase, phosphatidate phosphohydrolase, and diglyceride acyltransferase were similar, but glycerol-3-phosphate acyltransferase activity was slightly greater in females at certain substrate concentrations. Microsomal incorporation of [14C]oleate into total glycerolipids was not significantly greater in females. In further contrast to intact cells, microsomal incorporation of [14C]oleate into triglycerides, although significantly greater in female microsomes, accounted for only a small fraction of the fatty acid esterified. The binding affinity and stoichiometry of partially purified female hepatic fatty acid binding protein (FABP) were similar to

  12. Comparison of the Effects of Eicosapentaenoic Acid and Docosahexaenoic Acid on the Eradication of Helicobacter pylori Infection, Serum Inflammatory Factors and Total Antioxidant Capacity.

    PubMed

    Khandouzi, Nafiseh; Shidfar, Farzad; Agah, Shahram; Hosseini, Agha Fatemeh; Dehnad, Afsaneh

    2015-01-01

    Helicobacter pylori infection, the most common chronic bacterial infection in the world, and an important cause of gastrointestinal disorders, may be involved in the pathogenesis of some extra-gastrointestinal disturbances, as well as an increase in blood levels of certain inflammatory markers. Anti-bacterial activity against Helicobacter pylori and anti-inflammatory properties of omega-3 fatty acids have been studied in several research studies. The purpose of the present study was the comparison of the effects of Eicosapentaenoic Acid and Docosahexaenoic Acid supplementation on Helicobacter pylori eradication, serum levels of some inflammatory markers and total antioxidant capacity. In a randomized, double-blind, placebo-controlled clinical trial, 97 Helicobacter pylori positive patients (64 patients in the two intervention groups and 33 in the control group), received 2 grams daily of Eicosapentaenoic Acid, Docosahexaenoic Acid or Medium Chain Triglyceride oil as placebo, along with conventional tetra-drug Helicobacter pylori eradication regimen, for 12 weeks. Helicobacter pylori eradication test and measurement of concentration of interleukine-6, interleukine-8, high-sensitivity C-reactive protein and total antioxidant capacity were performed after the intervention. There was no significant difference in eradication rate of the infection, levels of interleukine-6 and total antioxidant capacity among the three groups, while the levels of interleukine-8 and high-sensitivity C-reactive protein were statistically different. Eicosapentaenoic Acid or Docosahexaenoic Acid supplementation had no significant differential impact on the eradication of Helicobacter pylori infection, and serum levels of interleukine-6 and total antioxidant capacity. However, it had a desirable effect on the levels of interleukine-8 and high-sensitivity C-reactive protein in Helicobacter pylori positive patients.

  13. Comparison of the Effects of Eicosapentaenoic Acid and Docosahexaenoic Acid on the Eradication of Helicobacter pylori Infection, Serum Inflammatory Factors and Total Antioxidant Capacity

    PubMed Central

    Khandouzi, Nafiseh; Shidfar, Farzad; Agah, Shahram; Hosseini, Agha Fatemeh; Dehnad, Afsaneh

    2015-01-01

    Helicobacter pylori infection, the most common chronic bacterial infection in the world, and an important cause of gastrointestinal disorders, may be involved in the pathogenesis of some extra-gastrointestinal disturbances, as well as an increase in blood levels of certain inflammatory markers. Anti-bacterial activity against Helicobacter pylori and anti-inflammatory properties of omega-3 fatty acids have been studied in several research studies. The purpose of the present study was the comparison of the effects of Eicosapentaenoic Acid and Docosahexaenoic Acid supplementation on Helicobacter pylori eradication, serum levels of some inflammatory markers and total antioxidant capacity. In a randomized, double-blind, placebo-controlled clinical trial, 97 Helicobacter pylori positive patients (64 patients in the two intervention groups and 33 in the control group), received 2 grams daily of Eicosapentaenoic Acid, Docosahexaenoic Acid or Medium Chain Triglyceride oil as placebo, along with conventional tetra-drug Helicobacter pylori eradication regimen, for 12 weeks. Helicobacter pylori eradication test and measurement of concentration of interleukine-6, interleukine-8, high-sensitivity C-reactive protein and total antioxidant capacity were performed after the intervention. There was no significant difference in eradication rate of the infection, levels of interleukine-6 and total antioxidant capacity among the three groups, while the levels of interleukine-8 and high-sensitivity C-reactive protein were statistically different. Eicosapentaenoic Acid or Docosahexaenoic Acid supplementation had no significant differential impact on the eradication of Helicobacter pylori infection, and serum levels of interleukine-6 and total antioxidant capacity. However, it had a desirable effect on the levels of interleukine-8 and high-sensitivity C-reactive protein in Helicobacter pylori positive patients. PMID:25561921

  14. Nucleic acid sensing with enzyme-DNA binding protein conjugates cascade and simple DNA nanostructures.

    PubMed

    Aktas, Gülsen Betül; Skouridou, Vasso; Masip, Lluis

    2017-03-22

    A versatile and universal DNA sensing platform is presented based on enzyme-DNA binding protein tags conjugates and simple DNA nanostructures. Two enzyme conjugates were thus prepared, with horseradish peroxidase linked to the dimeric single-chain bacteriophage Cro repressor protein (HRP-scCro) and glucose oxidase linked to the dimeric headpiece domain of Escherichia coli LacI repressor protein (GOx-dHP), and used in conjunction with a hybrid ssDNA-dsDNA detection probe. This probe served as a simple DNA nanostructure allowing first for target recognition through its target-complementary single-stranded DNA (ssDNA) part and then for signal generation after conjugate binding on the double-stranded DNA (dsDNA) containing the specific binding sites for the dHP and scCro DNA binding proteins. The DNA binding proteins chosen in this work have different sequence specificity, high affinity, and lack of cross-reactivity. The proposed sensing system was validated for the detection of model target ssDNA from high-risk human papillomavirus (HPV16) and the limits of detection of 45, 26, and 21 pM were achieved using the probes with scCro/dHP DNA binding sites ratio of 1:1, 2:1, and 1:2, respectively. The performance of the platform in terms of limit of detection was comparable to direct HRP systems using target-specific oligonucleotide-HRP conjugates. The ratio of the two enzymes can be easily manipulated by changing the number of binding sites on the detection probe, offering further optimization possibilities of the signal generation step. Moreover, since the signal is obtained in the absence of externally added hydrogen peroxide, the described platform is compatible with paper-based assays for molecular diagnostics applications. Finally, just by changing the ssDNA part of the detection probe, this versatile nucleic acid platform can be used for the detection of different ssDNA target sequences or in a multiplex detection configuration without the need to change any of the

  15. Single aromatic residue location alters nucleic acid binding and chaperone function of FIV nucleocapsid protein

    PubMed Central

    Wu, Hao; Wang, Wei; Naiyer, Nada; Fichtenbaum, Eric; Qualley, Dominic F.; McCauley, Micah J.; Gorelick, Robert J.; Rouzina, Ioulia; Musier-Forsyth, Karin; Williams, Mark C.

    2014-01-01

    Feline immunodeficiency virus (FIV) is a retrovirus that infects domestic cats, and is an excellent animal model for human immunodeficiency virus type 1 (HIV-1) pathogenesis. The nucleocapsid (NC) protein is critical for replication in both retroviruses. FIV NC has several structural features that differ from HIV-1 NC. While both NC proteins have a single conserved aromatic residue in each of the two zinc fingers, the aromatic residue on the second finger of FIV NC is located on the opposite C-terminal side relative to its location in HIV-1 NC. In addition, whereas HIV-1 NC has a highly charged cationic N-terminal tail and a relatively short C-terminal extension, the opposite is true for FIV NC. To probe the impact of these differences on the nucleic acid (NA) binding and chaperone properties of FIV NC, we carried out ensemble and single-molecule assays with wild-type (WT) and mutant proteins. The ensemble studies show that FIV NC binding to DNA is strongly electrostatic, with a higher effective charge than that observed for HIV-1 NC. The C-terminal basic domain contributes significantly to the NA binding capability of FIV NC. In addition, the non-electrostatic component of DNA binding is much weaker for FIV NC than for HIV-1 NC. Mutation of both aromatic residues in the zinc fingers to Ala (F12A/W44A) further increases the effective charge of FIV NC and reduces its non-electrostatic binding affinity. Interestingly, switching the location of the C-terminal aromatic residue to mimic the HIV-1 NC sequence (N31W/W44A) reduces the effective charge of FIV NC and increases its non-electrostatic binding affinity to values similar to HIV-1 NC. Consistent with the results of these ensemble studies, single-molecule DNA stretching studies show that while WT FIV NC has reduced stacking capability relative to HIV-1 NC, the aromatic switch mutant recovers the ability to intercalate between the DNA bases. Our results demonstrate that altering the position of a single aromatic

  16. Thermodynamic and solution state NMR characterization of the binding of secondary and conjugated bile acids to STARD5.

    PubMed

    Létourneau, Danny; Lorin, Aurélien; Lefebvre, Andrée; Cabana, Jérôme; Lavigne, Pierre; LeHoux, Jean-Guy

    2013-11-01

    STARD5 is a member of the STARD4 sub-family of START domain containing proteins specialized in the non-vesicular transport of lipids and sterols. We recently reported that STARD5 binds primary bile acids. Herein, we report on the biophysical and structural characterization of the binding of secondary and conjugated bile acids by STARD5 at physiological concentrations. We found that the absence of the 7α-OH group and its epimerization increase the affinity of secondary bile acids for STARD5. According to NMR titration and molecular modeling, the affinity depends mainly on the number and positions of the steroid ring hydroxyl groups and to a lesser extent on the presence or type of bile acid side-chain conjugation. Primary and secondary bile acids have different binding modes and display different positioning within the STARD5 binding pocket. The relative STARD5 affinity for the different bile acids studied is: DCA>LCA>CDCA>GDCA>TDCA>CA>UDCA. TCA and GCA do not bind significantly to STARD5. The impact of the ligand chemical structure on the thermodynamics of binding is discussed. The discovery of these new ligands suggests that STARD5 is involved in the cellular response elicited by bile acids and offers many entry points to decipher its physiological role.

  17. ATP binding cassette modulators control abscisic acid-regulated slow anion channels in guard cells

    PubMed Central

    Leonhardt, N; Vavasseur, A; Forestier, C

    1999-01-01

    In animal cells, ATP binding cassette (ABC) proteins are a large family of transporters that includes the sulfonylurea receptor and the cystic fibrosis transmembrane conductance regulator (CFTR). These two ABC proteins possess an ion channel activity and bind specific sulfonylureas, such as glibenclamide, but homologs have not been identified in plant cells. We recently have shown that there is an ABC protein in guard cells that is involved in the control of stomatal movements and guard cell outward K+ current. Because the CFTR, a chloride channel, is sensitive to glibenclamide and able to interact with K+ channels, we investigated its presence in guard cells. Potent CFTR inhibitors, such as glibenclamide and diphenylamine-2-carboxylic acid, triggered stomatal opening in darkness. The guard cell protoplast slow anion current that was recorded using the whole-cell patch-clamp technique was inhibited rapidly by glibenclamide in a dose-dependent manner; the concentration producing half-maximum inhibition was at 3 &mgr;M. Potassium channel openers, which bind to and act through the sulfonylurea receptor in animal cells, completely suppressed the stomatal opening induced by glibenclamide and recovered the glibenclamide-inhibited slow anion current. Abscisic acid is known to regulate slow anion channels and in our study was able to relieve glibenclamide inhibition of slow anion current. Moreover, in epidermal strip bioassays, the stomatal closure triggered by Ca2+ or abscisic acid was reversed by glibenclamide. These results suggest that the slow anion channel is an ABC protein or is tightly controlled by such a protein that interacts with the abscisic acid signal transduction pathway in guard cells. PMID:10368184

  18. Effect of Plasma Uric Acid on Antioxidant Capacity, Oxidative Stress, and Insulin Sensitivity in Obese Subjects

    PubMed Central

    Fabbrini, Elisa; Serafini, Mauro; Colic Baric, Irena; Hazen, Stanley L.; Klein, Samuel

    2014-01-01

    Oxidative stress is purported to be involved in the pathogenesis of obesity-associated insulin resistance. We evaluated whether alterations in levels of circulating uric acid (UA), a systemic antioxidant, affects the following: 1) systemic (plasma and saliva) nonenzymatic antioxidant capacity (NEAC); 2) markers of systemic (urinary 8-iso-prostaglandin-F2α) and muscle (carbonylated protein content) oxidative stress; and 3) whole-body insulin sensitivity (percentage increase in glucose uptake during a hyperinsulinemic-euglycemic clamp procedure). Thirty-one obese subjects (BMI 37.1 ± 0.7 kg/m2) with either high serum UA (HUA; 7.1 ± 0.4 mg/dL; n = 15) or normal serum UA (NUA; 4.5 ± 0.2 mg/dL; n = 16) levels were studied; 13 subjects with HUA levels were studied again after reduction of serum UA levels to 0 by infusing a recombinant urate oxidase. HUA subjects had 20–90% greater NEAC, but lower insulin sensitivity (40%) and levels of markers of oxidative stress (30%) than subjects in the NUA group (all P < 0.05). Acute UA reduction caused a 45–95% decrease in NEAC and a 25–40% increase in levels of systemic and muscle markers of oxidative stress (all P < 0.05), but did not affect insulin sensitivity (from 168 ± 25% to 156 ± 17%, P = NS). These results demonstrate that circulating UA is a major antioxidant and might help protect against free-radical oxidative damage. However, oxidative stress is not a major determinant of insulin action in vivo. PMID:24353177

  19. Relation between change in exercise capacity and change in blood amino acids in patients with chronic heart failure

    PubMed Central

    Morotomi, Nobuo; Saitoh, Masakazu; Ishii, Noriko; Ohno, Kayoko; Nagayama, Masatoshi; Kawate, Nobuyuki; Mizuma, Masazumi

    2017-01-01

    [Purpose] Although cardiac rehabilitation (CR) is recommended for patients with chronic heart failure (CHF), adequate exercise effect cannot be obtained in elderly patients. Administration of amino acids (AA) to CHF patients has been reported to improve exercise capacity, but the changes in AA composition in plasma before and after CR had not been reported. This study aimed to measure plasma levels of AA in CHF patients and compare with values of normal range. In addition the relationship between the change of exercise capacity and AA were examined. [Subjects and Methods] Twelve CHF patients (60% males, aged 68 ± 12 years) were studied. The correction between the rates of changes in exercise capacity parameters and in plasma AA levels was investigated. [Results] Anaerobic threshold (AT) and peak oxygen uptake (VO2) improved significantly after CR. The AA profile showed no specific pattern, and citrulline (Cit) was the amino acid showing a significant positive correlation with exercise capacity (∆Cit vs. ∆AT: r=0.602, ∆Cit vs. ∆AT-work rate (WR): r=0.681, ∆Cit vs. ∆VO2/WR: r=0.635). A tendency of positive correlation was observed between ∆Cit and ∆peak VO2 (r=0.456). [Conclusion] The AA profile showed no specific pattern, but a relationship between change in exercise capacity and Cit were found. PMID:28356624

  20. Relation between change in exercise capacity and change in blood amino acids in patients with chronic heart failure.

    PubMed

    Morotomi, Nobuo; Saitoh, Masakazu; Ishii, Noriko; Ohno, Kayoko; Nagayama, Masatoshi; Kawate, Nobuyuki; Mizuma, Masazumi

    2017-03-01

    [Purpose] Although cardiac rehabilitation (CR) is recommended for patients with chronic heart failure (CHF), adequate exercise effect cannot be obtained in elderly patients. Administration of amino acids (AA) to CHF patients has been reported to improve exercise capacity, but the changes in AA composition in plasma before and after CR had not been reported. This study aimed to measure plasma levels of AA in CHF patients and compare with values of normal range. In addition the relationship between the change of exercise capacity and AA were examined. [Subjects and Methods] Twelve CHF patients (60% males, aged 68 ± 12 years) were studied. The correction between the rates of changes in exercise capacity parameters and in plasma AA levels was investigated. [Results] Anaerobic threshold (AT) and peak oxygen uptake (VO2) improved significantly after CR. The AA profile showed no specific pattern, and citrulline (Cit) was the amino acid showing a significant positive correlation with exercise capacity (∆Cit vs. ∆AT: r=0.602, ∆Cit vs. ∆AT-work rate (WR): r=0.681, ∆Cit vs. ∆VO2/WR: r=0.635). A tendency of positive correlation was observed between ∆Cit and ∆peak VO2 (r=0.456). [Conclusion] The AA profile showed no specific pattern, but a relationship between change in exercise capacity and Cit were found.

  1. Pharmacokinetics, tissue distribution, and plasma protein binding study of chicoric acid by HPLC-MS/MS.

    PubMed

    Wang, Yutang; Xie, Guo; Liu, Qian; Duan, Xiang; Liu, Zhigang; Liu, Xuebo

    2016-09-15

    Chicoric acid is a major active constituent of Echinacea purpurea and has a variety of biological functions. In this study, a liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) approach was developed and validated for the determination of chicoric acid in rat plasma and various tissues using ferulic acid as an internal standard (IS). This method was successfully applied to pharmacokinetics, tissue distribution, and plasma protein binding (PPB) study of chicoric acid in Sprague-Dawley (SD) rats dosed with 50mg/kg by gastric gavage. The pharmacokinetic parameters were determined and showed a half-life (t1/2) of 4.53±1.44h, an apparent volume of mean residual time (MRT) of 18.58±4.43h, and an area under the curve (AUC) of 26.14 mghL(-1). The tissue distribution of chicoric acid in rats after gavage administration showed a decreasing tendency in different tissues (liver>lung>kidney>heart>spleen>brain). The PPB rates in rat plasma, human plasma, and bovine serum albumin were 98.3, 96.9, and 96.6%, respectively. These results provide insight for the further pharmacological investigation of chicoric acid.

  2. Determination of free bilirubin and its binding capacity by HSA using a microfluidic chip-capillary electrophoresis device with a multi-segment circular-ferrofluid-driven micromixing injection.

    PubMed

    Sun, Hui; Nie, Zhou; Fung, Ying Sing

    2010-09-01

    A PMMA microfluidic chip-CE device with a multi-segment circular-ferrofluid-driven micromixing injector has been developed for the determination of free bilirubin and its binding capacity by HSA at equilibrium. The design of the device and its fabrication by a low cost CO(2) laser are discussed for intended applications. Under optimized conditions, the total binding capacity of HSA for bilirubin was determined as 16.3±1.4 mg/l00 mL human serum (n=3) and residual binding capacity for bilirubin 9.8 mg/100 mL (n=3) in normal infants. To assess risk of hyperbilirubinemia, free bilirubin and residual binding capacity by HSA provide a better indicator than total bilirubin, as neonates with impaired bilirubin binding capacity could be detected. In addition, residual binding capacity provides an advanced indicator to predict the onset of hyperbilirubinemia before the appearance of free bilirubin. HSA down to 94 nL is used in each titration and a full assay of four titrations takes up 376 nL HSA, sufficient for newborns with HSA in microliter range. The device has shown capable to provide adequate margin of protection to detect an early rising level of bilirubin and impaired binding capacity prior to the onset of jaundice condition.

  3. Iron Oxide Surface Chemistry: Effect of Chemical Structure on Binding in Benzoic Acid and Catechol Derivatives.

    PubMed

    Korpany, Katalin V; Majewski, Dorothy D; Chiu, Cindy T; Cross, Shoronia N; Blum, Amy Szuchmacher

    2017-03-13

    The excellent performance of functionalized iron oxide nanoparticles (IONPs) in nanomaterial and biomedical applications often relies on achieving the attachment of ligands to the iron oxide surface both in sufficient number and with proper orientation. Toward this end, we determine relationships between the ligand chemical structure and surface binding on magnetic IONPs for a series of related benzoic acid and catechol derivatives. Ligand exchange was used to introduce the model ligands, and the resultant nanoparticles were characterized using Fourier transform infrared-attenuated internal reflectance spectroscopy, transmission electron microscopy, and nanoparticle solubility behavior. An in-depth analysis of ligand electronic effects and reaction conditions reveals that the nature of ligand binding does not solely depend on the presence of functional groups known to bind to IONPs. The structure of the resultant ligand-surface complex was primarily influenced by the relative positioning of hydroxyl and carboxylic acid groups within the ligand and whether or not HCl(aq) was added to the ligand-exchange reaction. Overall, this study will help guide future ligand-design and ligand-exchange strategies toward realizing truly custom-built IONPs.

  4. Evidence for pentagalloyl glucose binding to human salivary alpha-amylase through aromatic amino acid residues.

    PubMed

    Gyémánt, Gyöngyi; Zajácz, Agnes; Bécsi, Bálint; Ragunath, Chandran; Ramasubbu, Narayanan; Erdodi, Ferenc; Batta, Gyula; Kandra, Lili

    2009-02-01

    We demonstrate here that pentagalloyl glucose (PGG), a main component of gallotannins, was an effective inhibitor of HSA and it exerted similar inhibitory potency to Aleppo tannin used in this study. The inhibition of HSA by PGG was found to be non-competitive and inhibitory constants of K(EI)=2.6 microM and K(ESI)=3.9 microM were determined from Lineweaver-Burk secondary plots. PGG as a model compound for gallotannins was selected to study the inhibitory mechanism and to characterize the interaction of HSA with this type of molecules. Surface plasmon resonance (SPR) binding experiments confirmed the direct interaction of HSA and PGG, and it also established similar binding of Aleppo tannin to HSA. Saturation transfer difference (STD) experiment by NMR clearly demonstrated the aromatic rings of PGG may be involved in the interaction suggesting a possible stacking with the aromatic side chains of HSA. The role of aromatic amino acids of HSA in PGG binding was reinforced by kinetic studies with the W58L and Y151M mutants of HSA: the replacement of the active site aromatic amino acids with aliphatic ones decreased the PGG inhibition dramatically, which justified the importance of these residues in the interaction.

  5. Binding and detoxification of chlorpyrifos by lactic acid bacteria on rice straw silage fermentation.

    PubMed

    Wang, Yan-Su; Wu, Tian-Hao; Yang, Yao; Zhu, Cen-Ling; Ding, Cheng-Long; Dai, Chuan-Chao

    2016-01-01

    This investigation examined the reduction of pesticide residues on straw inoculated with lactic acid bacteria (LAB) during ensiling. Lactobacillus casei WYS3 was isolated from rice straw that contained pesticide residues. Non-sterilized rice straw, which was inoculated with L. casei WYS3, showed increased removal of chlorpyrifos after ensiling, compared with rice straw that was not inoculated with L. casei WYS3 or sterilized rice straw. In pure culture, these strains can bind chlorpyrifos as indicated by high-performance liquid chromatography analysis. Viable L. casei WYS3 was shown to bind 33.3-42% of exogenously added chlorpyrifos. These results are similar to those of acid-treated cells but less than those of heat-treated cells, which were found to bind 32.0% and 77.2% of the added chlorpyrifos respectively. Furthermore, gas chromatography-mass spectrometry analysis determined that L. casei WYS3 detoxified chlorpyrifos via P-O-C cleavage. Real-time polymerized chain reaction analysis determined that organophosphorus hydrolase gene expression tripled after the addition of chlorpyrifos to LAB cultures, compared with the control group (without chlorpyrifos). This paper highlights the potential use of LAB starter cultures for the detoxification and removal of chlorpyrifos residues in the environment.

  6. Analysis of amino acid sequence variations and immunoglobulin E-binding epitopes of German cockroach tropomyosin.

    PubMed

    Jeong, Kyoung Yong; Lee, Jongweon; Lee, In-Yong; Ree, Han-Il; Hong, Chein-Soo; Yong, Tai-Soon

    2004-09-01

    The allergenicities of tropomyosins from different organisms have been reported to vary. The cDNA encoding German cockroach tropomyosin (Bla g 7) was isolated, expressed, and characterized previously. In the present study, the amino acid sequence variations in German cockroach tropomyosin were analyzed in order to investigate its influence on allergenicity. We also undertook the identification of immunodominant peptides containing immunoglobulin E (IgE) epitopes which may facilitate the development of diagnostic and immunotherapeutic strategies based on the recombinant proteins. Two-dimensional gel electrophoresis and immunoblot analysis with mouse anti-recombinant German cockroach tropomyosin serum was performed to investigate the isoforms at the protein level. Reverse transcriptase PCR (RT-PCR) was applied to examine the sequence diversity. Eleven different variants of the deduced amino acid sequences were identified by RT-PCR. German cockroach tropomyosin has only minor sequence variations that did not seem to affect its allergenicity significantly. These results support the molecular basis underlying the cross-reactivities of arthropod tropomyosins. Recombinant fragments were also generated by PCR, and IgE-binding epitopes were assessed by enzyme-linked immunosorbent assay. Sera from seven patients revealed heterogeneous IgE-binding responses. This study demonstrates multiple IgE-binding epitope regions in a single molecule, suggesting that full-length tropomyosin should be used for the development of diagnostic and therapeutic reagents.

  7. Inosine 5'-monophosphate dehydrogenase binds nucleic acids in vitro and in vivo.

    PubMed Central

    McLean, Jeremy E; Hamaguchi, Nobuko; Belenky, Peter; Mortimer, Sarah E; Stanton, Martin; Hedstrom, Lizbeth

    2004-01-01

    Inosine 5'-monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in the de novo biosynthesis of guanine nucleotides. In addition to the catalytic domain, IMPDH contains a subdomain of unknown function composed of two cystathione beta-synthase domains. Our results, using three different assays, show that IMPDHs from Tritrichomonas foetus, Escherichia coli, and both human isoforms bind single-stranded nucleic acids with nanomolar affinity via the subdomain. Approx. 100 nucleotides are bound per IMPDH tetramer. Deletion of the subdomain decreases affinity 10-fold and decreases site size to 60 nucleotides, whereas substitution of conserved Arg/Lys residues in the subdomain with Glu decreases affinity by 20-fold. IMPDH is found in the nucleus of human cells, as might be expected for a nucleic-acid-binding protein. Lastly, immunoprecipitation experiments show that IMPDH binds both RNA and DNA in vivo. These experiments indicate that IMPDH has a previously unappreciated role in replication, transcription or translation that is mediated by the subdomain. PMID:14766016

  8. Fatty acid binding protein 7 and n-3 poly unsaturated fatty acid supply in early rat brain development.

    PubMed

    Maximin, Elise; Langelier, Bénédicte; Aïoun, Josiane; Al-Gubory, Kaïs H; Bordat, Christian; Lavialle, Monique; Heberden, Christine

    2016-03-01

    Fatty acid binding protein 7 (FABP7), abundant in the embryonic brain, binds with the highest affinity to docosahexaenoic acid (DHA) and is expressed in the early stages of embryogenesis. Here, we have examined the consequences of the exposure to different DHA levels and of the in utero depletion of FABP7 on early rat brain development. Neurodevelopment was evaluated through the contents of two proteins, connexin 43 (Cx43) and cyclin-dependent kinase 5 (CDK5), both involved in neuroblast proliferation, differentiation, and migration. The dams were fed with diets presenting different DHA contents, from deficiency to supplementation. DHA brain embryos contents already differed at embryonic day 11.5 and the differences kept increasing with time. Cx43 and CDK5 contents were positively associated with the brain DHA levels. When FABP7 was depleted in vivo by injections of siRNA in the telencephalon, the enhancement of the contents of both proteins was lost in supplemented animals, but FABP7 depletion did not modify phospholipid compositions regardless of the diets. Thus, FABP7 is a necessary mediator of the effect of DHA on these proteins synthesis, but its role in DHA uptake is not critical, although FABP7 is localized in phospholipid-rich areas. Our study shows that high contents of DHA associated with FABP7 are necessary to promote early brain development, which prompted us to recommend DHA supplementation early in pregnancy.

  9. Structural basis for the ligand-binding specificity of fatty acid-binding proteins (pFABP4 and pFABP5) in gentoo penguin.

    PubMed

    Lee, Chang Woo; Kim, Jung Eun; Do, Hackwon; Kim, Ryeo-Ok; Lee, Sung Gu; Park, Hyun Ho; Chang, Jeong Ho; Yim, Joung Han; Park, Hyun; Kim, Il-Chan; Lee, Jun Hyuck

    2015-09-11

    Fatty acid-binding proteins (FABPs) are involved in transporting hydrophobic fatty acids between various aqueous compartments of the cell by directly binding ligands inside their β-barrel cavities. Here, we report the crystal structures of ligand-unbound pFABP4, linoleate-bound pFABP4, and palmitate-bound pFABP5, obtained from gentoo penguin (Pygoscelis papua), at a resolution of 2.1 Å, 2.2 Å, and 2.3 Å, respectively. The pFABP4 and pFABP5 proteins have a canonical β-barrel structure with two short α-helices that form a cap region and fatty acid ligand binding sites in the hydrophobic cavity within the β-barrel structure. Linoleate-bound pFABP4 and palmitate-bound pFABP5 possess different ligand-binding modes and a unique ligand-binding pocket due to several sequence dissimilarities (A76/L78, T30/M32, underlining indicates pFABP4 residues) between the two proteins. Structural comparison revealed significantly different conformational changes in the β3-β4 loop region (residues 57-62) as well as the flipped Phe60 residue of pFABP5 than that in pFABP4 (the corresponding residue is Phe58). A ligand-binding study using fluorophore displacement assays shows that pFABP4 has a relatively strong affinity for linoleate as compared to pFABP5. In contrast, pFABP5 exhibits higher affinity for palmitate than that for pFABP4. In conclusion, our high-resolution structures and ligand-binding studies provide useful insights into the ligand-binding preferences of pFABPs based on key protein-ligand interactions.

  10. Model of β-Sheet of Muscle Fatty Acid Binding Protein of Locusta migratoria Displays Characteristic Topology.

    PubMed

    Kizilbash, Nadeem A; Hai, Abdul; Alruwaili, Jamal

    2013-01-01

    The β-sheet of muscle fatty acid binding protein of Locusta migratoria (Lm-FABP) was modeled by employing 2-D NMR data and the Rigid Body Assembly method. The model shows the β-sheet to comprise ten β-strands arranged anti-parallel to each other. There is a β-bulge between Ser 13 and Gln 14 which is a difference from the published structure of β-sheet of bovine heart Fatty Acid Binding Protein. Also, a hydrophobic patch consisting of Ile 45, Phe 51, Phe 64 and Phe 66 is present on the surface which is characteristic of most Fatty Acid Binding Proteins. A "gap" is present between βD and βE that provides evidence for the presence of a portal or opening between the polypeptide chains which allows ligand fatty acids to enter the protein cavity and bind to the protein.

  11. Structural analysis of binding functionality of folic acid-PEG dendrimers against folate receptor.

    PubMed

    Sampogna-Mireles, Diana; Araya-Durán, Ingrid D; Márquez-Miranda, Valeria; Valencia-Gallegos, Jesús A; González-Nilo, Fernando D

    2017-03-01

    Dendrimers functionalized with folic acid (FA) are drug delivery systems that can selectively target cancer cells with folate receptors (FR-α) overexpression. Incorporation of polyethylene glycol (PEG) can enhance dendrimers solubility and pharmacokinetics, but ligand-receptor binding must not be affected. In this work we characterized, at atomic level, the binding functionality of conventional site-specific dendrimers conjugated with FA with PEG 750 or PEG 3350 as a linker. After Molecular Dynamics simulation, we observed that both PEG's did not interfere over ligand-receptor binding functionality. Although binding kinetics could be notably affected, the folate fragment from both dendrimers remained exposed to the solvent before approaching selectively to FR-α. PEG 3350 provided better solubility and protection from enzymatic degradation to the dendrimer than PEG 750. Also, FA-PEG3350 dendrimer showed a slightly better interaction with FR-α than FA-PEG750 dendrimer. Therefore, theoretical evidence supports that both dendrimers are suitable as drug delivery systems for cancer therapies.

  12. Spectroscopic study on binding of gentisic acid to bovine serum albumin.

    PubMed

    Garzón, Andrés; Bravo, Iván; Carrión-Jiménez, M Rosario; Rubio-Moraga, Ángela; Albaladejo, José

    2015-01-01

    The interaction of (gentisic acid) GA with (bovine serum albumin) BSA has been studied by different spectroscopic techniques. GA is a monoanionic specie at the working pH of 7.4, it was determined by combining UV-Vis absorption spectroscopy and theoretical calculations. A set of fluorescence quenching experiments at different temperatures was carried out employing the native fluorescence of BSA. A Stern-Volmer constant (KSV) of (2.07±0.12)×10(4) mol(-1) L and a binding constant (Ka) of (8.47±4.39)×10(3) were determined at 310 K. The static quenching caused by the BSA-GA complex formation seems to play a significant role in the overall quenching process. A single binding site on BSA for GA was observed. ΔH=-55.6±0.2 kJ mol(-1) and ΔS=-104.3±0.6 J mol(-1) K(-1) were determined in a set of experiments on the dependence of Ka with the temperature. The binding process is, therefore, spontaneous and enthalpy-driven. Van der Waals forces and hydrogen bonds could also play the major role in the binding mode. The secondary structure changes of BSA in the absence and presence of GA were studied by FTIR and UV-Vis absorption spectroscopy.

  13. Isolation of dicarboxylic acid- and glucose-binding proteins from Pseudomonas aeruginosa.

    PubMed Central

    Stinson, M W; Cohen, M A; Merrick, J M

    1976-01-01

    Inducible binding proteins for C4-dicarboxylic acids (DBP) and glucose (GBP) were isolated from Pseudomonas aeruginosa by extraction of exponential-phase cells with 0.2 M MgC12 (pH 8.5) and by an osmotic shock procedure without affecting cell viability. DBP synthesis was induced by growth on aspartate, alpha-ketoglutarate, succinate, fumarate, malate, and malonate but not by growth on acetate, citrate, pyruvate, or glucose. Binding of succinate by DBP was competitively inhibited by 10-fold concentrations of fumarate and malate but not by a variety of related substances. GBP synthesis and transport of methyl alpha-glucoside by whole cells were induced by growth on glucose or pyruvate plus galactose, 2-deoxyglucose, or methyl alpha-glucoside but not by growth on gluconate, succinate, acetate, or pyruvate. The binding of radioactive glucose by GBP was significantly inhibited by 10-fold concentrations of glucose, galactose, and glucose-1-phosphate but not by the other carbohydrates tested. The binding of glucose by GBP or succinate by DBP did not result in any chemical alteration of the substrates. PMID:824281

  14. Binding of ring-substituted indole-3-acetic acids to human serum albumin.

    PubMed

    Soskić, Milan; Magnus, Volker

    2007-07-01

    The plant hormone, indole-3-acetic acid (IAA), and its ring-substituted derivatives have recently attracted attention as promising pro-drugs in cancer therapy. Here we present relative binding constants to human serum albumin for IAA and 34 of its derivatives, as obtained using the immobilized protein bound to a support suitable for high-performance liquid chromatography. We also report their octanol-water partition coefficients (logK(ow)) computed from retention data on a C(18) coated silica gel column. A four-parameter QSPR (quantitative structure-property relationships) model, based on physico-chemical properties, is put forward, which accounts for more than 96% of the variations in the binding affinities of these compounds. The model confirms the importance of lipophilicity as a global parameter governing interaction with serum albumin, but also assigns significant roles to parameters specifically related to the molecular topology of ring-substituted IAAs. Bulky substituents at ring-position 6 increase affinity, those at position 2 obstruct binding, while no steric effects were noted at other ring-positions. Electron-withdrawing substituents at position 5 enhance binding, but have no obvious effect at other ring positions.

  15. Binding of thyroglobulin to bovine thyroid membranes. Role of specific amino acids in receptor recognition.

    PubMed

    Shifrin, S; Kohn, L D

    1981-10-25

    Bovine thyroglobulin was treated with increasing ratios of succinic anhydride, trinitrobenzene sulfonic acid, tetranitromethane, and N-acetylimidazole in an attempt to assess the role of lysine or tyrosine residues in binding to thyroid membrane receptors. Extensive succinylation results in dissociation to 12 S thyroglobulin with retention of a considerable portion of the three-dimensional structure. Only 25% of the lysine residues can be modified by trinitrophenylation without affecting inter-subunit interactions. Succinylation as well as trinitrophenylation increases the affinity of thyroglobulin for the membrane receptor by a factor of 2. The binding of thyroglobulin to the membrane was reduced after nitration of 30% of the tyrosyl residues with tetranitromethane. O-Acetylation of 40-70% of the tyrosyl residues by N-acetylimidazole nearly abolished the ability of thyroglobulin to bind to the membrane. Removal of the O-acetyl group with hydroxylamine restored the binding properties. The results indicate that tyrosyl residues play an important role in thyroglobulin interactions with thyroid membranes.

  16. Spectroscopic evidence of a bidentate-binding of meso-2,3-dimercaptosuccinic acid on silver nanoclusters

    NASA Astrophysics Data System (ADS)

    Zaluzhna, Oksana; Brightful, Lyndsey; Allison, Thomas C.; Tong, YuYe J.

    2011-06-01

    New insight into the metal-ligand binding interaction in meso-2,3-dimercaptosuccinic acid (DMSA) protected silver nanoclusters (NCs) is presented in this work. IR, Raman and 13C NMR spectroscopic characterizations and DFT calculations suggest that DMSA forms a bidentate binding, rather than the originally-proposed monodentate binding, via two sulfur atoms with the underlying Ag7 NC, which is in agreement with recent ab initio calculations.

  17. Spectroscopic Evidence of a Bidentate-Binding of Meso-2,3-Dimercaptosuccinic Acid on Silver Nanoclusters

    SciTech Connect

    Zaluzhna, Oksana; Brightful, Lyndsey; Allison, Thomas C.; Tong, Yu ye J.

    2011-06-14

    New insight into the metal–ligand binding interaction in meso-2,3-dimercaptosuccinic acid (DMSA) protected silver nanoclusters (NCs) is presented in this work. IR, Raman and 13C NMR spectroscopic characterizations and DFT calculations suggest that DMSA forms a bidentate binding, rather than the originally- proposed monodentate binding, via two sulfur atoms with the underlying Ag7 NC, which is in agreement with recent ab initio calculations.

  18. Sacha inchi (Plukenetia volubilis): a seed source of polyunsaturated fatty acids, tocopherols, phytosterols, phenolic compounds and antioxidant capacity.

    PubMed

    Chirinos, Rosana; Zuloeta, Gledy; Pedreschi, Romina; Mignolet, Eric; Larondelle, Yvan; Campos, David

    2013-12-01

    Fatty acids (FA), phytosterols, tocopherols, phenolic compounds, total carotenoids and hydrophilic and lipophilic ORAC antioxidant capacities were evaluated in 16 cultivars of Sacha inchi (SI) seeds with the aim to valorise them and offer more information on the functional properties of SI seeds. A high α linolenic (α-Ln) fatty acid content was found in all cultivars (ω3, 12.8-16.0 g/100 g seed), followed by linoleic (L) fatty acid (ω6, 12.4-14.1g/100g seed). The ratio ω6/ω3 was within the 0.83-1.09 range. γ- and δ-tocopherols were the most important tocopherols, whereas the most representative phytosterols were β-sitosterol and stigmasterol. Contents of total phenolics, total carotenoids and hydrophilic and lipophilic antioxidant capacities ranged from 64.6 to 80 mg of gallic acid equivalent/100g seed; from 0.07 to 0.09 mg of β-carotene equivalent/100g of seed; from 4.3 to 7.3 and, from 1.0 to 2.8 μmol of Trolox equivalent/g of seed, respectively, among the evaluated SI cultivars. Results showed significant differences (p<0.05) among the evaluated SI cultivars in the contents of ω3, ω6, antioxidant capacities and other evaluated phytochemicals. SI seeds should be considered as an important dietary source of health promoting phytochemicals.

  19. Fatty Acids Profile, Phenolic Compounds and Antioxidant Capacity in Elicited Callus of Thevetia peruviana (Pers.) K. Schum.

    PubMed

    Rincón-Pérez, Jack; Rodríguez-Hernández, Ludwi; Ruíz-Valdiviezo, Víctor Manuel; Abud-Archila, Miguel; Luján-Hidalgo, María Celina; Ruiz-Lau, Nancy; González-Mendoza, Daniel; Gutiérrez-Miceli, Federico Antonio

    2016-01-01

    The aim of this study was analyze the effect of jasmonic acid (JA) and abscisic acid (ABA) as elicitors on fatty acids profile (FAP), phenolic compounds (PC) and antioxidant capacity (AC) in callus of Thevetia peruviana. Schenk & Hildebrandt (SH) medium, supplemented with 2 mg/L 2, 4-dichlorophenoxyacetic (2, 4-D) and 0.5 mg/L kinetin (KIN) was used for callus induction. The effect of JA (50, 75 and 100 μM) and ABA (10, 55 and 100 μM) on FAP, PC and AC were analyzed using a response surface design. A maximum of 2.8 mg/g of TPC was obtained with 100 plus 10 µM JA and ABA, respectively, whereas AC maximum (2.17 μg/mL) was obtained with 75 plus 100 µM JA and ABA, respectively. The FAP was affected for JA but not for ABA. JA increased cis-9, cis-12-octadecadienoic acid and decreased dodecanoic acid. Eight fatty acids were identified by GC-MS analysis and cis-9-octadecenoic acid (18:1) was the principal fatty acid reaching 76 % in treatment with 50 μM JA plus 55 μM ABA. In conclusion, JA may be used in T. peruviana callus culture for obtain oil with different fatty acids profile.

  20. Improving stability of virus-like particles by ion-exchange chromatographic supports with large pore size: advantages of gigaporous media beyond enhanced binding capacity.

    PubMed

    Yu, Mengran; Li, Yan; Zhang, Songping; Li, Xiunan; Yang, Yanli; Chen, Yi; Ma, Guanghui; Su, Zhiguo

    2014-02-28

    Limited binding capacity and low recovery of large size multi-subunits virus-like particles (VLPs) in conventional agarose-gel based chromatographic supports with small pores have long been a bottleneck limiting the large scale purification and application of VLPs. In this study, four anion exchange media including DEAE-Sepharose FF (DEAE-FF), DEAE-Capto, gigaporous DEAE-AP-120nm and DEAE-AP-280nm with average pore diameters of 32nm, 20nm, 120nm and 280nm, respectively, were applied for purification of hepatitis B virus surface antigen (HBsAg) VLPs. Pore size effects of media on the VLPs adsorption equilibrium, adsorption kinetics, dynamic binding capacity (DBC), and recovery were investigated in detail. According to the confocal laser scanning microscopy observation, adsorption of the VLPs in DEAE-FF and DEAE-Capto was mostly confined to a thin shell on the outer surface of the beads, leaving the underlying pore space and the binding sites inaccessibly, while the large pores in gigaporous media enabled the VLPs to access to the interior pore spaces by diffusion transport efficiently. Compared to the most widely used DEAE-FF, gigaporous media DEAE-AP-280nm gained about 12.9 times increase in static adsorption capacity, 8.0 times increase in DBC, and 11.4 times increase in effective pore diffusivity. Beyond increasing the binding capacity and enhancing the mass transfer, the gigaporous structure also significantly improved the stability of the VLPs during intensive adsorption-desorption process by lowing the multi-point interaction between the VLPs and binding sites in the pores. At 2.0mg/mL-media loading quantity, about 85.5% VLPs were correctly self-assembled after the chromatography with DEAE-AP-280nm media; oppositely about 85.2% VLPs lost their normal assembly with DEAE-FF due to irreversible disassembly. Comparative investigation was made to study the purifying performance of these four chromatographic media for actual VLPs purification from recombinant

  1. Metal loading effect on rare earth element binding to humic acid: Experimental and modelling evidence

    NASA Astrophysics Data System (ADS)

    Marsac, Rémi; Davranche, Mélanie; Gruau, Gérard; Dia, Aline

    2010-03-01

    The effect of metal loading on the binding of rare earth elements (REE) to humic acid (HA) was studied by combining ultrafiltration and Inductively Coupled Plasma Mass Spectrometry techniques. REE-HA complexation experiments were performed at pH 3 for REE/C molar ratios ranging from ca 4 × 10 -4 to 2.7 × 10 -2. Results show that the relative amount of REE bound to HA strongly increases with decreasing REE/C. A middle-REE (MREE) downward concavity is shown by patterns at high metal loading, whereas patterns at low metal loading display a regular increase from La to Lu. Humic Ion Model VI modelling are close to the experimental data variations, provided that (i) the ΔLK 2 parameter (i.e. the Model VI parameter taken into account the presence of strong but low density binding sites) is allowed to increase regularly from La to Lu (from 1.1 to 2.1) and (ii) the published log KMA values (i.e. the REE-HA binding constants specific to Model VI) are slightly modified, in particular with respect to heavy REE. Modelling approach provided evidence that logKdREE patterns with varying REE/C likely arises because REE binding to HA occurs through two types of binding sites in different density: (i) a few strong sites that preferentially complex the heavy REE and thus control the logKdREE atterns at low REE/C; (ii) a larger amount of weaker binding sites that preferentially complex the middle-REE and thus control the logKdREE pattern at high REE/C. Hence, metal loading exerts a major effect on HA-mediated REE binding, which could explain the diversity of published conditional constants for REE binding with HA. A literature survey suggests that the few strong sites activated at low REE/C could be multidentate carboxylic sites, or perhaps N-, or P-functional groups. Finally, an examination of the literature field data proposed that the described loading effect could account for much of the variation in REE patterns observed in natural organic-rich waters (DOC > 5 mg L -1 and 4

  2. Amino acid-based ionic liquids: using XPS to probe the electronic environment via binding energies.

    PubMed

    Hurisso, Bitu Birru; Lovelock, Kevin R J; Licence, Peter

    2011-10-21

    Here we report the synthesis and characterisation by X-ray photoelectron spectroscopy (XPS) of eight high purity amino acid-based ionic liquids (AAILs), each containing the 1-octyl-3-methylimidazolium, [C(8)C(1)Im](+), as a standard reference cation. All expected elements were observed and the electronic environments of these elements identified. A fitting model for the carbon 1s region of the AAILs is reported; the C aliphatic component of the cation was used as an internal reference to obtain a series of accurate and reproducible binding energies. Comparisons are made between XP spectra of the eight AAILs and selected non-functionalised ionic liquids. 1-octyl-3-methylimidazolium acetate was also studied as a model of the carboxyl containing amino acid anion. The influence of anionic substituent groups on the measured binding energies of all elements is presented, and communication between anion and cation is investigated. This data is interpreted in terms of hard and soft anions and compared to the Kamlet-Taft hydrogen bond acceptor ability, β, for the ionic liquids. A linear correlation is presented which suggests that the functional side chain, or R group, of the amino acid has little impact upon the electronic environment of the charge-bearing moieties within the anions and cations studied.

  3. cDNA structure, expression and nucleic acid-binding properties of three RNA-binding proteins in tobacco: occurrence of tissue-specific alternative splicing.

    PubMed Central

    Hirose, T; Sugita, M; Sugiura, M

    1993-01-01

    Three cDNAs encoding RNA-binding proteins were isolated from a tobacco (Nicotiana sylvestris) cDNA library. The predicted proteins (RGP-1) are homologous to each other and consist of a consensus-sequence type RNA-binding domain of 80 amino acids in the N-terminal half and a glycine-rich domain of 61-78 amino acids in the C-terminal half. Nucleic acid-binding assay using the in vitro synthesized RGP-1 protein confirmed that it is an RNA-binding protein. Based on its strong affinity for poly(G) and poly(U), the RGP-1 proteins are suggested to bind specifically to G and/or U rich sequences. All three genes are expressed in leaves, roots, flowers and cultured cells, however, the substantial amount of pre-mRNAs are accumulated especially in roots. Sequence analysis and ribonuclease protection assay indicated that significant amounts of alternatively spliced mRNAs, which are produced by differential selection of 5' splice sites, are also present in various tissues. Tissue-specific alternative splicing was found in two of the three genes. The alternatively spliced mRNAs are also detected in polysomal fractions and are suggested to produce truncated polypeptides. A possible role of this alternative splicing is discussed. Images PMID:8371974

  4. Screening nucleotide binding to amino acid-coated supports by surface plasmon resonance and nuclear magnetic resonance.

    PubMed

    Cruz, Carla; Cabrita, Eurico J; Queiroz, João A

    2011-08-01

    Here, we describe a rapid and efficient screening method using surface plasmon resonance (SPR) and saturation transfer difference-nuclear magnetic resonance (STD-NMR) spectroscopy to yield information regarding the residues involved in nucleotide binding to amino acid-coated supports. The aim of this work was to explore the use of these spectroscopic techniques to study amino acid-nucleotide interactions in order to improve the binding specificity of the amino acid ligands used to purify plasmid DNA. For SPR, we present a strategy that immobilizes arginine and lysine on a surface as model supports, and we analyze binding responses when synthetic homo-deoxyoligonucleotides are injected over the amino acid surface. The binding responses are detectable and reproducible despite the small size of the immobilized amino acids. Using STD-NMR, we performed epitope mapping of homo-deoxyoligonucleotides bound to L-arginine-bisoxyran-Sepharose and L-lysine-Sepharose supports. Polynucleotide binding preferences differed; for example, polyC interacted preferentially through its backbone with the two supports, whereas polyT bound the supports through its thymine moiety. STD-NMR combined with SPR measurements was successfully used to screen amino acid-nucleotide interactions and determine the binding affinities of the complexes.

  5. Tertiary structure of human alpha1-acid glycoprotein (orosomucoid). Straightforward fluorescence experiments revealing the presence of a binding pocket.

    PubMed

    Albani, Jihad R

    2004-02-25

    Binding of hemin to alpha1-acid glycoprotein has been investigated. Hemin binds to the hydrophobic pocket of hemoproteins. The fluorescent probe 2-(p-toluidino)-6-naphthalenesulfonate (TNS) binds to a hydrophobic domain in alpha1-acid glycoprotein with a dissociation constant equal to 60 microM. Addition of hemin to an alpha1-acid glycoprotein-TNS complex induces the displacement of TNS from its binding site. At saturation (1 hemin for 1 protein) all the TNS has been displaced from its binding site. The dissociation constant of hemin-alpha1-acid glycoprotein was found equal to 2 microM. Thus, TNS and hemin bind to the same hydrophobic site: the pocket of alpha1-acid glycoprotein. Energy-transfer studies performed between the Trp residues of alpha1-acid glycoprotein and hemin indicated that efficiency (E) of Trp fluorescence quenching was equal to 80% and the Förster distance, R0 at which the efficiency of energy transfer is 50% was calculated to be 26 A, revealing a very high energy transfer.

  6. The effects of calcium channel blocker benidipine and calmodulin antagonist W7 on GDP-binding capacity of brown adipose tissue in mice.

    PubMed

    Song, Yuh-Min; Lin, Pi-Yao; Chen, Ming-Der

    2009-03-01

    It has been suggested that increased dietary calcium intake can attenuate obesity. Calcium antagonists, such as benidipine, also have been shown to have an anti-obesity effect. However, the mechanism for calcium-related anti-obesity effect has not yet been established. A defective brown adipose tissue thermogenesis has been shown in obese rodents. This study was designed to examine the direct effects of calcium channel blocker benidipine and calmodulin antagonist W7 administration on the adaptive thermogenesis in brown adipose tissue taken from the genetically obese mice and their lean controls. The GDP binding to brown-fat cell mitochondria was used as a brown adipose tissue thermogenic index. The results show that benidipine treatment had no marked effect on brown-fat cell GDP-binding capacities in both obese and lean mice. However, GDP-binding capacities were significantly reduced in both obese and lean mice after the W7 administration. The results of this study support the previous finding that benidipine did not have direct thermogenic effect on brown adipose tissue and suggest that the change in intracellular calmodulin availability might contribute to the adaptive thermogenesis in brown adipose tissue.

  7. Catalytic and ligand binding properties of the FK506 binding protein FKBP12: effects of the single amino acid substitution of Tyr82 to Leu.

    PubMed Central

    Bossard, M J; Bergsma, D J; Brandt, M; Livi, G P; Eng, W K; Johnson, R K; Levy, M A

    1994-01-01

    The binding of FK506 and rapamycin to their cytosolic receptor FKBP12 is an intermediate step in the paths leading to their potent immunosuppressive properties. One of the amino acids defining the hydrophobic binding cleft for the macrocycles is Tyr82, which is thought to form a hydrogen bond with the amide oxygens of the common pipecolyl structural element within the two macrolides. To understand better the influence of this amino acid residue in catalytic activity (cis-trans peptidyl prolyl isomerization) and ligand binding properties, a Tyr82 to Leu site-specific modification of FKBP12 was prepared, purified and characterized. Kinetic experiments have demonstrated that the Tyr82 to Leu modification has a greater effect on catalytic properties than on ligand binding affinities, a result which indicates that these inhibitors may not be binding as true transition-state analogues. In an additional test for cellular function, expression of both wild-type and mutant human FKBP12 in a strain of Saccharomyces cerevisiae rendered resistant to rapamycin by deletion of the gene encoding a cytosolic rapamycin binding protein (RPB1), the yeast homologue of FKBP12, restored wild-type drug sensitivity. PMID:7507662

  8. d-Amino Acid Probes for Penicillin Binding Protein-based Bacterial Surface Labeling*

    PubMed Central

    Fura, Jonathan M.; Kearns, Daniel; Pires, Marcos M.

    2015-01-01

    Peptidoglycan is an essential and highly conserved mesh structure that surrounds bacterial cells. It plays a critical role in retaining a defined cell shape, and, in the case of pathogenic Gram-positive bacteria, it lies at the interface between bacterial cells and the host organism. Intriguingly, bacteria can metabolically incorporate unnatural d-amino acids into the peptidoglycan stem peptide directly from the surrounding medium, a process mediated by penicillin binding proteins (PBPs). Metabolic peptidoglycan remodeling via unnatural d-amino acids has provided unique insights into peptidoglycan biosynthesis of live bacteria and has also served as the basis of a synthetic immunology strategy with potential therapeutic implications. A striking feature of this process is the vast promiscuity displayed by PBPs in tolerating entirely unnatural side chains. However, the chemical space and physical features of this side chain promiscuity have not been determined systematically. In this report, we designed and synthesized a library of variants displaying diverse side chains to comprehensively establish the tolerability of unnatural d-amino acids by PBPs in both Gram-positive and Gram-negative organisms. In addition, nine Bacillus subtilis PBP-null mutants were evaluated with the goal of identifying a potential primary PBP responsible for unnatural d-amino acid incorporation and gaining insights into the temporal control of PBP activity. We empirically established the scope of physical parameters that govern the metabolic incorporation of unnatural d-amino acids into bacterial peptidoglycan. PMID:26499795

  9. Preferential lectin binding of cancer cells upon sialic acid treatment under nutrient deprivation.

    PubMed

    Badr, Haitham A; Elsayed, Abdelaleim I; Ahmed, Hafiz; Dwek, Miriam V; Li, Chen-Zhong; Djansugurova, Leyla B

    2013-10-01

    The terminal monosaccharide of glycoconjugates on a eukaryotic cell surface is typically a sialic acid (Neu5Ac). Increased sialylation usually indicates progression and poor prognosis of most carcinomas. Here, we utilize two human mammary epithelial cell lines, HB4A (breast normal cells) and T47D (breast cancer cells), as a model system to demonstrate differential surface glycans when treated with sialic acid under nutrient deprivation. Under a starved condition, sialic acid treatment of both cells resulted in increased activities of α2→3/6 sialyltransferases as demonstrated by solid phase assay using lectin binding. However, a very strong Maackia amurensis agglutinin I (MAL-I) staining on the membrane of sialic acid-treated T47D cells was observed, indicating an increase of Neu5Acα2→3Gal on the cell surface. To our knowledge, this is a first report showing the utility of lectins, particularly MAL-I, as a means to discriminate between normal and cancer cells after sialic acid treatment under nutrient deprivation. This method is sensitive and allows selective detection of glycan sialylation on a cancer cell surface.

  10. Betulin binds to gamma-aminobutyric acid receptors and exerts anticonvulsant action in mice.

    PubMed

    Muceniece, Ruta; Saleniece, Kristine; Rumaks, Juris; Krigere, Liga; Dzirkale, Zane; Mezhapuke, Rudolfs; Zharkova, Olga; Klusa, Vija

    2008-10-01

    The lupane type pentacyclic triterpenes: lupeol, betulin, and betulinic acid are widely distributed natural compounds. Recently, pharmaceutical compositions from plant extracts (family Marcgraviaceae) containing betulinic acid, have been patented as anxiolytic remedies. To extend our knowledge of the CNS effects of the triterpenes, we suggest here that the chemically related lupeol, betulin and betulinic acid may interact with the brain neurotransmitter gamma-aminobutyric acid (GABA) receptors in vitro and in vivo. Using radioligand receptor-binding assay, we showed that only betulin bound to the GABA(A)-receptor sites in mice brain in vitro and antagonised the GABA(A)-receptor antagonist bicuculline-induced seizures in mice after intracisternal and intraperitoneal administration. Neither betulinic acid nor lupeol bound to GABA(A) receptor nor did they inhibit bicuculline-induced seizures in vivo. These findings demonstrate for the first time the CNS effects of betulin in vivo, and they also show distinct GABA(A)-receptor-related properties of lupane type triterpenes. These findings may open new avenues in understanding the central effects of betulin, and they also indicate possibilities for novel drug design on the basis of betulin structure.

  11. In situ detection of salicylic acid binding sites in plant tissues.

    PubMed

    Liu, Jing-Wen; Deng, Da-Yi; Yu, Ying; Liu, Fang-Fei; Lin, Bi-Xia; Cao, Yu-Juan; Hu, Xiao-Gang; Wu, Jian-Zhong

    2015-02-01

    The determination of hormone-binding sites in plants is essential in understanding the mechanisms behind hormone function. Salicylic acid (SA) is an important plant hormone that regulates responses to biotic and abiotic stresses. In order to label SA-binding sites in plant tissues, a quantum dots (QDs) probe functionalized with a SA moiety was successfully synthesized by coupling CdSe QDs capped with 3-mercaptopropionic acid (MPA) to 4-amino-2-hydroxybenzoic acid (PAS), using 1-ethyl-3-(3-dimethyllaminopropyl) carbodiimide (EDC) as the coupling agent. The probe was then characterized by dynamic light scattering and transmission electron microscopy, as well as UV/vis and fluorescence spectrophotometry. The results confirmed the successful conjugation of PAS to CdSe QDs and revealed that the conjugates maintained the properties of the original QDs, with small core diameters and adequate dispersal in solution. The PAS-CdSe QDs were used to detect SA-binding sites in mung bean and Arabidopsis thaliana seedlings in vitro and in vivo. The PAS-CdSe QDs were effectively transported into plant tissues and specifically bound to SA receptors in vivo. In addition, the effects of the PAS-CdSe QDs on cytosolic Ca(2+) levels in the tips of A. thaliana seedlings were investigated. Both SA and PAS-CdSe QDs had similar effects on the trend in cytosolic-free Ca(2+) concentrations, suggesting that the PAS-CdSe QDs maintained the bioactivity of SA. To summarize, PAS-CdSe QDs have high potential as a fluorescent probe for the in vitro/in vivo labeling and imaging of SA receptors in plants.

  12. Structural analysis of site-directed mutants of cellular retinoic acid-binding protein II addresses the relationship between structural integrity and ligand binding

    SciTech Connect

    Vaezeslami, Soheila; Jia, Xiaofei; Vasileiou, Chrysoula; Borhan, Babak; Geiger, James H.

    2008-12-01

    A water network stabilizes the structure of cellular retionic acid binding protein II. The structural integrity of cellular retinoic acid-binding protein II (CRABPII) has been investigated using the crystal structures of CRABPII mutants. The overall fold was well maintained by these CRABPII mutants, each of which carried multiple different mutations. A water-mediated network is found to be present across the large binding cavity, extending from Arg111 deep inside the cavity to the α2 helix at its entrance. This chain of interactions acts as a ‘pillar’ that maintains the integrity of the protein. The disruption of the water network upon loss of Arg111 leads to decreased structural integrity of the protein. A water-mediated network can be re-established by introducing the hydrophilic Glu121 inside the cavity, which results in a rigid protein with the α2 helix adopting an altered conformation compared with wild-type CRABPII.

  13. Binding of Autotaxin to Integrins Localizes Lysophosphatidic Acid Production to Platelets and Mammalian Cells*

    PubMed Central

    Fulkerson, Zachary; Wu, Tao; Sunkara, Manjula; Kooi, Craig Vander; Morris, Andrew J.; Smyth, Susan S.

    2011-01-01

    Autotaxin (ATX) is a secreted lysophospholipase D that generates the bioactive lipid mediator lysophosphatidic acid (LPA). We and others have reported that ATX binds to integrins, but the function of ATX-integrin interactions is unknown. The recently reported crystal structure of ATX suggests a role for the solvent-exposed surface of the N-terminal tandem somatomedin B-like domains in binding to platelet integrin αIIbβ3. The opposite face of the somatomedin B-like domain interacts with the catalytic phosphodiesterase (PDE) domain to form a hydrophobic channel through which lysophospholipid substrates enter and leave the active site. Based on this structure, we hypothesize that integrin-bound ATX can access cell surface substrates and deliver LPA to cell surface receptors. To test this hypothesis, we investigated the integrin selectivity and signaling pathways that promote ATX binding to platelets. We report that both platelet β1 and β3 integrins interact in an activation-dependent manner with ATX via the SMB2 domain. ATX increases thrombin-stimulated LPA production by washed platelets ∼10-fold. When incubated under conditions to promote integrin activation, ATX generates LPA from CHO cells primed with bee venom phospholipase A2, and ATX-mediated LPA production is enhanced more than 2-fold by CHO cell overexpression of integrin β3. The effects of ATX on platelet and cell-associated LPA production, but not hydrolysis of small molecule or detergent-solubilized substrates, are attenuated by point mutations in the SMB2 that impair integrin binding. Integrin binding therefore localizes ATX activity to the cell surface, providing a mechanism to generate LPA in the vicinity of its receptors. PMID:21832043

  14. Acid Hydrolysis of Wheat Gluten Induces Formation of New Epitopes but Does Not Enhance Sensitizing Capacity by the Oral Route: A Study in “Gluten Free” Brown Norway Rats

    PubMed Central

    Kroghsbo, Stine; Andersen, Nanna B.; Rasmussen, Tina F.; Madsen, Charlotte B.

    2014-01-01

    Background Acid hydrolyzed wheat proteins (HWPs) are used in the food and cosmetic industry as emulsifiers. Cases of severe food allergic reactions caused by HWPs have been reported. Recent data suggest that these reactions are caused by HWPs produced by acid hydrolysis. Objectives To examine the sensitizing capacity of gluten proteins per se when altered by acid or enzymatic hydrolysis relative to unmodified gluten in rats naïve to gluten. Methods High IgE-responder Brown Norway (BN) rats bred on a gluten-free diet were sensitized without the use of adjuvant to three different gluten products (unmodified, acid hydrolyzed and enzymatic hydrolyzed). Rats were sensitized by intraperitoneal (i.p.) immunization three times with 200 µg gluten protein/rat or by oral dosing for 35 days with 0.2, 2 or 20 mg gluten protein/rat/day. Sera were analyzed for specific IgG and IgE and IgG-binding capacity by ELISA. IgE functionality was measured by rat basophilic leukemia (RBL) assay. Results Regardless of the route of dosing, all products had sensitizing capacity. When sensitized i.p., all three gluten products induced a strong IgG1 response in all animals. Acid hydrolyzed gluten induced the highest level of specific IgE but with a low functionality. Orally all three gluten products induced specific IgG1 and IgE but with different dose-response relations. Sensitizing rats i.p. or orally with unmodified or enzymatic hydrolyzed gluten induced specific IgG1 responses with similar binding capacity which was different from that of acid hydrolyzed gluten indicating that acid hydrolysis of gluten proteins induces formation of ‘new’ epitopes. Conclusions In rats not tolerant to gluten acid hydrolysis of gluten enhances the sensitizing capacity by the i.p. but not by the oral route. In addition, acid hydrolysis induces formation of new epitopes. This is in contrast to the enzymatic hydrolyzed gluten having an epitope pattern similar to unmodified gluten. PMID:25207551

  15. Clinical benefit using sperm hyaluronic acid binding technique in ICSI cycles: a systematic review and meta-analysis.

    PubMed

    Beck-Fruchter, Ronit; Shalev, Eliezer; Weiss, Amir

    2016-03-01

    The human oocyte is surrounded by hyaluronic acid, which acts as a natural selector of spermatozoa. Human sperm that express hyaluronic acid receptors and bind to hyaluronic acid have normal shape, minimal DNA fragmentation and low frequency of chromosomal aneuploidies. Use of hyaluronic acid binding assays in intracytoplasmic sperm injection (ICSI) cycles to improve clinical outcomes has been studied, although none of these studies had sufficient statistical power. In this systematic review and meta-analysis, electronic databases were searched up to June 2015 to identify studies of ICSI cycles in which spermatozoa able to bind hyaluronic acid was selected. The main outcomes were fertilization rate and clinical pregnancy rate. Secondary outcomes included cleavage rate, embryo quality, implantation rate, spontaneous abortion and live birth rate. Seven studies and 1437 cycles were included. Use of hyaluronic acid binding sperm selection technique yielded no improvement in fertilization and pregnancy rates. A meta-analysis of all available studies showed an improvement in embryo quality and implantation rate; an analysis of prospective studies only showed an improvement in embryo quality. Evidence does not support routine use of hyaluronic acid binding assays in all ICSI cycles. Identification of patients that might benefit from this technique needs further study.

  16. Premature capacity loss in lead/acid batteries: a discussion of the antimony-free effect and related phenomena

    NASA Astrophysics Data System (ADS)

    Hollenkamp, A. F.

    Instances of severe capacity loss in apparently healthy lead/acid batteries have been reported over a period of many years, and are still common today. In most cases, these phenomena are linked to the use of antimony-free positive grids and are invoked by repetitive deep-discharge duties. This situation represents probably the greatest barrier to the expansion of markets for lead/acid batteries. To date, research has focused on several possible explanations for capacity loss; notably, degradation of the positive active mass (e.g., relaxable insufficient mass utilization) and the development of electrical barriers around the grid. Although much of the evidence gathered is circumstantial, it does point to the key issues that must be addressed in future work.

  17. Amino Acids in the Basic Domain of Epstein-Barr Virus ZEBRA Protein Play Distinct Roles in DNA Binding, Activation of Early Lytic Gene Expression, and Promotion of Viral DNA Replication

    PubMed Central

    Heston, Lee; El-Guindy, Ayman; Countryman, Jill; Dela Cruz, Charles; Delecluse, Henri-Jacques; Miller, George

    2006-01-01

    The ZEBRA protein of Epstein-Barr virus (EBV) drives the viral lytic cycle cascade. The capacity of ZEBRA to recognize specific DNA sequences resides in amino acids 178 to 194, a region in which 9 of 17 residues are either lysine or arginine. To define the basic domain residues essential for activity, a series of 46 single-amino-acid-substitution mutants were examined for their ability to bind ZIIIB DNA, a high-affinity ZEBRA binding site, and for their capacity to activate early and late EBV lytic cycle gene expression. DNA binding was obligatory for the protein to activate the lytic cascade. Nineteen mutants that failed to bind DNA were unable to disrupt latency. A single acidic replacement of a basic amino acid destroyed DNA binding and the biologic activity of the protein. Four mutants that bound weakly to DNA were defective at stimulating the expression of Rta, the essential first target of ZEBRA in lytic cycle activation. Four amino acids, R183, A185, C189, and R190, are likely to contact ZIIIB DNA specifically, since alanine or valine substitutions at these positions drastically weakened or eliminated DNA binding. Twenty-three mutants were proficient in binding to ZIIIB DNA. Some DNA binding-proficient mutants were refractory to supershift by BZ-1 monoclonal antibody (epitope amino acids 214 to 230), likely as the result of the increased solubility of the mutants. Mutants competent to bind DNA could be separated into four functional groups: the wild-type group (eight mutants), a group defective at activating Rta (five mutants, all with mutations at the S186 site), a group defective at activating EA-D (three mutants with the R179A, S186T, and K192A mutations), and a group specifically defective at activating late gene expression (seven mutants). Three late mutants, with a Y180A, Y180E, or K188A mutation, were defective at stimulating EBV DNA replication. This catalogue of point mutants reveals that basic domain amino acids play distinct functions in binding

  18. Visualization of Iron-Binding Micelles in Acidic Recombinant Biomineralization Protein, MamC

    DOE PAGES

    Kashyap, Sanjay; Woehl, Taylor; Valverde-Tercedor, Carmen; ...

    2014-01-01

    Biological macromolecules are utilized in low-temperature synthetic methods to exert precise control over nanoparticle nucleation and placement. They enable low-temperature formation of a variety of functional nanostructured materials with properties often not achieved via conventional synthetic techniques. Here we report on the in situ visualization of a novel acidic bacterial recombinant protein, MamC, commonly present in the magnetosome membrane of several magnetotactic bacteria, including Magnetococcus marinus , strain MC-1. Our findings provide an insight into the self-assembly of MamC and point to formation of the extended protein surface, which is assumed to play an important role in the formationmore » of biotemplated inorganic nanoparticles. The self-organization of MamC is compared to the behavior of another acidic recombinant iron-binding protein, Mms6.« less

  19. Restoration of Ca2+ influx and degranulation capacity of variant RBL-2H3 cells upon implantation of isolated cromolyn binding protein

    PubMed Central

    Mazurek, N.; Bashkin, P.; Loyter, A.; Pecht, I.

    1983-01-01

    Recently, variants of the rat basophilic leukemia cells (RBL-2H3), deficient in their binding capacity for the antiallergic drug cromolyn but displaying unimpaired ability to bind IgE, were selected and cloned [Mazurek, N., Bashkin, P., Petrank, A. & Pecht, I. (1983) Nature (London) 303, 528-530]. Although the histamine content and the number of IgE receptors in these variants are similar to those of the parental cells, they cannot be stimulated immunologically to allow Ca2+ influx and to degranulate. In contrast, the Ca2+ ionophore A23187 causes these variants to degranulate, indicating that the mechanism distal to the Ca2+ gating is intact in the variants. The cromolyn binding protein (CBP), present in the membranes of RBL-2H3 cells, has recently been isolated by affinity chromatography under nondenaturing conditions. In the current study we have used Sendaivirus envelopes as fusogenic carriers to implant the purified CBP into the membrane of variant basophils that were defective in it. This fusion leads to the restoration of Ca2+ uptake and degranulation capacity of the variants after IgE-mediated stimulation. These restored activities seem to show a sigmoidal dependence on the amount of incorporated CBP. Saturation values comparable to those of the parental line are reached when the level of implanted CBP approaches its density on the latter line. The restored capacity is due to the implanted CBP, because the reinstated immunological response can be blocked by the inhibitory drug cromolyn and by monoclonal antibodies specific to CBP, both shown to prevent Ca2+ uptake and degranulation in mast cells and parental RBL-2H3 cells. These results point out that CBP plays an important role in the Ca2+ gating process resulting in degranulation. Images PMID:6577465

  20. Effects of feeding omega-3-fatty acids on fatty acid composition and quality of bovine sperm and on antioxidative capacity of bovine seminal plasma.

    PubMed

    Gürler, Hakan; Calisici, Oguz; Calisici, Duygu; Bollwein, Heinrich

    2015-09-01

    The aim of the present study was to examine the effects of feeding alpha-linolenic (ALA) acid on fatty acid composition and quality of bovine sperm and on antioxidative capacity of seminal plasma. Nine bulls (ALA bulls) were fed with 800 g rumen-resistant linseed oil with a content of 50% linolenic acid and eight bulls with 400 g palmitic acid (PA bulls). Sperm quality was evaluated for plasma membrane and acrosome intact sperm (PMAI), the amount of membrane lipid peroxidation (LPO), and the percentage of sperm with a high DNA fragmentation index (DFI). Fatty acid content of sperm was determined using gas chromatography. Total antioxidant capacity, glutathione peroxidase, and superoxide dismutase activity were determined in seminal plasma. Feeding ALA increased (P < 0.05) the docosahexaenoic acid (DHA) content in bulls whereas in PA bulls did not change. PMAI increased after cryopreservation in ALA bulls as well as in PA bulls during the experiment period (P < 0.005). LPO of sperm directly after thawing did not change during the study period in ALA group, but decreased in PA group (P < 0.006). After 3h of incubation LPO increased in the ALA group (P < 0.02), while LPO did not differ between phases within groups. In conclusion, feeding of neither saturated nor polyunsaturated fatty acids affect the antioxidant levels in seminal plasma. Both saturated as well as polyunsaturated fatty acids had positive effects on quality of cryopreserved bovine sperm, although the content of docosahexaenoic acid in sperm membranes increased only in ALA bulls.

  1. Synthesis and DNA-binding properties of novel DNA cyclo-intercalators containing purine-glucuronic acid hybrids.

    PubMed

    Zhang, Renshuai; Chen, Shaopeng; Wang, Xueting; Yu, Rilei; Li, Mingjing; Ren, Sumei; Jiang, Tao

    2016-06-24

    Novel DNA cyclo-intercalators, which incorporated two intercalator subunits linked by two bridges, were synthesized. Binding of the compounds to calf-thymus DNA was studied by fluorescence spectroscopy, and docking simulations were used to predict the binding modes of these cyclic compounds. The spectral data demonstrated that all of these compounds can interact with CT-DNA. The sugar moiety played an important role in the process of binding between the intercalators containing glucuronic acid and DNA. The length and flexibility of the connecting bridges affected the binding affinity of the resultant cyclo-intercalators. Docking simulations showed that compounds 7 and 8 interact with DNA as mono-intercalators.

  2. Peptide immobilized monolith containing tentacle-type functionalized polymer chains for high-capacity binding of immunoglobulin G.

    PubMed

    Du, Kaifeng

    2014-12-29

    A peptide immobilized tentacle-type monolith is developed here for high-performance IgG purification. In this work, the glucose-anchored GMA molecules serve as monomers to be grafted into the tentacle-type chains on highly porous monolith by a series of chemical reactions. While maintaining high column permeability, the tentacle grafting endows the monolith with lots of reactive handles to anchor more peptides. With that, the grafted monolith shows high peptide density of about 155μmolmL(-1), up to approximately 4.7 times higher over the ungrafted one (33μmolmL(-1)). As a result, the static adsorbing capacity and dynamic adsorption capacity at 50% breakthrough point reach 101.8 and 83.3mgmL(-1) for IgG adsorption, respectively. Regeneration, recycle and reuse of grafted monolith are highly successful for 25 runs without obvious capacity loss. By taking these advantages of high capacity and excellent structure stability, the affinity grafted monolith is evaluated by using cleared human blood supernatant. And the result shows the peptide immobilized tentacle type monolith displays excellent specificity and high effectiveness for IgG purification.

  3. Synthesis, characterisation and adsorption properties of a porous copper(II) 3D coordination polymer exhibiting strong binding enthalpy and adsorption capacity for carbon dioxide.

    PubMed

    Eckold, Pierre; Gee, William J; Hill, Matthew R; Batten, Stuart R

    2012-11-21

    The synthesis and characterisation of microporous coordination polymers containing copper(II) or cobalt(II) and 2-(pyridin-4-yl)malonaldehyde (Hpma) is described and the gas adsorption properties evaluated. Single-crystal X-ray structure determinations identified the structures as [M(pma)(2)]·2X (M = Cu, 1; Co, 2; X = MeOH, MeCN), which contain 3D networks with rutile topology and continuous 1D rectangular channels with diameters ranging from 3 to 4 Å. The materials exhibit low BET surface areas of 143 m(2) g(-1), but possess large capacities for carbon dioxide capture of 14.1 wt%. The small pore channels are shown to account for this, delivering a particularly strong binding enthalpy to adsorbed CO(2) of 38 kJ mol(-1), and a very large adsorption capacity relative to the low surface area.

  4. Expression of fatty acid binding proteins is altered in aged mouse brain.

    PubMed

    Pu, L; Igbavboa, U; Wood, W G; Roths, J B; Kier, A B; Spener, F; Schroeder, F

    1999-08-01

    Brain membrane lipid fatty acid composition and consequently membrane fluidity change with increasing age. Intracellular fatty acid binding proteins (FABPs) such as heart H-FABP and the brain specific B-FABP, detected by immunoblotting of brain tissue, are thought to be involved in fatty acid uptake, metabolism, and differentiation in brain. Yet, almost nothing is known regarding the effect of age on the expression of the cytosolic fatty acid binding proteins (FABPs) or their content in brain subfractions. Electrophoresis and quantitative immunoblotting were used to examine the content of these FABPs in synaptosomes in brains from 4, 15, and 25 month old C57BL/6NNia male mice. Brain H-FABP and B-FABP were differentially expressed in mouse brain subcellular fractions. Brain H-FABP was highly concentrated in synaptosomal cytosol. The level of brain H-FABP in synaptosomes, synaptosomal cytosol, and intrasynaptosomal membranes was decreased 33, 35, and 43%, respectively, in 25 month old mice. B-FABP was detected in lower quantity than H-FABP. More important, B-FABP decreased in synaptosomes, synaptic plasma membranes, and synaptosomal cytosol from brains of 25 month old mice. In contrast to H-FABP, B-FABP was not detectable in the intrasynaptosomal membranes in any of the three age groups of mice. In conclusion, expression of both H-FABP and B-FABP was markedly reduced in aged mouse brain. Age differences in brain H-FABP and B-FABP levels in synaptosomal plasma membranes and synaptosomal cytosol may be important factors modulating neuronal differentiation and function.

  5. Structural and biochemical characterization of the lungfish (Lepidosiren paradoxa) liver basic fatty acid binding protein.

    PubMed

    Di Pietro, S M; Santomé, J A

    2001-04-01

    Only one fatty acid-binding protein (FABP) from the liver of the lungfish (Lepidosiren paradoxa) was isolated and characterized. The sequence comparison of lungfish FABP with that of the known members of the liver FABP (L-FABP) and liver basic FABP (Lb-FABP) subfamilies indicates that it is more closely related to chicken, iguana, frog, axolotl, catfish, and shark Lb-FABPs than to mammalian and axolotl L-FABPs. Lungfish liver expression of this single Lb-FABP contrasts with the other fish studied so far which coexpress an Lb-FABP with heart-adipocyte and/or intestinal FABP types. The lungfish liver FABP expression pattern resembles that of tetrapods, which only expresses liver type FABPs. Lungfish Lb-FABP is one of the two FABPs reported to have a disulfide bridge. The molecular modeling of lungfish Lb-FABP predicts that nine of the conserved residues of Lb-FABPs are oriented toward the binding cavity, thus suggesting they are related to the protein binding characteristics.

  6. Introduction of Ca(2+)-binding amino-acid sequence into the T4 lysozyme.

    PubMed

    Leontiev, V V; Uversky, V N; Permyakov, E A; Murzin, A G

    1993-03-05

    The 51-62 loop of T4 phage lysozyme was altered by site-directed mutagenesis to obtain maximal homology with the typical EF-hand motif. A Ca(2+)-binding site was designed and created by replacing both Gly-51 and Asn-53 with aspartic acid. The mutant T4 lysozyme (G51D/N53D) was expressed in Escherichia coli. The activity of the G51D/N53D-mutant was about 60% of that of the wild-type protein. This mutant can bind Ca2+ ions specifically, while the effective dissociation constant was essentially greater than that of the EF-hand proteins. Stability of the G51D/N53D-mutant apo-form to urea- or temperature-induced denaturation was the same as that of the wild-type protein. In the presence of Ca2+ ions in solution the stability of the mutant T4 phage lysozyme was less than that of the wild-type protein. It is suggested that the binding of Ca2+ by the mutant is accompanied by the considerable conformational changes in the 'corrected' loop, which can lead to the Ca(2+)-induced destabilization of the protein.

  7. Competitive Binding to Cuprous Ions of Protein and BCA in the Bicinchoninic Acid Protein Assay

    PubMed Central

    Huang, Tao; Long, Mian; Huo, Bo

    2010-01-01

    Although Bicinchoninic acid (BCA) has been widely used to determine protein concentration, the mechanism of interaction between protein, copper ion and BCA in this assay is still not well known. Using the Micro BCA protein assay kit (Pierce Company), we measured the absorbance at 562 nm of BSA solutions with different concentrations of protein, and also varied the BCA concentration. When the concentration of protein was increased, the absorbance exhibited the known linear and nonlinear increase, and then reached an unexpected plateau followed by a gradual decrease. We introduced a model in which peptide chains competed with BCA for binding to cuprous ions. Formation of the well-known chromogenic complex of BCA-Cu1+-BCA was competed with the binding of two peptide bonds (NTPB) to cuprous ion, and there is the possibility of the existence of two new complexes. A simple equilibrium equation was established to describe the correlations between the substances in solution at equilibrium, and an empirical exponential function was introduced to describe the reduction reaction. Theoretical predictions of absorbance from the model were in good agreement with the measurements, which not only validated the competitive binding model, but also predicted a new complex of BCA-Cu1+-NTPB that might exist in the final solution. This work provides a new insight into understanding the chemical bases of the BCA protein assay and might extend the assay to higher protein concentration. PMID:21625379

  8. DNA binding mode of novel tetradentate amino acid based 2-hydroxybenzylidene-4-aminoantipyrine complexes

    NASA Astrophysics Data System (ADS)

    Raman, N.; Sobha, S.; Selvaganapathy, M.; Mahalakshmi, R.

    2012-10-01

    Few transition metal complexes of tetradentate N2O2 donor Schiff base ligands containing 2-hydroxybenzylidene-4-aminoantipyrine and amino acids (alanine/valine) abbreviated to KHL1/KHL2 have been synthesized. All the metal complexes have been fully characterized with the help of elemental analyses, molecular weights, molar conductance values, magnetic moments and spectroscopic data. The Schiff bases KHL1/KHL2 are found to act as tetradentate ligands using N2O2 donor set of atoms leading to a square-planar geometry for the complexes around the metal ions. The binding behaviors of the complexes to calf thymus DNA have been investigated by absorption spectra, viscosity measurements and cyclic voltammetry. The DNA binding constants reveal that all these complexes interact with DNA through minor groove binding mode. The studies on mechanism of photocleavage reveal that singlet oxygen (1O2) and superoxide anion radical (O2rad -) may play an important role in the photocleavage. The Schiff bases and their metal complexes have been screened for their in vitro antibacterial activities against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epidermidis, Klebsiella pneumoniae and antifungal activities against Aspergillus niger, Fusarium solani, Culvularia lunata, Rhizoctonia bataicola and Candida albicans by MIC method.

  9. Competitive Binding to Cuprous Ions of Protein and BCA in the Bicinchoninic Acid Protein Assay.

    PubMed

    Huang, Tao; Long, Mian; Huo, Bo

    2010-01-01

    Although Bicinchoninic acid (BCA) has been widely used to determine protein concentration, the mechanism of interaction between protein, copper ion and BCA in this assay is still not well known. Using the Micro BCA protein assay kit (Pierce Company), we measured the absorbance at 562 nm of BSA solutions with different concentrations of protein, and also varied the BCA concentration. When the concentration of protein was increased, the absorbance exhibited the known linear and nonlinear increase, and then reached an unexpected plateau followed by a gradual decrease. We introduced a model in which peptide chains competed with BCA for binding to cuprous ions. Formation of the well-known chromogenic complex of BCA-Cu(1+)-BCA was competed with the binding of two peptide bonds (NTPB) to cuprous ion, and there is the possibility of the existence of two new complexes. A simple equilibrium equation was established to describe the correlations between the substances in solution at equilibrium, and an empirical exponential function was introduced to describe the reduction reaction. Theoretical predictions of absorbance from the model were in good agreement with the measurements, which not only validated the competitive binding model, but also predicted a new complex of BCA-Cu(1+)-NTPB that might exist in the final solution. This work provides a new insight into understanding the chemical bases of the BCA protein assay and might extend the assay to higher protein concentration.

  10. Polymorphic Nucleic Acid Binding of Bioactive Isoquinoline Alkaloids and Their Role in Cancer

    PubMed Central

    Maiti, Motilal; Kumar, Gopinatha Suresh

    2010-01-01

    Bioactive alkaloids occupy an important position in applied chemistry and play an indispensable role in medicinal chemistry. Amongst them, isoquinoline alkaloids like berberine, palmatine and coralyne of protoberberine group, sanguinarine of the benzophenanthridine group, and their derivatives represent an important class of molecules for their broad range of clinical and pharmacological utility. In view of their extensive occurrence in various plant species and significantly low toxicities, prospective development and use of these alkaloids as effective anticancer agents are matters of great current interest. This review has focused on the interaction of these alkaloids with polymorphic nucleic acid structures (B-form, A-form, Z-form, HL-form, triple helical form, quadruplex form) and their topoisomerase inhibitory activity reported by several research groups using various biophysical techniques like spectrophotometry, spectrofluorimetry, thermal melting, circular dichroism, NMR spectroscopy, electrospray ionization mass spectroscopy, viscosity, isothermal titration calorimetry, differential scanning calorimetry, molecular modeling studies, and so forth, to elucidate their mode and mechanism of action for structure-activity relationships. The DNA binding of the planar sanguinarine and coralyne are found to be stronger and thermodynamically more favoured compared to the buckled structure of berberine and palmatine and correlate well with the intercalative mechanism of sanguinarine and coralyne and the partial intercalation by berberine and palmatine. Nucleic acid binding properties are also interpreted in relation to their anticancer activity. PMID:20814427

  11. Expression Pattern of Fatty Acid Binding Proteins in Celiac Disease Enteropathy

    PubMed Central

    Bottasso Arias, Natalia M.; García, Marina; Bondar, Constanza; Guzman, Luciana; Redondo, Agustina; Chopita, Nestor; Córsico, Betina; Chirdo, Fernando G.

    2015-01-01

    Celiac disease (CD) is an immune-mediated enteropathy that develops in genetically susceptible individuals following exposure to dietary gluten. Severe changes at the intestinal mucosa observed in untreated CD patients are linked to changes in the level and in the pattern of expression of different genes. Fully differentiated epithelial cells express two isoforms of fatty acid binding proteins (FABPs): intestinal and liver, IFABP and LFABP, respectively. These proteins bind and transport long chain fatty acids and also have other important biological roles in signaling pathways, particularly those related to PPARγ and inflammatory processes. Herein, we analyze the serum levels of IFABP and characterize the expression of both FABPs at protein and mRNA level in small intestinal mucosa in severe enteropathy and normal tissue. As a result, we observed higher levels of circulating IFABP in untreated CD patients compared with controls and patients on gluten-free diet. In duodenal mucosa a differential FABPs expression pattern was observed with a reduction in mRNA levels compared to controls explained by the epithelium loss in severe enteropathy. In conclusion, we report changes in FABPs' expression pattern in severe enteropathy. Consequently, there might be alterations in lipid metabolism and the inflammatory process in the small intestinal mucosa. PMID:26346822

  12. Attenuated murine cytomegalovirus binds to N-acetylglucosamine, and shift to virulence may involve recognition of sialic acids.

    PubMed Central

    Ravindranath, R M; Graves, M C

    1990-01-01

    Treatment of cells with lectins specific for N-acetylglucosamine (GlcNAc) blocked infection by mouse cytomegalovirus (MCMV), and GlcNAc pretreatment of the lectin blocked this effect. MCMV failed to infect N-acetylglucosaminidase (GlcNAcase)-treated mouse embryo fibroblasts (MEF). GlcNAc and GlcNAc-containing synthetic oligosaccharides directly inhibited viral infectivity. Ulex lectin inhibition of infection was shown to be due to inhibition of surface adsorption of 35S-labeled virus. Also, GlcNAcase eluted 35S-labeled virus adsorbed to MEF at 4 degrees C and inhibited plaque formation if added after adsorption at this temperature. These findings indicate that GlcNAc binding is involved in attachment rather than in some later step in infection. High-performance thin-layer chromatography overlay of [35S]MCMV indicated that it binds to a GlcNAc-containing asialoglycolipid. Analogous experiments indicated that MCMV made virulent by in vivo salivary gland passage binds to sialic acids in addition to GlcNAc. Treatment of MEF with sialic acid-binding lectins blocked infectivity. Incubation of virus with sialic acids also prevented infection. N-acetylneuraminic acid was 10(3)-fold more potent than N-glycolylneuraminic acid. Sialidase-treated target cells were not efficiently infected by the virus. Thus, MCMV binds to GlcNAc on the cell surface, and the shift to virulence (by in vivo salivary gland passage) correlates with viral recognition of sialic acids. Images PMID:2170680

  13. The antiviral drug acyclovir is a slow-binding inhibitor of (D)-amino acid oxidase.

    PubMed

    Katane, Masumi; Matsuda, Satsuki; Saitoh, Yasuaki; Sekine, Masae; Furuchi, Takemitsu; Koyama, Nobuhiro; Nakagome, Izumi; Tomoda, Hiroshi; Hirono, Shuichi; Homma, Hiroshi

    2013-08-20

    d-Amino acid oxidase (DAO) is a degradative enzyme that is stereospecific for d-amino acids, including d-serine and d-alanine, which are believed to be coagonists of the N-methyl-d-aspartate (NMDA) receptor. To identify a new class of DAO inhibitor(s) that can be used to elucidate the molecular details of the active site environment of DAO, manifold biologically active compounds of microbial origin and pre-existing drugs were screened for their ability to inhibit DAO activity, and several compounds were identified as candidates. One of these compounds, acyclovir (ACV), a well-known antiviral drug used for the treatment of herpesvirus infections, was characterized and evaluated as a novel DAO inhibitor in vitro. Analysis showed that ACV acts on DAO as a reversible slow-binding inhibitor, and interestingly, the time required to achieve equilibrium between DAO, ACV, and the DAO/ACV complex was highly dependent on temperature. The binding mechanism of ACV to DAO was investigated in detail by several approaches, including kinetic analysis, structural modeling of DAO complexed with ACV, and site-specific mutagenesis of an active site residue postulated to be involved in the binding of ACV. The results confirm that ACV is a novel, active site-directed inhibitor of DAO that can be a valuable tool for investigating the structure-function relationships of DAO, including the molecular details of the active site environment of DAO. In particular, it appears that ACV can serve as an active site probe to study the structural basis of temperature-induced conformational changes of DAO.

  14. A putative carbohydrate-binding domain of the lactose-binding Cytisus sessilifolius anti-H(O) lectin has a similar amino acid sequence to that of the L-fucose-binding Ulex europaeus anti-H(O) lectin.

    PubMed

    Konami, Y; Yamamoto, K; Osawa, T; Irimura, T

    1995-04-01

    The complete amino acid sequence of a lactose-binding Cytisus sessilifolius anti-H(O) lectin II (CSA-II) was determined using a protein sequencer. After digestion of CSA-II with endoproteinase Lys-C or Asp-N, the resulting peptides were purified by reversed-phase high performance liquid chromatography (HPLC) and then subjected to sequence analysis. Comparison of the complete amino acid sequence of CSA-II with the sequences of other leguminous seed lectins revealed regions of extensive homology. The amino acid sequence of a putative carbohydrate-binding domain of CSA-II was found to be similar to those of several anti-H(O) leguminous lectins, especially to that of the L-fucose-binding Ulex europaeus lectin I (UEA-I).

  15. Brain energy metabolism spurns fatty acids as fuel due to their inherent mitotoxicity and potential capacity to unleash neurodegeneration.

    PubMed

    Schönfeld, Peter; Reiser, Georg

    2017-03-30

    The brain uses long-chain fatty acids (LCFAs) to a negligible extent as fuel for the mitochondrial energy generation, in contrast to other tissues that also demand high energy. Besides this generally accepted view, some studies using cultured neural cells or whole brain indicate a moderately active mitochondrial β-oxidation. Here, we corroborate the conclusion that brain mitochondria are unable to oxidize fatty acids. In contrast, the combustion of liver-derived ketone bodies by neural cells is long-known. Furthermore, new insights indicate the use of odd-numbered medium-chain fatty acids as valuable source for maintaining the level of intermediates of the citric acid cycle in brain mitochondria. Non-esterified LCFAs or their activated forms exert a large variety of harmful side-effects on mitochondria, such as enhancing the mitochondrial ROS generation in distinct steps of the β-oxidation and therefore potentially increasing oxidative stress. Hence, the question arises: Why do in brain energy metabolism mitochondria selectively spurn LCFAs as energy source? The most likely answer are the relatively higher content of peroxidation-sensitive polyunsaturated fatty acids and, the low antioxidative defense in brain tissue. There are two remarkable peroxisomal defects, one relating to α-oxidation of phytanic acid and the other to uptake of very long-chain fatty acids (VLCFAs) which lead to pathologically high tissue levels of such fatty acids. Both, the accumulation of phytanic acid and that of VLCFAs give an enlightening insight into harmful activities of fatty acids on neural cells, which possibly explain why evolution has prevented brain mitochondria from the equipment of with significant β-oxidation enzymatic capacity.

  16. Characterization of the binding strengths between boronic acids and cis-diol-containing biomolecules by affinity capillary electrophoresis.

    PubMed

    Lü, Chenchen; Liu, Zhen

    2015-01-01

    The affinity of boronic acids toward cis-diol-containing biomolecules has found wide applications in many fields, such as sensing, separation, drug delivery, and functional materials. A sound understanding of the binding interactions will greatly facilitate exquisite applications of this chemistry. Traditional techniques are associated with some apparent drawbacks, so they are only applicable to a limited range of boronic acids and cis-diol-containing biomolecules. This chapter describes an affinity capillary electrophoresis (ACE) method for the characterization of the binding strengths between boronic acids and cis-diol-containing biomolecules. As compared with existing approaches, such as (11)B NMR, the ACE method exhibits several significant advantages: (1) possibility of simultaneous study of multiple interactions, (2) low requirement on the purity of the binding species, (3) widely applicable to almost all types of cis-diol-containing compounds and boronic acids, and (4) high accuracy and precision.

  17. The development and amino acid binding ability of nano-materials based on azo derivatives: theory and experiment.

    PubMed

    Shang, Xuefang; Du, Jinge; Yang, Wancai; Liu, Yun; Fu, Zhiyuan; Wei, Xiaofang; Yan, Ruifang; Yao, Ningcong; Guo, Yaping; Zhang, Jinlian; Xu, Xiufang

    2014-05-01

    Two nano-material-containing azo groups have been designed and developed, and the binding ability of nano-materials with various amino acids has been characterized by UV-vis and fluorescence titrations. Results indicated that two nano-materials showed the strongest binding ability for homocysteine among twenty normal kinds of amino acids (alanine, valine, leucine, isoleucine, methionine, aspartic acid, glutamic acid, arginine, glycine, serine, threonine, asparagine, phenylalanine, histidine, tryptophan, proline, lysine, glutamine, tyrosine and homocysteine). The reason for the high sensitivity for homocysteine was that two nano-materials containing an aldehyde group reacted with SH in homocysteine and afforded very stable thiazolidine derivatives. Theoretical investigation further illustrated the possible binding mode in host-guest interaction and the roles of molecular frontier orbitals in molecular interplay. Thus, the two nano-materials can be used as optical sensors for the detection of homocysteine.

  18. Identification of multiple salicylic acid-binding proteins using two high throughput screens

    PubMed Central

    Manohar, Murli; Tian, Miaoying; Moreau, Magali; Park, Sang-Wook; Choi, Hyong Woo; Fei, Zhangjun; Friso, Giulia; Asif, Muhammed; Manosalva, Patricia; von Dahl, Caroline C.; Shi, Kai; Ma, Shisong; Dinesh-Kumar, Savithramma P.; O'Doherty, Inish; Schroeder, Frank C.; van Wijk, Klass J.; Klessig, Daniel F.

    2014-01-01

    Salicylic acid (SA) is an important hormone involved in many diverse plant processes, including floral induction, stomatal closure, seed germination, adventitious root initiation, and thermogenesis. It also plays critical functions during responses to abiotic and biotic stresses. The role(s) of SA in signaling disease resistance is by far the best studied process, although it is still only partially understood. To obtain insights into how SA carries out its varied functions, particularly in activating disease resistance, two new high throughput screens were developed to identify novel SA-binding proteins (SABPs). The first utilized crosslinking of the photo-reactive SA analog 4-AzidoSA (4AzSA) to proteins in an Arabidopsis leaf extract, followed by immuno-selection with anti-SA antibodies and then mass spectroscopy-based identification. The second utilized photo-affinity crosslinking of 4AzSA to proteins on a protein microarray (PMA) followed by detection with anti-SA antibodies. To determine whether the candidate SABPs (cSABPs) obtained from these screens were true SABPs, recombinantly-produced proteins were generated and tested for SA-inhibitable crosslinking to 4AzSA, which was monitored by immuno-blot analysis, SA-inhibitable binding of the SA derivative 3-aminoethylSA (3AESA), which was detected by a surface plasmon resonance (SPR) assay, or SA-inhibitable binding of [3H]SA, which was detected by size exclusion chromatography. Based on our criteria that true SABPs must exhibit SA-binding activity in at least two of these assays, nine new SABPs are identified here; nine others were previously reported. Approximately 80 cSABPs await further assessment. In addition, the conflicting reports on whether NPR1 is an SABP were addressed by showing that it bound SA in all three of the above assays. PMID:25628632

  19. Evolutionary diversification of retinoic acid receptor ligand-binding pocket structure by molecular tinkering

    PubMed Central

    Gutierrez-Mazariegos, Juliana; Nadendla, Eswar Kumar; Studer, Romain A.; Alvarez, Susana; de Lera, Angel R.; Kuraku, Shigehiro; Bourguet, William; Laudet, Vincent

    2016-01-01

    Whole genome duplications (WGDs) have been classically associated with the origin of evolutionary novelties and the so-called duplication–degeneration–complementation model describes the possible fates of genes after duplication. However, how sequence divergence effectively allows functional changes between gene duplicates is still unclear. In the vertebrate lineage, two rounds of WGDs took place, giving rise to paralogous gene copies observed for many gene families. For the retinoic acid receptors (RARs), for example, which are members of the nuclear hormone receptor (NR) superfamily, a unique ancestral gene has been duplicated resulting in three vertebrate paralogues: RARα, RARβ and RARγ. It has previously been shown that this single ancestral RAR was neofunctionalized to give rise to a larger substrate specificity range in the RARs of extant jawed vertebrates (also called gnathostomes). To understand RAR diversification, the members of the cyclostomes (lamprey and hagfish), jawless vertebrates representing the extant sister group of gnathostomes, provide an intermediate situation and thus allow the characterization of the evolutionary steps that shaped RAR ligand-binding properties following the WGDs. In this study, we assessed the ligand-binding specificity of cyclostome RARs and found that their ligand-binding pockets resemble those of gnathostome RARα and RARβ. In contrast, none of the cyclostome receptors studied showed any RARγ-like specificity. Together, our results suggest that cyclostome RARs cover only a portion of the specificity repertoire of the ancestral gnathostome RARs and indicate that the establishment of ligand-binding specificity was a stepwise event. This iterative process thus provides a rare example for the diversification of receptor–ligand interactions of NRs following WGDs. PMID:27069642

  20. Nucleic Acid Binding by Mason-Pfizer Monkey Virus CA Promotes Virus Assembly and Genome Packaging

    PubMed Central

    Füzik, Tibor; Píchalová, Růžena; Schur, Florian K. M.; Strohalmová, Karolína; Křížová, Ivana; Hadravová, Romana; Rumlová, Michaela; Briggs, John A. G.

    2016-01-01

    ABSTRACT The Gag polyprotein of retroviruses drives immature virus assembly by forming hexameric protein lattices. The assembly is primarily mediated by protein-protein interactions between capsid (CA) domains and by interactions between nucleocapsid (NC) domains and RNA. Specific interactions between NC and the viral RNA are required for genome packaging. Previously reported cryoelectron microscopy analysis of immature Mason-Pfizer monkey virus (M-PMV) particles suggested that a basic region (residues RKK) in CA may serve as an additional binding site for nucleic acids. Here, we have introduced mutations into the RKK region in both bacterial and proviral M-PMV vectors and have assessed their impact on M-PMV assembly, structure, RNA binding, budding/release, nuclear trafficking, and infectivity using in vitro and in vivo systems. Our data indicate that the RKK region binds and structures nucleic acid that serves to promote virus particle assembly in the cytoplasm. Moreover, the RKK region appears to be important for recruitment of viral genomic RNA into Gag particles, and this function could be linked to changes in nuclear trafficking. Together these observations suggest that in M-PMV, direct interactions between CA and nucleic acid play important functions in the late stages of the viral life cycle. IMPORTANCE Assembly of retrovirus particles is driven by the Gag polyprotein, which can self-assemble to form virus particles and interact with RNA to recruit the viral genome into the particles. Generally, the capsid domains of Gag contribute to essential protein-protein interactions during assembly, while the nucleocapsid domain interacts with RNA. The interactions between the nucleocapsid domain and RNA are important both for identifying the genome and for self-assembly of Gag molecules. Here, we show that a region of basic residues in the capsid protein of the betaretrovirus Mason-Pfizer monkey virus (M-PMV) contributes to interaction of Gag with nucleic acid. This

  1. Influence of fatty acid on lipase-catalyzed synthesis of ascorbyl esters and their free radical scavenging capacity.

    PubMed

    Stojanović, Marija; Carević, Milica; Mihailović, Mladen; Veličković, Dušan; Dimitrijević, Aleksandra; Milosavić, Nenad; Bezbradica, Dejan

    2015-01-01

    Fatty acid (FA) ascorbyl esters are recently emerging food, cosmetic, and pharmaceutical additives, which can be prepared in an eco-friendly way by using lipases as catalysts. Because they are amphiphilic molecules, which possess high free radical scavenging capacity, they can be applied as liposoluble antioxidants as well as emulsifiers and biosurfactants. In this study, the influence of a wide range of acyl donors on ester yield in lipase-catalyzed synthesis and ester antioxidant activity was examined. Among saturated acyl donors, higher yields and antioxidant activities of esters were achieved when short-chain FAs were used. Oleic acid gave the highest yield overall and its ester exhibited a high antioxidant activity. Optimization of experimental factors showed that the highest conversion (60.5%) in acetone was achieved with 5 g L(-1) of lipase, 50 mM of vitamin C, 10-fold molar excess of oleic acid, and 0.7 mL L(-1) of initial water. Obtained results showed that even short- and medium-chain ascorbyl esters could be synthesized with high yields and retained (or even exceeded) free radical scavenging capacity of l-ascorbic acid, indicating prospects of broadening their application in emulsions and liposomes.

  2. Fatty acid binding protein facilitates sarcolemmal fatty acid transport but not mitochondrial oxidation in rat and human skeletal muscle

    PubMed Central

    Holloway, Graham P; Lally, Jamie; Nickerson, James G; Alkhateeb, Hakam; Snook, Laelie A; Heigenhauser, George J F; Calles-Escandon, Jorge; Glatz, Jan F C; Luiken, Joost J F P; Spriet, Lawrence L; Bonen, Arend

    2007-01-01

    The transport of long-chain fatty acids (LCFAs) across mitochondrial membranes is regulated by carnitine palmitoyltransferase I (CPTI) activity. However, it appears that additional fatty acid transport proteins, such as fatty acid translocase (FAT)/CD36, influence not only LCFA transport across the plasma membrane, but also LCFA transport into mitochondria. Plasma membrane-associated fatty acid binding protein (FABPpm) is also known to be involved in sacrolemmal LCFA transport, and it is also present on the mitochondria. At this location, it has been identified as mitochondrial aspartate amino transferase (mAspAT), despite being structurally identical to FABPpm. Whether this protein is also involved in mitochondrial LCFA transport and oxidation remains unknown. Therefore, we have examined the ability of FABPpm/mAspAT to alter mitochondrial fatty acid oxidation. Muscle contraction increased (P < 0.05) the mitochondrial FAT/CD36 content in rat (+22%) and human skeletal muscle (+33%). By contrast, muscle contraction did not alter the content of mitochondrial FABPpm/mAspAT protein in either rat or human muscles. Electrotransfecting rat soleus muscles, in vivo, with FABPpm cDNA increased FABPpm protein in whole muscle (+150%; P < 0.05), at the plasma membrane (+117%; P < 0.05) and in mitochondria (+80%; P < 0.05). In these FABPpm-transfected muscles, palmitate transport into giant vesicles was increased by +73% (P < 0.05), and fatty acid oxidation in intact muscle was increased by +18% (P < 0.05). By contrast, despite the marked increase in mitochondrial FABPpm/mAspAT protein content (+80%), the rate of mitochondrial palmitate oxidation was not altered (P > 0.05). However, electrotransfection increased mAspAT activity by +70% (P < 0.05), and the mitochondrial FABPpm/mAspAT protein content was significantly correlated with mAspAT activity (r= 0.75). It is concluded that FABPpm has two distinct functions depending on its subcellular location: (a) it contributes to

  3. A single amino acid change humanizes long-chain fatty acid binding and activation of mouse peroxisome proliferator-activated receptor α

    PubMed Central

    Oswal, Dhawal P.; Alter, Gerald M.; Rider, S. Dean; Hostetler, Heather A.

    2014-01-01

    Peroxisome proliferator-activated receptor α (PPARα) is an important regulator of hepatic lipid metabolism which functions through ligand binding. Despite high amino acid sequence identity (>90%), marked differences in PPARα ligand binding, activation and gene regulation have been noted across species. Similar to previous observations with synthetic agonists, we have recently reported differences in ligand affinities and extent of activation between human PPARα (hPPARα) and mouse PPARα (mPPARα) in response to long chain fatty acids (LCFA). The present study was aimed to determine if structural alterations could account for these differences. The binding of PPARα to LCFA was examined through in silico molecular modeling and docking simulations. Modeling suggested that variances at amino acid position 272 are likely to be responsible for differences in saturated LCFA binding to hPPARα and mPPARα. To confirm these results experimentally, LCFA binding, circular dichroism, and transactivation studies were performed using a F272I mutant form of mPPARα. Experimental data correlated with in silico docking simulations, further confirming the importance of amino acid 272 in LCFA binding. Although the driving force for evolution of species differences at this position are yet unidentified, this study enhances our understanding of ligand-induced regulation by PPARα and demonstrates the efficacy of molecular modeling and docking simulations. PMID:24858253

  4. Structural and functional interaction of fatty acids with human liver fatty acid-binding protein (L-FABP) T94A variant.

    PubMed

    Huang, Huan; McIntosh, Avery L; Martin, Gregory G; Landrock, Kerstin K; Landrock, Danilo; Gupta, Shipra; Atshaves, Barbara P; Kier, Ann B; Schroeder, Friedhelm

    2014-05-01

    The human liver fatty acid-binding protein (L-FABP) T94A variant, the most common in the FABP family, has been associated with elevated liver triglyceride levels. How this amino acid substitution elicits these effects is not known. This issue was addressed using human recombinant wild-type (WT) and T94A variant L-FABP proteins as well as cultured primary human hepatocytes expressing the respective proteins (genotyped as TT, TC and CC). The T94A substitution did not alter or only slightly altered L-FABP binding affinities for saturated, monounsaturated or polyunsaturated long chain fatty acids, nor did it change the affinity for intermediates of triglyceride synthesis. Nevertheless, the T94A substitution markedly altered the secondary structural response of L-FABP induced by binding long chain fatty acids or intermediates of triglyceride synthesis. Finally, the T94A substitution markedly decreased the levels of induction of peroxisome proliferator-activated receptor α-regulated proteins such as L-FABP, fatty acid transport protein 5 and peroxisome proliferator-activated receptor α itself meditated by the polyunsaturated fatty acids eicosapentaenoic acid and docosahexaenoic acid in cultured primary human hepatocytes. Thus, although the T94A substitution did not alter the affinity of human L-FABP for long chain fatty acids, it significantly altered human L-FABP structure and stability, as well as the conformational and functional response to these ligands.

  5. Absorption Spectroscopy Study of Acid-Base and Metal-Binding Properties of Flavanones

    NASA Astrophysics Data System (ADS)

    Shubina, V. S.; Shatalina, Yu. V.

    2013-11-01

    We have used absorption spectroscopy to study the acid-base and metal-binding properties of two structurally similar flavanones: taxifolin and naringenin. We have determined the acid dissociation constants for taxifolin (pKa1 = 7.10 ± 0.05, pKa2 = 8.60 ± 0.09, pKa3 = 8.59 ± 0.19, pKa4 = 11.82 ± 0.36) and naringenin (pKa1 = 7.05 ± 0.05, pKa2 = 8.85 ± 0.09, pKa3 = 12.01 ± 0.38). The appearance of new absorption bands in the visible wavelength region let us determine the stoichiometric composition of the iron (II) complexes of the flavanones. We show that at pH 5, in solution there is a mixture of complexes between taxifolin and iron (II) ions in stoichiometric ratio 2:1 and 1:2, while at pH 7.4 and pH 9, we detect a 1:1 taxifolin:Fe(II) complex. We established that at these pH values, naringenin forms a 2:1 complex with iron (II) ions. We propose structures for the complexes formed. Comprehensive study of the acid-base properties and the metal-binding capability of the two structurally similar flavanones let us determine the structure-properties relation and the conditions under which antioxidant activity of the polyphenols appears, via chelation of variable-valence metal ions.

  6. Allosteric Regulation in the Ligand Binding Domain of Retinoic Acid Receptorγ

    PubMed Central

    Amal, Ismail; Lutzing, Régis; Stote, Roland H.; Rochette-Egly, Cécile; Rochel, Natacha; Dejaegere, Annick

    2017-01-01

    Retinoic acid (RA) plays key roles in cell differentiation and growth arrest through nuclear retinoic acid receptors (RARs), which are ligand-dependent transcription factors. While the main trigger of RAR activation is the binding of RA, phosphorylation of the receptors has also emerged as an important regulatory signal. Phosphorylation of the RARγ N-terminal domain (NTD) is known to play a functional role in neuronal differentiation. In this work, we investigated the phosphorylation of RARγ ligand binding domain (LBD), and present evidence that the phosphorylation status of the LBD affects the phosphorylation of the NTD region. We solved the X-ray structure of a phospho-mimetic mutant of the LBD (RARγ S371E), which we used in molecular dynamics simulations to characterize the consequences of the S371E mutation on the RARγ structural dynamics. Combined with simulations of the wild-type LBD, we show that the conformational equilibria of LBD salt bridges (notably R387-D340) are affected by the S371E mutation, which likely affects the recruitment of the kinase complex that phosphorylates the NTD. The molecular dynamics simulations also showed that a conservative mutation in this salt bridge (R387K) affects the dynamics of the LBD without inducing large conformational changes. Finally, cellular assays showed that the phosphorylation of the NTD of RARγ is differentially regulated by retinoic acid in RARγWT and in the S371N, S371E and R387K mutants. This multidisciplinary work highlights an allosteric coupling between phosphorylations of the LBD and the NTD of RARγ and supports the importance of structural dynamics involving electrostatic interactions in the regulation of RARs activity. PMID:28125680

  7. Origin of low sodium capacity in graphite and generally weak substrate binding of Na and Mg among alkali and alkaline earth metals.

    PubMed

    Liu, Yuanyue; Merinov, Boris V; Goddard, William A

    2016-04-05

    It is well known that graphite has a low capacity for Na but a high capacity for other alkali metals. The growing interest in alternative cation batteries beyond Li makes it particularly important to elucidate the origin of this behavior, which is not well understood. In examining this question, we find a quite general phenomenon: among the alkali and alkaline earth metals, Na and Mg generally have the weakest chemical binding to a given substrate, compared with the other elements in the same column of the periodic table. We demonstrate this with quantum mechanics calculations for a wide range of substrate materials (not limited to C) covering a variety of structures and chemical compositions. The phenomenon arises from the competition between trends in the ionization energy and the ion-substrate coupling, down the columns of the periodic table. Consequently, the cathodic voltage for Na and Mg is expected to be lower than those for other metals in the same column. This generality provides a basis for analyzing the binding of alkali and alkaline earth metal atoms over a broad range of systems.

  8. Origin of low sodium capacity in graphite and generally weak substrate binding of Na and Mg among alkali and alkaline earth metals

    NASA Astrophysics Data System (ADS)

    Liu, Yuanyue; Merinov, Boris V.; Goddard, William A., III

    2016-04-01

    It is well known that graphite has a low capacity for Na but a high capacity for other alkali metals. The growing interest in alternative cation batteries beyond Li makes it particularly important to elucidate the origin of this behavior, which is not well understood. In examining this question, we find a quite general phenomenon: among the alkali and alkaline earth metals, Na and Mg generally have the weakest chemical binding to a given substrate, compared with the other elements in the same column of the periodic table. We demonstrate this with quantum mechanics calculations for a wide range of substrate materials (not limited to C) covering a variety of structures and chemical compositions. The phenomenon arises from the competition between trends in the ionization energy and the ion-substrate coupling, down the columns of the periodic table. Consequently, the cathodic voltage for Na and Mg is expected to be lower than those for other metals in the same column. This generality provides a basis for analyzing the binding of alkali and alkaline earth metal atoms over a broad range of systems.

  9. Amino acids outside of the loops that define the agonist binding site are important for ligand binding to insect nicotinic acetylcholine receptors.

    PubMed

    Liu, Zewen; Han, Zhaojun; Liu, Shuhua; Zhang, Yixi; Song, Feng; Yao, Xiangmei; Gu, Jianhua

    2008-07-01

    Nicotinic acetylcholine (ACh) receptors (nAChRs) are the targets of several kinds of insecticides. Based on the mutagenesis studies of Torpedo californica nAChRs and solved structure of a molluscan, glial-derived soluble ACh-binding protein, a model of the agonist site was constructed with contributing amino acids from three distinct loops (A, B, and C) of the alpha subunits and another three loops (D, E, and F) of the non-alpha subunits. According to this model, most insect nAChR subunits can form the functional heteromeric or homomeric receptors. Actually, insect subunits themselves did not form any functional receptor at various combinations as yet, and only part of them can form the functional receptors with vertebrate non-alpha subunits. These findings suggested that the agonist binding for insect nAChRs was not only contributed by those key amino acids in six loops, but also some unidentified amino acids from other regions. In our previous studies on nAChRs for Nilaparvata lugens, a target-site mutation (Y151S) was found within two alpha subunits (Nlalpha1 and Nlalpha3). In Drosophila S2 cells and Xenopus oocytes, Nlalpha1 can form functional receptors with rat beta2 subunit. However, the same thing was not observed in Nlalpha3. In the present paper, by exchanging the corresponding regions between Nlalpha1 and Nlalpha3 to generate different chimeras, amino acid residues or residue clusters in the regions outside the six loops were found to play essential roles in agonist binding, especially for the amino acid clusters between loop B and C. This result indicated that the residues in the six loops could be necessary, but not enough for the activity of agonist binding.

  10. Uncoupling of gamma-aminobutyric acid B receptors from GTP-binding proteins by N-ethylmaleimide: effect of N-ethylmaleimide on purified GTP-binding proteins

    SciTech Connect

    Asano, T.; Ogasawara, N.

    1986-03-01

    Treatment of membranes from bovine cerebral cortex with N-ethylmaleimide (NEM) resulted in inhibition of gamma-aminobutyric acid (GABA) binding to GABAB receptors. The binding curve for increasing concentrations of agonist was shifted to the right by NEM treatment. Guanine nucleotide had little effect on the binding of GABA to NEM-treated membranes. The addition of purified GTP-binding proteins, which were the substrates of islet-activating protein (IAP), pertussis toxin, to the NEM-treated membranes caused a shift of the binding curve to the left, suggesting modification of GTP-binding proteins rather than receptors by NEM. The effect of NEM on two purified GTP-binding proteins, Gi (composed of three subunits with molecular weight of alpha, 41,000; beta, 35,000; gamma, 10,000) and Go (alpha, 39,000; beta, 35,000; gamma, 10,000) was studied. NEM did not significantly change guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) binding and GTPase activity of these two proteins. NEM-treated Gi and Go were not ADP-ribosylated by IAP and did not increase GABA binding to NEM-treated membranes. When alpha and beta gamma subunits were treated with NEM and then mixed with nontreated alpha and beta gamma to form Gi or Go, respectively, both oligomers with NEM-treated alpha-subunits lost their abilities to be IAP substrates and to couple to receptors. Results indicate that NEM uncoupled GTP-binding proteins from receptors by modifying alpha-subunits of GTP-binding proteins, and the site seemed to be on or near the site of ADP-ribosylation by IAP. When alpha and beta gamma subunits were treated with NEM and then mixed to form Gi or Go, GTP gamma S binding in the absence of Mg2+ and GTPase activity were changed, although they were not affected when oligomers were treated with NEM. Results suggest the existence of another sulfhydryl group which is protected from NEM by the association of subunits.

  11. Phenolic acids, anthocyanins, and antioxidant capacity in rice (Oryza sativa L.) grains at four stages of development after flowering.

    PubMed

    Shao, Yafang; Xu, Feifei; Sun, Xiao; Bao, Jinsong; Beta, Trust

    2014-01-15

    This study investigated differences in total phenolic content (TPC), antioxidant capacity, and phenolic acids in free, conjugated and bound fractions of white (unpolished), red and black rice at 1-, 2-, and 3-weeks of grain development after flowering and at maturity. Unlike the TPC (mg/100g) of white rice (14.6-33.4) and red rice (66.8-422.2) which was significantly higher at 1-week than at later stages, the TPC of black rice (56.5-82.0) was highest at maturity. The antioxidant capacity measured by DPPH radical scavenging and ORAC methods generally followed a similar trend as TPC. Only black rice had detectable anthocyanins (26.5-174.7mg/100g). Cyanidin-3-glucoside (C3G) and peonidin-3-glucoside (P3G) were the main anthocyanins in black rice showing significantly higher levels at 2- and 3-weeks than at 1-week development and at maturity. At all stages, the phenolic acids existed mainly in the bound form as detected by HPLC and confirmed by LC-MS/MS. Black rice (20.1-31.7mg/100g) had higher total bound phenolic acids than white rice and red rice (7.0-11.8mg/100g). Protocatechuic acid was detected in red rice and black rice with relatively high levels at 1-week development (1.41mg/100g) and at maturity (4.48mg/100g), respectively. Vanillic acid (2.4-5.4mg/100g) was detected only in black rice where it peaked at maturity. p-Coumaric acid (<3.5mg/100g) did not differ significantly at most stages with somewhat high levels at 1-week for red and black rice. Ferulic acid (4.0-17.9mg/100g), the most abundant bound phenolic acid, had an inconsistent trend with higher levels being observed in black rice where it peaked at maturity. Isoferulic acid levels (0.8-1.6mg/100g) were generally low with slightly elevated values being observed at maturity. Overall black rice had higher total bound phenolic acids than white and red rice while white rice at all stages of development after flowering.

  12. NOVEL POLY-GLUTAMIC ACID FUNCTIONALIZED MICROFILTRATION MEMBRANES FOR SORPTION OF HEAVY METALS AT HIGH CAPACITY

    EPA Science Inventory

    Various sorbent/ion exchange materials have been reported in the literature for metal ion entrapment. We have developed a highly innovative and new approach to obtain high metal pick-up utilizing poly-amino acids (poly-L-glutamic acid, 14,000 MW) covalently attached to membrane p...

  13. Chemically binding carboxylic acids onto TiO2 nanoparticles with adjustable coverage by solvothermal strategy.

    PubMed

    Qu, Qiyun; Geng, Hongwei; Peng, Ruixiang; Cui, Qi; Gu, Xiaohong; Li, Fanqing; Wang, Mingtai

    2010-06-15

    This paper presents a solvothermal strategy for chemical modification of TiO(2) nanoparticles with carboxylic acids. Solvothermal reaction between the TiO(2) nanoparticles and carboxylic acid molecules in an autoclave at 100 degrees C provides carboxylic acid-modified TiO(2) particles with a modification efficiency much higher than the conventional immersion method. TiO(2) nanoparticles were prepared by hydrolysis of titanium isopropoxide in nitric acid solution; the modified nanoparticles were characterized by powder X-ray diffraction pattern, scanning electron microscopy, absorption and Fourier transform infrared spectra, and thermogravimetric analysis. Results show that the binding form of the modifier molecules on TiO(2) surface is in a bidentate chelating mode, the crystalline phase composition and morphological structure of the preformed TiO(2) nanoparticles are not affected by the solvothermal reaction, and the surface coverage of the modifier molecules can be adjusted by the weight ratio of modifier/TiO(2) during feeding. It is evident that the reaction processes in the solvothermal strategy involve the formation of double hydrogen bondings between carboxylic acid molecule and TiO(2) at the same Ti site and the coordination at solvothermal temperature by dehydration from the hydrogen bondings. The solvothermal strategy for modifying TiO(2) nanoparticles is expected to find potential applications in many fields; for example, our results demonstrate that the photovoltaic performance of the TiO(2) nanoparticles can be improved by the solvothermal modification even with an insulating modifier and controlled by the modifier coverage.

  14. Molecular cloning and analysis of functional cDNA and genomic clones encoding bovine cellular retinoic acid-binding protein.

    PubMed Central

    Shubeita, H E; Sambrook, J F; McCormick, A M

    1987-01-01

    A recombinant cDNA clone, pCRABP-HS1, encoding cellular retinoic acid-binding protein was isolated from a bovine adrenal cDNA library. COS-7 cells transfected with pCRABP-HS1 produced a biologically active retinoic acid-binding protein molecule of the expected molecular mass (15.5 kDa). RNA blot hybridization analysis using pCRABP-HS1 as a probe revealed a single 1050-nucleotide mRNA species in bovine adrenal, uterus, and testis, tissues that contain the highest levels of retinoic acid-binding activity. No hybridization was detected in RNA extracted from ovary, spleen, kidney, or liver, which contain relatively low levels of cellular retinoic acid-binding protein activity. Analysis of genomic clones isolated from an EcoRI bovine genomic library demonstrated that the bovine cellular retinoic acid-binding protein gene is composed of four exons and three introns. Two putative promoter sequences were identified in the cloned 5' sequence of the gene. Images PMID:3039499

  15. Molecular cloning and analysis of functional cDNA and genomic clones encoding bovine cellular retinoic acid-binding protein.

    PubMed

    Shubeita, H E; Sambrook, J F; McCormick, A M

    1987-08-01

    A recombinant cDNA clone, pCRABP-HS1, encoding cellular retinoic acid-binding protein was isolated from a bovine adrenal cDNA library. COS-7 cells transfected with pCRABP-HS1 produced a biologically active retinoic acid-binding protein molecule of the expected molecular mass (15.5 kDa). RNA blot hybridization analysis using pCRABP-HS1 as a probe revealed a single 1050-nucleotide mRNA species in bovine adrenal, uterus, and testis, tissues that contain the highest levels of retinoic acid-binding activity. No hybridization was detected in RNA extracted from ovary, spleen, kidney, or liver, which contain relatively low levels of cellular retinoic acid-binding protein activity. Analysis of genomic clones isolated from an EcoRI bovine genomic library demonstrated that the bovine cellular retinoic acid-binding protein gene is composed of four exons and three introns. Two putative promoter sequences were identified in the cloned 5' sequence of the gene.

  16. Simultaneous determination of cation exchange capacity and surface area of acid activated bentonite powders by methylene blue sorption

    NASA Astrophysics Data System (ADS)

    Yener, Nilgün; Biçer, Cengiz; Önal, Müşerref; Sarıkaya, Yüksel

    2012-01-01

    To distinguish the ion exchanged and physically adsorbed methylene blue cations (MB+) on ionic surfaces, acid activated bentonite samples were used as porous adsorbents. A natural calcium bentonite (CaB) sample from Enez/Edirne, Turkey, was acid activated at 90 °C for 16 h with various HCl/CaB ratios. The irreversible exchange and physical adsorption of MB+ cations on the ionic solids have simultaneously occurred. The ion exchanged (mex) and physically adsorbed (mad) MB+ contents were obtained as the values of sorption capacity at c = 0 and the increase to a plateaus of adsorption isotherms, respectively. The mad value was taken to be monolayer adsorption capacity. Cation exchange capacity (CEC) and specific surface area (SMB) for each sample were calculated from the mex and mad values, respectively. Also, the BET specific surface areas (SBET) and pore size distribution were determined from low temperature nitrogen adsorption/desorption data. A linear correlation between the SMB and SBET values was found.

  17. Effects of polymannuronate on performance, antioxidant capacity, immune status, cecal microflora, and volatile fatty acids in broiler chickens.

    PubMed

    Zhu, Wenhui; Li, Defa; Wang, Jianhong; Wu, Hui; Xia, Xuan; Bi, Wanghua; Guan, Huashi; Zhang, Liying

    2015-03-01

    The aim of this study was to assess the effects of purified polymannuronate (PM) obtained from marine brown algae on the performance, antioxidant capacity, immune status, and cecal fermentation profile of broiler chickens. In a 42 d experiment, 540 (average BW 43.77±1.29 g) 1-d-old Arbor Acres male broilers were randomly divided into 5 treatments with 6 replicates of 18 chicks and fed a corn and soybean meal (SBM)-based diet supplemented with 0, 1, 2, 3, or 4 g/kg polymannuronate. Adding polymannuronate to the broiler chickens' diets resulted in a significantly increased ADG and improved feed conversion compared with the control treatment. From d 1 to 42, the ADG of broilers fed 1, 2, 3, or 4 g/kg of polymannuronate was increased by 2.58, 4.33, 4.20, and 3.47%, respectively. Furthermore, parameters related to immune status, antioxidant capacity, and composition of the cecal microflora in broiler chickens fed the polymannuronate-containing diets were altered compared with broiler chickens fed a diet without polymannuronate. Supplementation with polymannuronate significantly increased the concentrations of lactic acid and acetic acid in the cecum compared with the control group. The results indicate that polymannuronate has the potential to improve broiler chicken immune status, antioxidant capacity, and performance.

  18. Selective binding of C-6 OH sulfated hyaluronic acid to the angiogenic isoform of VEGF(165).

    PubMed

    Lim, Dong-Kwon; Wylie, Ryan G; Langer, Robert; Kohane, Daniel S

    2016-01-01

    Vascular endothelial growth factor 165 (VEGF165) is an important extracellular protein involved in pathological angiogenesis in diseases such as cancer, wet age-related macular degeneration (wet-AMD) and retinitis pigmentosa. VEGF165 exists in two different isoforms: the angiogenic VEGF165a, and the anti-angiogenic VEGF165b. In some angiogenic diseases the proportion of VEGF165b may be equal to or higher than that of VEGF165a. Therefore, developing therapeutics that inhibit VEGF165a and not VEGF165b may result in greater anti-angiogenic activity and therapeutic benefit. To this end, we report the selective binding properties of sulfated hyaluronic acid (s-HA). Selective biopolymers offer several advantages over antibodies or aptamers including cost effective and simple synthesis, and the ability to make nanoparticles or hydrogels for drug delivery applications or VEGF165a sequestration. Limiting sulfation to the C-6 hydroxyl (C-6 OH) in the N-acetyl-glucosamine repeat unit of hyaluronic acid (HA) resulted in a polymer with strong affinity for VEGF165a but not VEGF165b. Increased sulfation beyond the C-6 OH (i.e. greater than 1 sulfate group per HA repeat unit) resulted in s-HA polymers that bound both VEGF165a and VEGF165b. The C-6 OH sulfated HA (Mw 150 kDa) showed strong binding properties to VEGF165a with a fast association rate constant (Ka; 2.8 × 10(6) M(-1) s(-1)), slow dissociation rate constant (Kd; 2.8 × 10(-3) s(-1)) and strong equilibrium binding constant (KD; ∼1.0 nM)), which is comparable to the non-selective VEGF165 binding properties of the commercialized therapeutic anti-VEGF antibody (Avastin(®)). The C-6 OH sulfated HA also inhibited human umbilical vein endothelial cell (HUVEC) survival and proliferation and human dermal microvascular endothelial cell (HMVEC) tube formation. These results demonstrate that the semi-synthetic natural polymer, C-6 OH sulfated HA, may be a promising biomaterial for the treatment of angiogenesis

  19. Coexistence of multiple minor states of fatty acid binding protein and their functional relevance

    PubMed Central

    Yu, Binhan; Yang, Daiwen

    2016-01-01

    Proteins are dynamic over a wide range of timescales, but determining the number of distinct dynamic processes and identifying functionally relevant dynamics are still challenging. Here we present the study on human intestinal fatty acid binding protein (hIFABP) using a novel analysis of 15N relaxation dispersion (RD) and chemical shift saturation transfer (CEST) experiments. Through combined analysis of the two types of experiments, we found that hIFABP exists in a four-state equilibrium in which three minor states interconvert directly with the major state. According to conversion rates from the major “closed” state to minor states, these minor states are irrelevant to the function of fatty acid transport. Based on chemical shifts of the minor states which could not be determined from RD data alone but were extracted from a combined analysis of RD and CEST data, we found that all the minor states are native-like. This conclusion is further supported by hydrogen-deuterium exchange experiments. Direct conversions between the native state and native-like intermediate states may suggest parallel multitrack unfolding/folding pathways of hIFABP. Moreover, hydrogen-deuterium exchange data indicate the existence of another locally unfolded minor state that is relevant to the fatty acid entry process. PMID:27677899

  20. Unfolding pathway in red kidney bean acid phosphatase is dependent on ligand binding.

    PubMed

    Cashikar, A G; Rao, N M

    1996-03-01

    Structural basis for ligand-induced protein stabilization was investigated in the case of an acid phosphatase (red kidney bean purple acid phosphatase (KBPAP)) from red kidney bean. Phosphate, a physiological ligand, increases the stability against solvent denaturation by 3.5 kcal/mol. Generality of phosphate stabilization was shown by similar effects with other KBPAP ligands viz. adenosine 5'-O-(thiotriphosphate), a nonhydrolyzable ligand, and arsenate, an inhibitor. The dissociation constant of phosphate obtained from denaturation curves matches with the dissociation constant estimated by conventional methods. The guanidinium chloride-mediated denaturation of KBPAP was monitored by several structural and functional parameters viz. activity, tryptophan fluorescence, 8-anilinonaphthalene 1-sulfonic acid binding, circular dichroism, and size exclusion chromatography, in the presence and absence of 10 mm phosphate. In the presence of phosphate, profiles of all the parameters shift to a higher guanidinium chloride concentration. Noncoincidence of these profiles in the absence of phosphate indicates multistate unfolding pathway for KBPAP; however, in the presence of phosphate, KBPAP unfolds with a single intermediate. Based on the crystal structure, we propose that the Arg258 may have an important role to play in stabilization mediated by phosphate.

  1. Vanadium-fulvic acid chemistry: conformational and binding studies by electron spin probe techniques

    NASA Astrophysics Data System (ADS)

    Templeton, G. Daniel, III; Chasteen, N. Dennis

    1980-05-01

    Two fractions of soil fulvic acid (FA) were separated by gel filtration chromatography. An observed increase in volume of the heavier fraction (FA I) with increasing pH was attributed to aggregation, intramolecular negative charge repulsions and the rupture of hydrogen bonds, which control molecular conformation. Optical absorption properties and elemental analyses of both fractions were determined. The stability constants and stoichiometries of FA complexes with vanadyl, VO 2+, at pH 5.0 and ionic strength of 0.04 M were measured by electron paramagnetic resonance (EPR) spectroscopy. EPR spectra of model VO 2+ complexes with phthalic and salicylic acids, which are the probable functional groups present in FA, are identical to those of the VO 2+-FA complexes. Aggregation of FA I occurs in the presence of VO 2+ to form a complex that can be approximated as '(VO) 2(FA I) 6'. The average site distance between vanadyl ions in this complex is shown t