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Sample records for acid dopac binds

  1. At low concentrations, 3,4-dihydroxyphenylacetic acid (DOPAC) binds non-covalently to alpha-synuclein and prevents its fibrillation.

    Science.gov (United States)

    Zhou, Wenbo; Gallagher, Amy; Hong, Dong-Pyo; Long, Chunmei; Fink, Anthony L; Uversky, Vladimir N

    2009-05-01

    Several studies have shown that catecholamines can inhibit the fibrillation of alpha-synuclein (alpha-Syn), a small presynaptic protein whose aggregation is believed to be a critical step in the etiology of Parkinson's disease and several other neurodegenerative disorders. However, the mechanism of this inhibition is uncertain. We show here that substoichiometric concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC), a normal product of the metabolism of dopamine, can inhibit the fibrillation of alpha-Syn, due to non-covalent binding of DOPAC to alpha-Syn monomer. Intriguingly, the presence of alpha-Syn accelerates the spontaneous oxidation of DOPAC, and the oxidized form of DOPAC (the quinone) is responsible for the fibrillation inhibition. In addition, the presence of DOPAC leads to the oxidation of the methionine residues of alpha-Syn, probably due to the H(2)O(2) production as a by-product of DOPAC oxidation. The lack of fibrillation results from the formation of stable oligomers, which are very similar to those observed transiently at early stages of the alpha-Syn fibrillation. A possible explanation for this phenomenon is that DOPAC stabilizes the normally transient oligomers and prevents them from subsequent fibril formation. The analysis of the alpha-Syn Y39W variant suggests that DOPAC binds non-covalently to the same N-terminal region of alpha-Syn as lipid vesicles, probably in the vicinity of residue 39. In contrast to the compounds with 1,2-dihydroxyphenyl groups (DOPAC and catechol), their 1,4-dihydroxyphenyl isomers (hydroquinone and homogentisic acid) are able to modify alpha-Syn covalently, probably due to the less steric hindrance in the Michael addition.

  2. Ascorbate reduces morphine-induced extracellular DOPAC level in the nucleus accumbens: A microdialysis study in rats.

    Science.gov (United States)

    Rajaei, Z; Alaei, H; Nasimi, A; Amini, H; Ahmadiani, A

    2005-08-16

    Most drugs of abuse increase dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) release in the shell of the nucleus accumbens. The effects of ascorbate, which is known to modulate dopamine neurotransmission, on the extracellular level of DOPAC in the nucleus accumbens of naive rats and of rats treated acutely with morphine were studied by using in vivo microdialysis and high performance liquid chromatography with electrochemical detection (HPLC-ECD). Acute morphine (20 mg/kg ip) treatment increased the level of DOPAC in the nucleus accumbens to approximately 170% of basal level. Acute treatment with ascorbate (500 mg/kg ip) alone did not alter nucleus accumbens' DOPAC level, but pretreatment with ascorbate (500 mg/kg ip) 30 min before morphine administration attenuated the effects of acute morphine on the level of DOPAC. These results suggest that ascorbate modulates the mesolimbic dopaminergic pathway.

  3. Solution Structure and Backbone Dynamics of Human Liver Fatty Acid Binding Protein: Fatty Acid Binding Revisited

    OpenAIRE

    Cai, Jun; Lücke, Christian; Chen, Zhongjing; Qiao, Ye; Klimtchuk, Elena; Hamilton, James A.

    2012-01-01

    Liver fatty acid binding protein (L-FABP), a cytosolic protein most abundant in liver, is associated with intracellular transport of fatty acids, nuclear signaling, and regulation of intracellular lipolysis. Among the members of the intracellular lipid binding protein family, L-FABP is of particular interest as it can i), bind two fatty acid molecules simultaneously and ii), accommodate a variety of bulkier physiological ligands such as bilirubin and fatty acyl CoA. To better understand the p...

  4. Zinc Binding by Lactic Acid Bacteria

    Directory of Open Access Journals (Sweden)

    Jasna Mrvčić

    2009-01-01

    Full Text Available Zinc is an essential trace element in all organisms. A common method for the prevention of zinc deficiency is pharmacological supplementation, especially in a highly available form of a metalloprotein complex. The potential of different microbes to bind essential and toxic heavy metals has recently been recognized. In this work, biosorption of zinc by lactic acid bacteria (LAB has been investigated. Specific LAB were assessed for their ability to bind zinc from a water solution. Significant amount of zinc ions was bound, and this binding was found to be LAB species-specific. Differences among the species in binding performance at a concentration range between 10–90 mg/L were evaluated with Langmuir model for biosorption. Binding of zinc was a fast process, strongly influenced by ionic strength, pH, biomass concentration, and temperature. The most effective metal-binding LAB species was Leuconostoc mesenteroides (27.10 mg of Zn2+ per gram of dry mass bound at pH=5 and 32 °C, during 24 h. FT-IR spectroscopy analysis and electron microscopy demonstrated that passive adsorption and active uptake of the zinc ions were involved.

  5. Binding of straight-chain saturated dicarboxylic acids to albumin.

    OpenAIRE

    Tonsgard, J H; Mendelson, S A; Meredith, S C

    1988-01-01

    Dicarboxylic acids are prominent features of several diseases, including Reye's syndrome. Long-chain dicarboxylic acids have profound effects on the function and structure of isolated mitochondria, suggesting that they could contribute to the mitochondrial dysfunction in Reye's syndrome. Binding of fatty acids to albumin and the intracellular fatty acid-binding proteins is important in regulating the transport and metabolism of fatty acids and protects against the toxic effects of unbound fat...

  6. Binding of straight-chain saturated dicarboxylic acids to albumin.

    Science.gov (United States)

    Tonsgard, J H; Mendelson, S A; Meredith, S C

    1988-11-01

    Dicarboxylic acids are prominent features of several diseases, including Reye's syndrome. Long-chain dicarboxylic acids have profound effects on the function and structure of isolated mitochondria, suggesting that they could contribute to the mitochondrial dysfunction in Reye's syndrome. Binding of fatty acids to albumin and the intracellular fatty acid-binding proteins is important in regulating the transport and metabolism of fatty acids and protects against the toxic effects of unbound fatty acids. We studied the binding of dicarboxylic acids to defatted albumin using equilibrium dialysis to assess to what extent dicarboxylic acids are likely to be bound in the plasma of patients. Dicarboxylic acids bind weakly to albumin in a molar ratio of 3.8, 4.2, 1.6, 0.8, and 0.7 to 1 for octadecanedioic, hexadecanedioic, tetradecanedioic, dodecanedioic, and decanedioic acid, respectively. The dissociation constants for long-chain dicarboxylic acids are 100-1,000-fold larger than those of comparable monocarboxylic acids. Oleate competes with dicarboxylic acid and reduces the moles of dicarboxylic acid bound per mol of albumin to less than 1. Octanoate inhibits dicarboxylic acid binding. Our observations indicate that in Reye's syndrome, substantial concentrations of dicarboxylic acids of patients may be free and potentially toxic to mitochondria and other cellular processes.

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

    Energy Technology Data Exchange (ETDEWEB)

    Gangi Setty, Thanuja [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Cho, Christine [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Govindappa, Sowmya [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Apicella, Michael A. [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Ramaswamy, S., E-mail: ramas@instem.res.in [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India)

    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.

  8. Peptide Nucleic Acids Having Enhanced Binding Affinity and Sequence Specificity

    DEFF Research Database (Denmark)

    1998-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary DNA and RNA strands more strongly than a corresponding DNA strand, and exhibit increased sequence specificity and binding affinity. Methods of increasing binding affinity and sequence specificity of peptide nucleic aci...

  9. MK-801诱导的精神分裂症发育模型大鼠脑组织DA,DOPAC,Glu和GABA浓度的变化%Concentration change of DA, DOPAC, Glu and GABA in brain tissues in schizophrenia developmental model rats induced by MK-801

    Institute of Scientific and Technical Information of China (English)

    刘勇; 唐亚梅; 蒲唯丹; 张向晖; 赵靖平

    2011-01-01

    Objective To explore the related neurobiochemical mechanism by comparing the concentration change of dopamine (DA),dihydroxy-phenyl acetic acid (DOPAC),glutamate (Glu),and γ-aminobutyric acid (GABA) in the brain tissues in schizophrenia (SZ) developmental model rats and chronic medication model rats.Methods A total of 60 neonatal male Spragur-Dawley (SD) rats were randomly assigned to 3 groups at the postnatal day 6:an SZ developmental rat model group (subcutaneous injection with MK-801 at the postnatal day 7-10,0.1 mg/kg,Bid),a chronic medication model group (intraperitoneal injection at the postnatal day 47-60,0.2 mg/kg,Qd),and a normal control group (injection with O.9% normal saline during the corresponding periods).DA,DOPAC,Glu,and GABA of the tissue homogenate from the medial prefrontal cortex (mPFC) and hippocampus were examined with Coularray electrochemic detection by high performance liquid chromatogram technique.The utilization rate of DA and Glu was calculated.Results Compared with the normal control group,the concentration of DA and DOPAC in the mPFC and the hippocampus in the SZ developmental model group significantly decreased ( P < 0.05 ),and the GABA concentration and Glu utilization rate in the mPFC also decreased (P < 0.05 ).Compared with the chronic medication model group,the DA concentration of the mPFC in the SZ developmental group decreased ( P < 0.05 ),and the DOPAC concentration and the utility rate of DA in the hippocampus also decreased (P <0.01,P <0.05,respectively).Conclusion The activities of DA,Glu and GABA system decrease in the mPFC and the DA system function reduces in the hippocampus of SZ developmental rats.%目的:通过对MK-801诱导的精神分裂症(schizophrenia,SZ)发育模型大鼠和慢性给药模型大鼠脑组织多巴胺(dopamine,DA)、二羟苯乙酸(dihydroxy-phenyl acetic acid,DOPAC)、谷氨酸盐(glutamate,Glu)和γ-氨基丁酸(γ-aminobutyric acid,GABA)浓度的比较研究,探讨

  10. Echinococcus granulosus fatty acid binding proteins subcellular localization.

    Science.gov (United States)

    Alvite, Gabriela; Esteves, Adriana

    2016-05-01

    Two fatty acid binding proteins, EgFABP1 and EgFABP2, were isolated from the parasitic platyhelminth Echinococcus granulosus. These proteins bind fatty acids and have particular relevance in flatworms since de novo fatty acids synthesis is absent. Therefore platyhelminthes depend on the capture and intracellular distribution of host's lipids and fatty acid binding proteins could participate in lipid distribution. To elucidate EgFABP's roles, we investigated their intracellular distribution in the larval stage by a proteomic approach. Our results demonstrated the presence of EgFABP1 isoforms in cytosolic, nuclear, mitochondrial and microsomal fractions, suggesting that these molecules could be involved in several cellular processes.

  11. Thermodynamics of Molybdate Binding to Humic Acid

    Science.gov (United States)

    Thalhammer, K.; Gilbert, B.

    2016-12-01

    Molybdenum is an essential nutrient for diazotrophic bacteria that use nitrogenase I to fix atmospheric nitrogen in soils into bioavailable forms such as ammonia. This metalloid is released during rock weathering processes and at neutral pH it exists primarily as the soluble oxyanion molybdate, MoO42-. It has been established that molybdate mobility and bioavailability in soils is influenced by sorption to mineral surfaces and complexation by natural organic matter (NOM). The molybdate ion is readily bound by ortho dihydroxybenzene molecules such as catechol and catechol groups in siderophores. Humic acids (HA) found in NOM contain abundant phenolic groups and extended X-ray absorption fine structure (EXAFS) spectroscopy demonstrated that molybdate is bound by catechol-containing molecules in soil organic matter1. However, to our knowledge no quantitative determination of the affinity of molybdate to HA has been reported. We studied the interactions of molybdate with Suwannee River HA using ultraviolet-visible (UV-vis) absorption spectroscopy and isothermal titration calorimetry (ITC) to determine the conditional equilibrium constant for complexation at neutral pH. We further used ITC to investigate the thermodynamic contributions to complexation and the interaction kinetics. Addition of molybdate to HA caused the formation of complexes with UV-vis absorption spectra in good agreement with molybdate-catechol species indicating catechol groups to be the primary ligands in HA. ITC data revealed that binding enthalpies and kinetics were strongly influenced by ionic strength, suggesting a role for macromolecular reorganization driven by metalloid addition. 1. Wichard et al., Nature Geoscience 2, 625 - 629 (2009).

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  13. Nucleic acids encoding a cellulose binding domain

    Energy Technology Data Exchange (ETDEWEB)

    Shoseyov, Oded (Karmey Yosef, IL); Shpiegl, Itai (Rehovot, IL); Goldstein, Marc A. (Davis, CA); Doi, Roy H. (Davis, CA)

    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.

  14. Predicting nucleic acid binding interfaces from structural models of proteins.

    Science.gov (United States)

    Dror, Iris; Shazman, Shula; Mukherjee, Srayanta; Zhang, Yang; Glaser, Fabian; Mandel-Gutfreund, Yael

    2012-02-01

    The function of DNA- and RNA-binding proteins can be inferred from the characterization and accurate prediction of their binding interfaces. However, the main pitfall of various structure-based methods for predicting nucleic acid binding function is that they are all limited to a relatively small number of proteins for which high-resolution three-dimensional structures are available. In this study, we developed a pipeline for extracting functional electrostatic patches from surfaces of protein structural models, obtained using the I-TASSER protein structure predictor. The largest positive patches are extracted from the protein surface using the patchfinder algorithm. We show that functional electrostatic patches extracted from an ensemble of structural models highly overlap the patches extracted from high-resolution structures. Furthermore, by testing our pipeline on a set of 55 known nucleic acid binding proteins for which I-TASSER produces high-quality models, we show that the method accurately identifies the nucleic acids binding interface on structural models of proteins. Employing a combined patch approach we show that patches extracted from an ensemble of models better predicts the real nucleic acid binding interfaces compared with patches extracted from independent models. Overall, these results suggest that combining information from a collection of low-resolution structural models could be a valuable approach for functional annotation. We suggest that our method will be further applicable for predicting other functional surfaces of proteins with unknown structure. Copyright © 2011 Wiley Periodicals, Inc.

  15. Chiral morphology of calcite through selective binding of amino acids

    Science.gov (United States)

    Orme, Christine

    2002-03-01

    Many living organisms contain biominerals and composites with finely tuned properties, reflecting a remarkable level of control over the nucleation, growth and shape of the constituent crystals. Peptides and proteins play an important role in achieving this control. Using in situ AFM we find that site-specific binding of amino acid residues to surface steps changes the step-edge free energies, giving rise to direction-specific binding energies unique to individual amino acid enantiomers and leading to chiral modifications that propagate from atomic length scales to macroscopic length scales. Molecular modeling studies support an energetic basis for the differences in binding. Our results emphasize that the mechanism under-lying crystal modification through organic molecules is best understood by considering both stereochemical recognition as well as the effects of binding on the interfacial energies of the growing crystal.

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

    Directory of Open Access Journals (Sweden)

    Roviello GN

    2016-11-01

    Full Text Available Giovanni N Roviello,1 Caterina Vicidomini,1 Vincenzo Costanzo,1 Valentina Roviello2 1CNR Istituto di Biostrutture e Bioimmagini, Via Mezzocannone site and Headquarters, 2Centro Regionale di Competenza (CRdC Tecnologie, Via Nuova Agnano, Napoli, Italy Abstract: 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. Keywords: nucleic acid binding, polyinosinic acid, double-stranded nucleic acids, oligolysine, circular dichroism

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

    Science.gov (United States)

    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. Study of binding glycyrrhetic acid to AT1 receptor

    Institute of Scientific and Technical Information of China (English)

    ZHANG; Fengyun; (张凤云); YUE; Baozhen; (岳保珍); HE; Shipeng; (贺师鹏)

    2003-01-01

    To analyze the binding of glycyrrhetic acid (GA) to angiotensin II type I (AT1) receptor and to explore the mechanisms underlying the binding, primary cell culture of rat vascular smooth muscle cell (VSMC), radioactive ligand-receptor binding assay, lascer confocal scanning microscope (LCSM), Northern blot, 3H-TdR incorporation DNA assay were used in this study. The results suggest that specific binding of GA to AT1 receptor (IC50 value was 35.0 μmol/L) increases intracellular [Ca2+]i of VSMC, activates transcription factor c-myc and promotes the proliferation of VSMC, therefore GA was probably an agonist of AT1 receptor, providing a new target for GA's pharmaceutical effects.

  19. Analysis of the ligand binding properties of recombinant bovine liver-type fatty acid binding protein

    DEFF Research Database (Denmark)

    Rolf, B; Oudenampsen-Krüger, E; Börchers, T

    1995-01-01

    The coding part of the cDNA for bovine liver-type fatty acid binding protein (L-FABP) has been amplified by RT-PCR, cloned and used for the construction of an Escherichia coli (E. coli) expression system. The recombinant protein made up to 25% of the soluble E. coli proteins and could be isolated...

  20. Modeling lanthanide series binding sites on humic acid.

    Science.gov (United States)

    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.

  1. The inhibition of anti-DNA binding to DNA by nucleic acid binding polymers.

    Directory of Open Access Journals (Sweden)

    Nancy A Stearns

    Full Text Available Antibodies to DNA (anti-DNA are the serological hallmark of systemic lupus erythematosus (SLE and can mediate disease pathogenesis by the formation of immune complexes. Since blocking immune complex formation can attenuate disease manifestations, the effects of nucleic acid binding polymers (NABPs on anti-DNA binding in vitro were investigated. The compounds tested included polyamidoamine dendrimer, 1,4-diaminobutane core, generation 3.0 (PAMAM-G3, hexadimethrine bromide, and a β-cylodextrin-containing polycation. As shown with plasma from patients with SLE, NABPs can inhibit anti-DNA antibody binding in ELISA assays. The inhibition was specific since the NABPs did not affect binding to tetanus toxoid or the Sm protein, another lupus autoantigen. Furthermore, the polymers could displace antibody from preformed complexes. Together, these results indicate that NABPs can inhibit the formation of immune complexes and may represent a new approach to treatment.

  2. Kinetics of fatty acid binding ability of glycated human serum albumin

    Indian Academy of Sciences (India)

    Eiji Yamazaki; Minoru Inagaki; Osamu Kurita; Tetsuji Inoue

    2005-09-01

    Kinetics of fatty acid binding ability of glycated human serum albumin (HSA) were investigated by fluorescent displacement technique with 1-anilino-8-naphtharene sulphonic acid (ANS method), and photometric detection of nonesterified-fatty-acid (NEFA method). Changing of binding affinities of glycated HSA toward oleic acid, linoleic acid, lauric acid, and caproic acid, were not observed by the ANS method. However, decreases of binding capacities after 55 days glycation were confirmed by the NEFA method in comparison to control HSA. The decrease in binding affinities was: oleic acid (84%), linoleic acid (84%), lauric acid (87%), and caproic acid (90%), respectively. The decreases were consistent with decrease of the intact lysine residues in glycated HSA. The present observation indicates that HSA promptly loses its binding ability to fatty acid as soon as the lysine residues at fatty acid binding sites are glycated.

  3. Nucleic acid-binding glycoproteins which solubilize nucleic acids in dilute acid: re-examination of the Ustilago maydis glycoproteins

    Energy Technology Data Exchange (ETDEWEB)

    Unrau, P.; Champ, D.R.; Young, J.L.; Grant, C.E.

    1980-01-01

    Holloman reported the isolation from Ustilago maydis of a glycoprotein which prevented the precipitation of nucleic acids in cold 5% trichloroacetic acid. Two glycoprotein fractions from U. maydis with this nucleic acid-solubilizing activity were isolated in our laboratory using improved purification procedures. The activity was not due to nuclease contamination. The glycoproteins are distinguished by: their ability to bind to concanavalin A-Sepharose; their differential binding to double- and single-stranded deoxyribonucleic acid, and to ribonucleic acid; their molecular weights (46,000 and 69,000); and the relative amounts present in growing versus nongrowing cells. Both fractions required sulfhydryl-reducing conditions for optimal yields, specific activity, and stability. Nucleic acid binding was cooperative, the minimum number of glycoproteins required to make a native T7 DNA molecule soluble in dilute acid being estimated at 2 and 15, respectively.

  4. Cellular nucleic acid binding protein binds G-rich single-stranded nucleic acids and may function as a nucleic acid chaperone.

    Science.gov (United States)

    Armas, Pablo; Nasif, Sofía; Calcaterra, Nora B

    2008-02-15

    Cellular nucleic acid binding protein (CNBP) is a small single-stranded nucleic acid binding protein made of seven Zn knuckles and an Arg-Gly rich box. CNBP is strikingly conserved among vertebrates and was reported to play broad-spectrum functions in eukaryotic cells biology. Neither its biological function nor its mechanisms of action were elucidated yet. The main goal of this work was to gain further insights into the CNBP biochemical and molecular features. We studied Bufo arenarum CNBP (bCNBP) binding to single-stranded nucleic acid probes representing the main reported CNBP putative targets. We report that, although bCNBP is able to bind RNA and single-stranded DNA (ssDNA) probes in vitro, it binds RNA as a preformed dimer whereas both monomer and dimer are able to bind to ssDNA. A systematic analysis of variant probes shows that the preferred bCNBP targets contain unpaired guanosine-rich stretches. These data expand the knowledge about CNBP binding stoichiometry and begins to dissect the main features of CNBP nucleic acid targets. Besides, we show that bCNBP presents a highly disordered predicted structure and promotes the annealing and melting of nucleic acids in vitro. These features are typical of proteins that function as nucleic acid chaperones. Based on these data, we propose that CNBP may function as a nucleic acid chaperone through binding, remodeling, and stabilizing nucleic acids secondary structures. This novel CNBP biochemical activity broadens the field of study about its biological function and may be the basis to understand the diverse ways in which CNBP controls gene expression.

  5. Bile salt recognition by human liver fatty acid binding protein.

    Science.gov (United States)

    Favretto, Filippo; Santambrogio, Carlo; D'Onofrio, Mariapina; Molinari, Henriette; Grandori, Rita; Assfalg, Michael

    2015-04-01

    Fatty acid binding proteins (FABPs) act as intracellular carriers of lipid molecules, and play a role in global metabolism regulation. Liver FABP (L-FABP) is prominent among FABPs for its wide ligand repertoire, which includes long-chain fatty acids as well as bile acids (BAs). In this work, we performed a detailed molecular- and atomic-level analysis of the interactions established by human L-FABP with nine BAs to understand the binding specificity for this important class of cholesterol-derived metabolites. Protein-ligand complex formation was monitored using heteronuclear NMR, steady-state fluorescence spectroscopy, and mass spectrometry. BAs were found to interact with L-FABP with dissociation constants in the narrow range of 0.6-7 μm; however, the diverse substitution patterns of the sterol nucleus and the presence of side-chain conjugation resulted in complexes endowed with various degrees of conformational heterogeneity. Trihydroxylated BAs formed monomeric complexes in which single ligand molecules occupied similar internal binding sites, based on chemical-shift perturbation data. Analysis of NMR line shapes upon progressive addition of taurocholate indicated that the binding mechanism departed from a simple binary association equilibrium, and instead involved intermediates along the binding path. The co-linear chemical shift behavior observed for L-FABP complexes with cholate derivatives added insight into conformational dynamics in the presence of ligands. The observed spectroscopic features of L-FABP/BA complexes, discussed in relation to ligand chemistry, suggest possible molecular determinants of recognition, with implications regarding intracellular BA transport. Our findings suggest that human L-FABP is a poorly selective, universal BA binder.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1991-08-01

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

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

    NARCIS (Netherlands)

    Xiong, J.; Koopal, L.K.; Tan, W.; Fang, L.; Wang, W.; Zhao, W.; Liu, T.; Zhang, J.; Weng, L.

    2013-01-01

    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

  10. The clinical significance of fatty acid binding proteins

    Directory of Open Access Journals (Sweden)

    Barbara Choromańska

    2011-11-01

    Full Text Available Excessive levels of free fatty acids are toxic to cells. The human body has evolved a defense mechanism in the form of small cytoplasmic proteins called fatty acid binding proteins (FABPs that bind long-chain fatty acids (LCFA, and then refer them to appropriate intracellular disposal sites (oxidation in mitochondria and peroxisomes or storage in the endoplasmic reticulum. So far, nine types of these proteins have been described, and their name refers to the place in which they were first identified or where they can be found in the greatest concentration. The most important FABPs were isolated from the liver (L-FABP, heart (H-FABP, intestine (I-FABP, brain (B-FABP, epidermis (E-FABP and adipocytes (A-FABP. Determination of H-FABP is used in the diagnosis of myocardial infarction, and L-FABP in kidney lesions of different etiologies. It is postulated that FABPs play an important role in the pathogenesis of metabolic diseases. Elevated levels of A-FABP have been found in the pericardial fat tissue and were associated with cardiac dysfunction in obese people. A rise in A-FABP has been observed in patients with type II diabetes. I-FABP is known as a marker of cell damage in the small intestine. Increased concentration of B-FABP has been associated with human brain tumors such as glioblastoma and astrocytoma, as well as with neurodegenerative diseases (Alzheimer’s, Parkinson’s and other disorders of cognitive function. The aim of this work was to present current data on the clinical significance of fatty acid binding proteins.

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

    Science.gov (United States)

    Tonsgard, J H; Meredith, S C

    1991-06-15

    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.

  12. Structural and binding properties of two paralogous fatty acid binding proteins of Taenia solium metacestode.

    Directory of Open Access Journals (Sweden)

    Seon-Hee Kim

    Full Text Available BACKGROUND: Fatty acid (FA binding proteins (FABPs of helminths are implicated in acquisition and utilization of host-derived hydrophobic substances, as well as in signaling and cellular interactions. We previously demonstrated that secretory hydrophobic ligand binding proteins (HLBPs of Taenia solium metacestode (TsM, a causative agent of neurocysticercosis (NC, shuttle FAs in the surrounding host tissues and inwardly transport the FAs across the parasite syncytial membrane. However, the protein molecules responsible for the intracellular trafficking and assimilation of FAs have remained elusive. METHODOLOGY/PRINCIPAL FINDINGS: We isolated two novel TsMFABP genes (TsMFABP1 and TsMFABP2, which encoded 133- and 136-amino acid polypeptides with predicted molecular masses of 14.3 and 14.8 kDa, respectively. They shared 45% sequence identity with each other and 15-95% with other related-members. Homology modeling demonstrated a characteristic β-barrel composed of 10 anti-parallel β-strands and two α-helices. TsMFABP2 harbored two additional loops between β-strands two and three, and β-strands six and seven, respectively. TsMFABP1 was secreted into cyst fluid and surrounding environments, whereas TsMFABP2 was intracellularly confined. Partially purified native proteins migrated to 15 kDa with different isoelectric points of 9.2 (TsMFABP1 and 8.4 (TsMFABP2. Both native and recombinant proteins bound to 11-([5-dimethylaminonaphthalene-1-sulfonyl]aminoundecannoic acid, dansyl-DL-α-amino-caprylic acid, cis-parinaric acid and retinol, which were competitively inhibited by oleic acid. TsMFABP1 exhibited high affinity toward FA analogs. TsMFABPs showed weak binding activity to retinol, but TsMFABP2 showed relatively high affinity. Isolation of two distinct genes from an individual genome strongly suggested their paralogous nature. Abundant expression of TsMFABP1 and TsMFABP2 in the canal region of worm matched well with the histological distributions

  13. Structural and Binding Properties of Two Paralogous Fatty Acid Binding Proteins of Taenia solium Metacestode

    Science.gov (United States)

    Yang, Hyun-Jong; Shin, Joo-Ho; Diaz-Camacho, Sylvia Paz; Nawa, Yukifumi; Kang, Insug; Kong, Yoon

    2012-01-01

    Background Fatty acid (FA) binding proteins (FABPs) of helminths are implicated in acquisition and utilization of host-derived hydrophobic substances, as well as in signaling and cellular interactions. We previously demonstrated that secretory hydrophobic ligand binding proteins (HLBPs) of Taenia solium metacestode (TsM), a causative agent of neurocysticercosis (NC), shuttle FAs in the surrounding host tissues and inwardly transport the FAs across the parasite syncytial membrane. However, the protein molecules responsible for the intracellular trafficking and assimilation of FAs have remained elusive. Methodology/Principal Findings We isolated two novel TsMFABP genes (TsMFABP1 and TsMFABP2), which encoded 133- and 136-amino acid polypeptides with predicted molecular masses of 14.3 and 14.8 kDa, respectively. They shared 45% sequence identity with each other and 15–95% with other related-members. Homology modeling demonstrated a characteristic β-barrel composed of 10 anti-parallel β-strands and two α-helices. TsMFABP2 harbored two additional loops between β-strands two and three, and β-strands six and seven, respectively. TsMFABP1 was secreted into cyst fluid and surrounding environments, whereas TsMFABP2 was intracellularly confined. Partially purified native proteins migrated to 15 kDa with different isoelectric points of 9.2 (TsMFABP1) and 8.4 (TsMFABP2). Both native and recombinant proteins bound to 11-([5-dimethylaminonaphthalene-1-sulfonyl]amino)undecannoic acid, dansyl-DL-α-amino-caprylic acid, cis-parinaric acid and retinol, which were competitively inhibited by oleic acid. TsMFABP1 exhibited high affinity toward FA analogs. TsMFABPs showed weak binding activity to retinol, but TsMFABP2 showed relatively high affinity. Isolation of two distinct genes from an individual genome strongly suggested their paralogous nature. Abundant expression of TsMFABP1 and TsMFABP2 in the canal region of worm matched well with the histological distributions of lipids

  14. Simultaneous radioenzymatic assay of dopamine and dihydroxyphenylacetic acid: an index of in vivo dopamine release

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, C.H.; Wooten, G.F.

    1981-03-01

    The relative brain tissue concentrations of dopamine (DA) and its deaminated metabolite, dihydroxyphenylacetic acid (DOPAC), appears to be a reliable index of the functional activity of dopaminergic neurons. In order to apply this approach to the assessment of dopaminergic neuronal activity in small regions of brain, we have developed a sensitive radioenzymatic assay for simultaneous measurement of DA and DOPAC. The sensitivity of the assay for DA is approximately 10 pg and for DOPAC 100 pg. In addition, the assay is highly specific, simple, and relatively inexpensive. The concurrent estimation of tissue DA and DOPAC concentrations seems to be a reliable means of evaluating the rate of DA turnover or release in behavioral, electrical stimulation, and certain drug paradigms. However, the release or turnover of DA as induced by D-amphetamine (and perhaps other indirectly-acting dopaminemimetic drugs) cannot be meaningfully assessed by measurement of DA and DOPAC alone.

  15. Transcriptional regulation of muscle fatty acid-binding protein.

    Science.gov (United States)

    Carey, J O; Neufer, P D; Farrar, R P; Veerkamp, J H; Dohm, G L

    1994-03-15

    Heart fatty acid-binding protein (H-FABP) is present in a wide variety of tissues but is found in the highest concentration in cardiac and red skeletal muscle. It has been proposed that the expression of H-FABP correlates directly with the fatty acid-oxidative capacity of the tissue. In the present study, the expression of H-FABP was measured in red and white skeletal muscle under two conditions in which fatty acid utilization is known to be increased: streptozotocin-induced diabetes and fasting. Protein concentration, mRNA concentration and transcription rate were measured under both conditions. The level of both protein and mRNA increased approximately 2-fold under each condition. The transcription rate was higher in red skeletal muscle than in white muscle, was increased 2-fold during fasting, but was unchanged by streptozotocin-induced diabetes. In addition to supporting the hypothesis that H-FABP is induced during conditions of increased fatty acid utilization, these findings demonstrate that the regulation of H-FABP expression may or may not be at the level of transcription depending on the stimulus.

  16. Raisin dietary fiber composition and in vitro bile acid binding.

    Science.gov (United States)

    Camire, Mary E; Dougherty, Michael P

    2003-01-29

    Raisins are dried grapes that are popular shelf-stable snacks. Three commercially important types of raisins were studied: sun-dried (natural), artificially dried (dipped), and sulfur dioxide-treated (golden) raisins. Dietary fiber composition was analyzed by AACC method 32-25. Polysaccharides were hydrolyzed, and the resulting sugars were analyzed by colorimetric and gas chomatographic methods. Fructans were measured with a colorimetric kit assay. Total dietary fiber values agreed with published values, with pectins and neutral polysaccharides of mannose and glucose residues predominating. Dipped raisins had over 8% fructans. No fructans were found in fresh grapes. Raisin types varied in their ability to bind bile acids in vitro. Coarsely chopped raisins bound more bile than did finely chopped or whole raisins.

  17. Peptide nucleic acid (PNA) binding-mediated gene regulation

    Institute of Scientific and Technical Information of China (English)

    2004-01-01

    Peptide nucleic acids (PNAs) are synthetic oligonucleotides with chemically modified backbones. PNAs can bind to both DNA and RNA targets in a sequence-specific manner to form PNA/DNA and PNA/RNA duplex structures. When bound to double-stranded DNA (dsDNA) targets, the PNA molecule replaces one DNA strand in the duplex by strand invasion to form a PNA/DNA/PNA [or (PNA)2/DNA] triplex structure and the displaced DNA strand exists as a singlestranded D-loop. PNA has been used in many studies as research tools for gene regulation and gene targeting. The Dloops generated from the PNA binding have also been demonstrated for its potential in initiating transcription and inducing gene expression. PNA provides a powerful tool to study the mechanism of transcription and an innovative strategy to regulate target gene expression. An understanding of the PNA-mediated gene regulation will have important clinical implications in treatment of many human diseases including genetic, cancerous, and age-related diseases.

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

    Science.gov (United States)

    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.

  19. In Vitro Bile Acid Binding Capacities of Red Leaf Lettuce and Cruciferous Vegetables.

    Science.gov (United States)

    Yang, Isabelle F; Jayaprakasha, Guddadarangavvanahally K; Patil, Bhimanagouda S

    2017-09-13

    In the present study, we tested the bile acid binding capacity of red leaf lettuce, red cabbage, red kale, green kale, and Brussels sprouts through in vitro digestion process by simulating mouth, gastric, and intestinal digestion using six bile acids at physiological pH. Green and red kale exhibited significantly higher (86.5 ± 2.9 and 89.7 ± 0.9%, respectively) bile acid binding capacity compared to the other samples. Further, three different compositions of bile acids were tested to understand the effect on different health conditions. To predict the optimal dose for bile acid binding, we established a logistic relationship between kale dose and bile acid binding capacity. The results indicated that kale showed significantly higher bile acid binding capacity (82.5 ± 2.9% equivalent to 72.06 mg) at 1.5 g sample and remained constant up to 2.5 g. In addition, minimally processed (microwaved 3 min or steamed 8 min) green kale showed significantly enhanced bile acid binding capacity (91.1 ± 0.3 and 90.2 ± 0.7%, respectively) compared to lyophilized kale (85.5 ± 0.24%). Among the six bile acids tested, kale preferentially bound hydrophobic bile acids chenodeoxycholic acid and deoxycholic acid. Therefore, regular consumption of kale, especially minimally processed kale, can help excrete more bile acids and, thus, may lower the risk of hypercholesterolemia.

  20. Value of heart-type fatty acid-binding protein (H-FABP) for ...

    African Journals Online (AJOL)

    Key Words: heart-type fatty acid-binding protein, acute coronary syndrome, biomarker. ... facilitating the intracellular cytoplasmic transport of the fatty acids, and are highly expressed ..... nary syndrome: systematic review and critical appraisal.

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

    Science.gov (United States)

    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.

  2. Dietary Fat Content Effects on Concentrations of Liver and Intestinal Fatty Acid Binding Proteins

    OpenAIRE

    Murakami, Hiroshi; Sakai, Yasuo; Ohta, Kazutoshi; Hatakeyama, Katsuyoshi

    1998-01-01

    Two fatty acid binding proteins, liver and intestinal, have been identified in the rat intestine. Both are thought to be closely related to the absorption and metabolism of fatty acids in the intestinal epithelium. However, the underlying mechanism is not clearly understood. The purpose of this study was therefore to investigate the roles of these two fatty acid binding proteins in the intestinal absorption of fatty acids. Rats were fed diets varying in fat content for two or four weeks. Live...

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    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.

  6. Branched peptide boronic acids (BPBAs): a novel mode of binding towards RNA.

    Science.gov (United States)

    Zhang, Wenyu; Bryson, David I; Crumpton, Jason B; Wynn, Jessica; Santos, Webster L

    2013-03-25

    We report branched peptide boronic acids (BPBAs) that bind to RRE IIB from an on-bead high-throughput screening of a 3.3.4-library (46 656 compounds). We demonstrate that boronic acids are tunable moieties that afford a novel binding mode towards RNA.

  7. A Large-Scale Assessment of Nucleic Acids Binding Site Prediction Programs

    OpenAIRE

    Miao, Zhichao; Westhof, Eric

    2015-01-01

    Computational prediction of nucleic acid binding sites in proteins are necessary to disentangle functional mechanisms in most biological processes and to explore the binding mechanisms. Several strategies have been proposed, but the state-of-the-art approaches display a great diversity in i) the definition of nucleic acid binding sites; ii) the training and test datasets; iii) the algorithmic methods for the prediction strategies; iv) the performance measures and v) the distribution and avail...

  8. A Large-Scale Assessment of Nucleic Acids Binding Site Prediction Programs.

    OpenAIRE

    Zhichao Miao; Eric Westhof

    2015-01-01

    Computational prediction of nucleic acid binding sites in proteins are necessary to disentangle functional mechanisms in most biological processes and to explore the binding mechanisms. Several strategies have been proposed, but the state-of-the-art approaches display a great diversity in i) the definition of nucleic acid binding sites; ii) the training and test datasets; iii) the algorithmic methods for the prediction strategies; iv) the performance measures and v) the distribution and avail...

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Leenheer, J.A.; Brown, G.K.; Cabaniss, S.E. [Geological Survey, Denver, CO (United States); MacCarthy, P. [Colorado School of Mines, Golden, CO (United States). Dept. of Chemistry

    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. Binding of lipoic acid induces conformational change and appearance of a new binding site in methylglyoxal modified serum albumin.

    Science.gov (United States)

    Suji, George; Khedkar, Santosh A; Singh, Sreelekha K; Kishore, Nand; Coutinho, Evans C; Bhor, Vikrant M; Sivakami, S

    2008-06-01

    The binding of lipoic acid (LA), to methylglyoxal (MG) modified BSA was studied using isothermal titration calorimetry in combination with enzyme kinetics and molecular modelling. The binding of LA to BSA was sequential with two sites, one with higher binding constant and another comparatively lower. In contrast the modified protein showed three sequential binding sites with a reduction in affinity at the high affinity binding site by a factor of 10. CD results show appreciable changes in conformation of the modified protein as a result of binding to LA. The inhibition of esterase like activity of BSA by LA revealed that it binds to site II in domain III of BSA. The pH dependence of esterase activity of native BSA indicated a catalytic group with a pK(a) = 7.9 +/- 0.1, assigned to Tyr411 with the conjugate base stabilised by interaction with Arg410. Upon modification by MG, this pK(a) increased to 8.13. A complex obtained by docking of LA to BSA and BSA in which Arg410 is modified to hydroimidazolone showed that the long hydrocarbon chain of lipoic acid sits in a cavity different from the one observed for unmodified BSA. The molecular electrostatic potential showed that the modification of Arg410 reduced the positive electrostatic potential around the protein-binding site. Thus it can be concluded that the modification of BSA by MG resulted in altered ligand binding characteristics due to changes in the internal geometry and electrostatic potential at the binding site.

  12. Phylogenetic distribution of [3H]kainic acid receptor binding sites in neuronal tissue.

    Science.gov (United States)

    London, E D; Klemm, N; Coyle, J T

    1980-06-23

    The phylogenetic distribution of specific binding sites for kainic acid was determined in 14 species including invertebrates and vertebrates. The highest level of binding was observed in brains of the frog (Xenopus laevis), followed by the spiny dogfish (Heterodontus francisci), the goldfish (Carasius auratus) and the chick (Gallus domesticus). Although significant specific binding was noted in some of the lowest forms tested (e.g. Hydra littoralis), this was not a consistent observation in the invertebrates. In most cases, specific binding to both high and low affinity sites was detected; notable exceptions were the cockroach brain (Periplaneta americana), which had negligible high affinity binding, and the crayfish brain (Procambarus) which had negligible low affinity binding. In the spiny dogfish, the smooth dogfish and the chick, the highest level of binding occurred in cerebellum with less in the forebrain and the least in the medulla; in the mammalian species, the highest level of binding occurred in the forebrain structures with less in the cerebellum and least in the medulla. Eadie plots of the saturation isotherms for [3H]kainic acid revealed similar kinetics of binding for frog whole brain, rat forebrain and human parietal cortex with two apparent populations of binding sites: KD1 = 25--50 nM and KD2 = 3--14 nM. While binding in the spiny dogfish forebrain and human caudate nucleus occurred exclusively at a high affinity component, the cerebella of chick, rat and man exhibited only a low affinity binding site. In the 3 species studied most extensively, frog, rat and man, unlabeled kainic acid was the most potent inhibitor of the specific binding of [3H]-kainic acid. L-Glutamic acid was 20--20-fold less potent than kainic acid, and D-glutamic acid was 4--2500-fold less potent than its L-isomer. Reduction of the isopropylene side chain of kainic acid to form dihydrokainic acid decreased the affinity of the derivative 115--30,000-fold. Hill coefficients

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    CERN Document Server

    Ropo, Matti; Baldauf, Carsten

    2016-01-01

    We derive structural and binding energy trends for twenty amino acids, their dipeptides, and their interactions with the divalent cations Ca$^{2+}$, Ba$^{2+}$, Sr$^{2+}$, Cd$^{2+}$, Pb$^{2+}$, and Hg$^{2+}$. The underlying data set consists of 45,892 first-principles predicted conformers with relative energies up to about 4 eV (about 400kJ/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 C$_5$ or equatorial C$_7$. 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 ...

  16. Comparative analysis of the sialic acid binding activity of four different IBV strains

    OpenAIRE

    2009-01-01

    Abstract Avian infectious bronchitis virus (IBV) is a major pathogen in commercial poultry flocks. Recently we demonstrated, that sialic acid serves as a receptor determinant for IBV on the tracheal epithelium. Here we compared the IBV strains Beaudette, 4/91, Italy02, and QX for their sialic acid binding properties. We demonstrate that sialic acid binding is important for the infection of primary chicken kidney cells and the tracheal epithelium by all four strains. There were only...

  17. Cerebrospinal fluid 3,4-dihydroxyphenylacetic acid level after tolcapone administration as an indicator of nigrostriatal degeneration.

    Science.gov (United States)

    Thiffault, Christine; Langston, J William; Di Monte, Donato A

    2003-09-01

    The development of reliable biological markers of nigrostriatal degeneration has important implications from both experimental and clinical viewpoints, since such biomarkers could be used for diagnostic and monitoring purposes in models of parkinsonism as well as in Parkinson's disease patients. In this study, levels of the dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) were measured in the cerebrospinal fluid (CSF) of normal and parkinsonian squirrel monkeys in order to assess their reliability as indicators of nigrostriatal injury. In particular, we tested the hypothesis that these measurements may become more accurate by inhibiting catecholamine-O-methyltransferase (COMT) activity and therefore blocking the conversion of DOPAC to homovanillic acid. Oral administration of the COMT inhibitor tolcapone (2 doses of 15 mg/kg each with a 4-h interval) significantly reduced enzyme activity in the monkey brain. Tolcapone treatment enhanced CSF DOPAC concentrations in unlesioned animals (by approximately four times) as well as monkeys rendered parkinsonian after severe nigrostriatal dopaminergic injury caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Importantly, however, COMT inhibition greatly magnified the differences in CSF DOPAC levels between control and parkinsonian monkeys, since MPTP-induced DOPAC depletion was 35% in the absence vs >60% in the presence of tolcapone. Thus, tolcapone administration enhances the detection of DOPAC in the CSF and, by doing so, improves the reliability of CSF DOPAC as a marker of nigrostriatal degeneration.

  18. Thermodynamic parameters for binding of fatty acids to human serum albumin

    DEFF Research Database (Denmark)

    Pedersen, A O; Honoré, B; Brodersen, R

    1990-01-01

    Binding of laurate and myristate anions to human serum albumin has been studied over a range of temperatures, 5-37 degrees C, at pH 7.4. The binding curves indicate that the strength of binding of the first few molecules of fatty acid to albumin (r less than 5) decreases with increasing temperature...... constant, it was possible to calculate values for the changes in enthalpy and entropy during the initial binding step. For the medium-chain fatty acids, laurate and myristate, binding of the first molecule to albumin appeared to be enthalpic, with a tendency to an increasing contribution of entropy...... to binding energy with increasing chain length of the fatty acid. Udgivelsesdato: 1990-Jul-5...

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

    Science.gov (United States)

    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.

  20. Natural flavonoids as antidiabetic agents. The binding of gallic and ellagic acids to glycogen phosphorylase b.

    Science.gov (United States)

    Kyriakis, Efthimios; Stravodimos, George A; Kantsadi, Anastassia L; Chatzileontiadou, Demetra S M; Skamnaki, Vassiliki T; Leonidas, Demetres D

    2015-07-08

    We present a study on the binding of gallic acid and its dimer ellagic acid to glycogen phosphorylase (GP). Ellagic acid is a potent inhibitor with Kis of 13.4 and 7.5 μM, in contrast to gallic acid which displays Kis of 1.7 and 3.9 mM for GPb and GPa, respectively. Both compounds are competitive inhibitors with respect to the substrate, glucose-1-phoshate, and non-competitive to the allosteric activator, AMP. However, only ellagic acid functions with glucose in a strongly synergistic mode. The crystal structures of the GPb-gallic acid and GPb-ellagic acid complexes were determined at high resolution, revealing that both ligands bind to the inhibitor binding site of the enzyme and highlight the structural basis for the significant difference in their inhibitory potency.

  1. Interaction of Palmitic Acid with Metoprolol Succinate at the Binding Sites of Bovine Serum Albumin

    OpenAIRE

    Mashiur Rahman; Farzana Prianka; Mohammad Shohel; Md. Abdul Mazid

    2014-01-01

    Purpose: The aim of this study was to characterize the binding profile as well as to notify the interaction of palmitic acid with metoprolol succinate at its binding site on albumin. Methods: The binding of metoprolol succinate to bovine serum albumin (BSA) was studied by equilibrium dialysis method (ED) at 27°C and pH 7.4, in order to have an insight in the binding chemistry of the drug to BSA in presence and absence of palmitic acid. The study was carried out using ranitidine as site-1 a...

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

    Science.gov (United States)

    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...

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Directory of Open Access Journals (Sweden)

    Xin Peng

    Full Text Available 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.

  5. Unexpected tricovalent binding mode of boronic acids within the active site of a penicillin-binding protein.

    Science.gov (United States)

    Zervosen, Astrid; Herman, Raphael; Kerff, Frédéric; Herman, Alexandre; Bouillez, André; Prati, Fabio; Pratt, R F; Frère, Jean-Marie; Joris, Bernard; Luxen, André; Charlier, Paulette; Sauvage, Eric

    2011-07-20

    Boronic acids bearing appropriate side chains are good inhibitors of serine amidohydrolases. The boron usually adopts a tetrahedral conformation, bound to the nucleophilic serine of the active site and mimicking the transition state of the enzymatic reaction. We have solved the structures of complexes of a penicillin-binding protein, the DD-peptidase from Actinomadura sp. R39, with four amidomethylboronic acids (2,6-dimethoxybenzamidomethylboronic acid, phenylacetamidomethylboronic acid, 2-chlorobenzamidomethylboronic acid, and 2-nitrobenzamidomethylboronic acid) and the pinacol ester derived from phenylacetamidomethylboronic acid. We found that, in each case, the boron forms a tricovalent adduct with Oγ of Ser49, Ser298, and the terminal amine group of Lys410, three key residues involved in the catalytic mechanism of penicillin-binding proteins. This represents the first tricovalent enzyme-inhibitor adducts observed by crystallography. In two of the five R39-boronate structures, the boronic acid is found as a tricovalent adduct in two monomers of the asymmetric unit and as a monocovalent adduct with the active serine in the two remaining monomers of the asymmetric unit. Formation of the tricovalent complex from a classical monocovalent complex may involve rotation around the Ser49 Cα-Cβ bond to place the boron in a position to interact with Ser298 and Lys410, and a twisting of the side-chain amide such that its carbonyl oxygen is able to hydrogen bond to the oxyanion hole NH of Thr413. Biphasic kinetics were observed in three of the five cases, and details of the reaction between R39 and 2,6-dimethoxybenzamidomethylboronic acid were studied. Observation of biphasic kinetics was not, however, thought to be correlated to formation of tricovalent complexes, assuming that the latter do form in solution. On the basis of the crystallographic and kinetic results, a reaction scheme for this unexpected inhibition by boronic acids is proposed.

  6. Role of fatty acid binding proteins and long chain fatty acids in modulating nuclear receptors and gene transcription.

    Science.gov (United States)

    Schroeder, Friedhelm; Petrescu, Anca D; Huang, Huan; Atshaves, Barbara P; McIntosh, Avery L; Martin, Gregory G; Hostetler, Heather A; Vespa, Aude; Landrock, Danilo; Landrock, Kerstin K; Payne, H Ross; Kier, Ann B

    2008-01-01

    Abnormal energy regulation may significantly contribute to the pathogenesis of obesity, diabetes mellitus, cardiovascular disease, and cancer. For rapid control of energy homeostasis, allosteric and posttranslational events activate or alter activity of key metabolic enzymes. For longer impact, transcriptional regulation is more effective, especially in response to nutrients such as long chain fatty acids (LCFA). Recent advances provide insights into how poorly water-soluble lipid nutrients [LCFA; retinoic acid (RA)] and their metabolites (long chain fatty acyl Coenzyme A, LCFA-CoA) reach nuclei, bind their cognate ligand-activated receptors, and regulate transcription for signaling lipid and glucose catabolism or storage: (i) while serum and cytoplasmic LCFA levels are in the 200 mircroM-mM range, real-time imaging recently revealed that LCFA and LCFA-CoA are also located within nuclei (nM range); (ii) sensitive fluorescence binding assays show that LCFA-activated nuclear receptors [peroxisome proliferator-activated receptor-alpha (PPARalpha) and hepatocyte nuclear factor 4alpha (HNF4alpha)] exhibit high affinity (low nM KdS) for LCFA (PPARalpha) and/or LCFA-CoA (PPARalpha, HNF4alpha)-in the same range as nuclear levels of these ligands; (iii) live and fixed cell immunolabeling and imaging revealed that some cytoplasmic lipid binding proteins [liver fatty acid binding protein (L-FABP), acyl CoA binding protein (ACBP), cellular retinoic acid binding protein-2 (CRABP-2)] enter nuclei, bind nuclear receptors (PPARalpha, HNF4alpha, CRABP-2), and activate transcription of genes in fatty acid and glucose metabolism; and (iv) studies with gene ablated mice provided physiological relevance of LCFA and LCFA-CoA binding proteins in nuclear signaling. This led to the hypothesis that cytoplasmic lipid binding proteins transfer and channel lipidic ligands into nuclei for initiating nuclear receptor transcriptional activity to provide new lipid nutrient signaling pathways that

  7. The human fatty acid-binding protein family: Evolutionary divergences and functions

    Directory of Open Access Journals (Sweden)

    Smathers Rebecca L

    2011-03-01

    Full Text Available Abstract Fatty acid-binding proteins (FABPs are members of the intracellular lipid-binding protein (iLBP family and are involved in reversibly binding intracellular hydrophobic ligands and trafficking them throughout cellular compartments, including the peroxisomes, mitochondria, endoplasmic reticulum and nucleus. FABPs are small, structurally conserved cytosolic proteins consisting of a water-filled, interior-binding pocket surrounded by ten anti-parallel beta sheets, forming a beta barrel. At the superior surface, two alpha-helices cap the pocket and are thought to regulate binding. FABPs have broad specificity, including the ability to bind long-chain (C16-C20 fatty acids, eicosanoids, bile salts and peroxisome proliferators. FABPs demonstrate strong evolutionary conservation and are present in a spectrum of species including Drosophila melanogaster, Caenorhabditis elegans, mouse and human. The human genome consists of nine putatively functional protein-coding FABP genes. The most recently identified family member, FABP12, has been less studied.

  8. Combined fluorescence and electrochemical investigation on the binding interaction between organic acid and human serum albumin

    Institute of Scientific and Technical Information of China (English)

    CHEN Yan-Min; GUO Liang-Hong

    2009-01-01

    Human serum albumin (HSA) is a plasma protein responsible for the binding and transport of fatty acids and a variety of exogenous chemicals such as drugs and environmental pollutants. Such binding plays a crucial role in determining the ADME (absorption, distribution, metabolism, and excretion) and bioavailability of the pollutants. We report investigation on the binding interaction between HSA and acetic acid (C2), octanoic acid (C8) and dodecanoic acid (C12) by the combination of site-specific fluorescent probe, tryptophan intrinsic fluorescence and tyrosine electrochemistry. Two fluorescent probes, dansylamide and dansyl-L-proline, were employed in the displacement measurement to study fatty acid interaction with the two drug-binding sites on HSA. Intrinsic fluorescence of tryptophan in HSA was monitored upon addition of the fatty acids into HSA. Electrocatalyzed response of the tyrosine residues in HSA by a redox mediator was used to investigate the binding interaction. Qualitatively, observations made by the three approaches are very similar. HSA did not show any change in either fluorescence or electrochemistry after mixing with C2, suggesting there is no significant interaction with the short-chain fatty acid. For C8, the measured signal dropped in a single-exponential fashion, indicative of independent and non-cooperative binding. The calculated association constant and binding ratio is 3.1×106 L/mol and 1 with drug binding Site I, 1.1×107 L/mol and 1 with Site II, and 7.0×104 L/mol and 4 with the tryptophan site. The measurement with C12 displayed multiple phases of fluorescence change, suggesting cooperativity and allosteric effect of C12 binding. These results correlate well with those obtained by the established methods, and validate the new approach as a viable tool to study the interactions of environmental pollutants with biological molecules.

  9. A novel ligand-binding domain involved in allosteric regulation of amino acid metabolism in prokaryotes

    NARCIS (Netherlands)

    Ettema, T.J.G.; Brinkman, A.B.; Tani, T.H.; Rafferty, J.B.; Oost, van der J.

    2002-01-01

    A combination of sequence profile searching and structural protein analysis has revealed a novel type of small molecule binding domain that is involved in the allosteric regulation of prokaryotic amino acid metabolism. This domain, designated RAM, has been found to be fused to the DNA-binding domain

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

    Science.gov (United States)

    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...

  11. Peptide Nucleic Acids Having Enhanced Binding Affinity, Sequence Specificity and Solubility

    DEFF Research Database (Denmark)

    1998-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary DNA and RNA strands more strongly than a corresponding DNA strand, and exhibit increased sequence specificity and solubility. The peptide nucleic acids comprise ligands selected from a group consisting of naturally......-occurring nucleobases and non-naturally-occurring nucleobases attached to a polyamide backbone, and contain C1-C8 alkylamine side chains. Methods of enhancing the solubility, binding affinity and sequence specificity of PNAs are provided....

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

    Science.gov (United States)

    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. Interaction of Palmitic Acid with Metoprolol Succinate at the Binding Sites of Bovine Serum Albumin

    Directory of Open Access Journals (Sweden)

    Mashiur Rahman

    2014-12-01

    Full Text Available Purpose: The aim of this study was to characterize the binding profile as well as to notify the interaction of palmitic acid with metoprolol succinate at its binding site on albumin. Methods: The binding of metoprolol succinate to bovine serum albumin (BSA was studied by equilibrium dialysis method (ED at 27°C and pH 7.4, in order to have an insight in the binding chemistry of the drug to BSA in presence and absence of palmitic acid. The study was carried out using ranitidine as site-1 and diazepam as site-2 specific probe. Results: Different analysis of binding of metoprolol succinate to bovine serum albumin suggested two sets of association constants: high affinity association constant (k1 = 11.0 x 105 M-1 with low capacity (n1 = 2 and low affinity association (k2 = 4.0×105 M-1 constant with high capacity (n2 = 8 at pH 7.4 and 27°C. During concurrent administration of palmitic acid and metoprolol succinate in presence or absence of ranitidine or diazepam, it was found that palmitic acid displaced metoprolol succinate from its binding site on BSA resulting reduced binding of metoprolol succinate to BSA. The increment in free fraction of metoprolol succinate was from 26.27% to 55.08% upon the addition of increased concentration of palmitic acid at a concentration of 0×10-5 M to 16×10-5 M. In presence of ranitidine and diazepam, palmitic acid further increases the free fraction of metoprolol succinate from 33.05% to 66.95% and 40.68% to 72.88%, respectively. Conclusion: This data provided the evidence of interaction at higher concentration of palmitic acid at the binding sites on BSA, which might change the pharmacokinetic properties of metoprolol succinate.

  14. Antioxidant activity of bovine serum albumin binding amino acid Schiff-bases metal complexes

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Glutamic acid-salicylaldehyde Schiff-base metal complexes are bound into bovine serum albumin (BSA), which afforded BSA binding Schiff-base metal complexes (BSA-SalGluM, M=Cu, Co, Ni, Zn). The BSA binding metal complexes were characterized by UV-vis spectra and Native PAGE. It showed that the protein structures of BSA kept after coordinating amino acid Schiff-bases metal complexes. The effect of the antioxidant activity was investigated. The results indicate that the antioxidant capacity of BSA increased more than 10 times after binding Schiff-base metal complexes.

  15. NMR Studies of a New Binding Mode of the Amino Acid Esters by Porphyrinatozinc(Ⅱ)

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The binding mode of the amino acid ethyl esters(guest) by 5-(2-carboxylphenyl)-10,15,20-triphenylporphyrinatozinc(Ⅱ)(host 1) was studied by means of 1H NMR spectra. The binding mode is the hydrogen-bonding between the amino group of the guest and the carboxyl group of host 1 plus the coordination between the zinc atom of porphyrinatozinc(Ⅱ) and the carbonyl group of the guest. This is a novel binding mode of the metalloporphyrin to amino acid derivatives.

  16. Fluorescence Quenching and Binding Interaction of l0-Methylacridinium Iodide to Nucleic Acids

    Institute of Scientific and Technical Information of China (English)

    孙险峰; 江致勤; 丁兵林

    2003-01-01

    Interaction of 10-methylacridinium iodide (MAI) as fluorescence probe with nucleobases, nucleosides and nucleic acids has been studied by UV-visible absorption and fluorescence spectroscopy. It was found that fluorescence of MAI is strongly quenched by the nucleobases, nucleosides and nucleic acids, respectively. The quenching follows the Stern-Volmer linear equation. The fluorescence quenching rate constant (kq) was measured to be 109-1010 (L/mol)/s within the range of diffusion-controlled rate limit, indicating that the interaction between MAI and nucleic acid and their precursors is characteristic of electron transfer mechanism. In addition, the binding interaction model of MAI to calf thymus DNA (ct-DNA) was further investigated. Apparent hypochromism in the absorption spectra of MAI was observed when MAI binds to ct-DNA.Three spectroscopic methods, which include (1) UV spectroscopy, (2) fluorescence quenching of MAI, (3) competitive dual-probe method of MAI and ethidium bromide (EB), were utilized to determine the affinity binding constants (K)of MAI and ct-DNA. The binding constants K obtained from the above methods gave consistent data in the same range (1.0-5.5) ×104 L/mol, which lend credibility to these measurements. The binding site number was determined to be 1.9. The influence of thermal denaturation and phosphate concentration on the binding was examined. The binding model of MAI to ct-DNA including intercalation and outside binding was investigated.

  17. Lipid binding protein response to a bile acid library: a combined NMR and statistical approach.

    Science.gov (United States)

    Tomaselli, Simona; Pagano, Katiuscia; Boulton, Stephen; Zanzoni, Serena; Melacini, Giuseppe; Molinari, Henriette; Ragona, Laura

    2015-11-01

    Primary bile acids, differing in hydroxylation pattern, are synthesized from cholesterol in the liver and, once formed, can undergo extensive enzyme-catalysed glycine/taurine conjugation, giving rise to a complex mixture, the bile acid pool. Composition and concentration of the bile acid pool may be altered in diseases, posing a general question on the response of the carrier (bile acid binding protein) to the binding of ligands with different hydrophobic and steric profiles. A collection of NMR experiments (H/D exchange, HET-SOFAST, ePHOGSY NOESY/ROESY and (15) N relaxation measurements) was thus performed on apo and five different holo proteins, to monitor the binding pocket accessibility and dynamics. The ensemble of obtained data could be rationalized by a statistical approach, based on chemical shift covariance analysis, in terms of residue-specific correlations and collective protein response to ligand binding. The results indicate that the same residues are influenced by diverse chemical stresses: ligand binding always induces silencing of motions at the protein portal with a concomitant conformational rearrangement of a network of residues, located at the protein anti-portal region. This network of amino acids, which do not belong to the binding site, forms a contiguous surface, sensing the presence of the bound lipids, with a signalling role in switching protein-membrane interactions on and off.

  18. Tetranectin-binding site on plasminogen kringle 4 involves the lysine-binding pocket and at least one additional amino acid residue

    DEFF Research Database (Denmark)

    Graversen, Jonas Heilskov; Sigurskjold, B W; Thøgersen, H C

    2000-01-01

    that all amino acid residues of plasminogen kringle 4 found to be involved in t-AMCHA binding are also involved in binding tetranectin. Notably, one amino acid residue of plasminogen kringle 4, Arg 32, not involved in binding t-AMCHA, is critical for binding tetranectin. We also find that Asp 57 and Asp 55......, we analyze the interaction of wild-type and six single-residue mutants of recombinant plasminogen kringle 4 expressed in Escherichia coli with the recombinant C-type lectin domain of tetranectin and trans-aminomethyl-cyclohexanoic acid (t-AMCHA) using isothermal titration calorimetry. We find...

  19. Binding modes of aromatic ligands to mammalian heme peroxidases with associated functional implications: crystal structures of lactoperoxidase complexes with acetylsalicylic acid, salicylhydroxamic acid, and benzylhydroxamic acid.

    Science.gov (United States)

    Singh, Amit K; Singh, Nagendra; Sinha, Mau; Bhushan, Asha; Kaur, Punit; Srinivasan, Alagiri; Sharma, Sujata; Singh, Tej P

    2009-07-24

    The binding and structural studies of bovine lactoperoxidase with three aromatic ligands, acetylsalicylic acid (ASA), salicylhydoxamic acid (SHA), and benzylhydroxamic acid (BHA) show that all the three compounds bind to lactoperoxidase at the substrate binding site on the distal heme side. The binding of ASA occurs without perturbing the position of conserved heme water molecule W-1, whereas both SHA and BHA displace it by the hydroxyl group of their hydroxamic acid moieties. The acetyl group carbonyl oxygen atom of ASA forms a hydrogen bond with W-1, which in turn makes three other hydrogen-bonds, one each with heme iron, His-109 N(epsilon2), and Gln-105 N(epsilon2). In contrast, in the complexes of SHA and BHA, the OH group of hydroxamic acid moiety in both complexes interacts with heme iron directly with Fe-OH distances of 3.0 and 3.2A respectively. The OH is also hydrogen bonded to His-109 N(epsilon2) and Gln-105N(epsilon2). The plane of benzene ring of ASA is inclined at 70.7 degrees from the plane of heme moiety, whereas the aromatic planes of SHA and BHA are nearly parallel to the heme plane with inclinations of 15.7 and 6.2 degrees , respectively. The mode of ASA binding provides the information about the mechanism of action of aromatic substrates, whereas the binding characteristics of SHA and BHA indicate the mode of inhibitor binding.

  20. Molecular mechanism of recombinant liver fatty acid binding protein's antioxidant activity

    OpenAIRE

    YAN, JING; Gong, Yuewen; She, Yi-Min; Wang, Guqi; Roberts, Michael S; Burczynski, Frank J.

    2009-01-01

    Hepatocytes expressing liver fatty acid binding protein (L-FABP) are known to be more resistant to oxidative stress than those devoid of this protein. The mechanism for the observed antioxidant activity is not known. We examined the antioxidant mechanism of a recombinant rat L-FABP in the presence of a hydrophilic (AAPH) or lipophilic (AMVN) free radical generator. Recombinant L-FABP amino acid sequence and its amino acid oxidative products following oxidation were identified by MALDI quadrup...

  1. Identification of amino acid residues in PEPHC1 important for binding to the tumor-specific receptor EGFRvIII

    DEFF Research Database (Denmark)

    Hansen, Charlotte Lund; Hansen, Paul Robert; Pedersen, Nina

    2008-01-01

    to identify the amino acid residues important for binding of PEPHC1 to EGFRvIII. The results indicate that the amino acid residues at the N-terminus of PEPHC1 are essential for the binding to the mutated receptor. One analog, [Ala(12)]PEPHC1, showed higher selective binding to EGFRvIII than PEPHC1...

  2. Binding of Ca2+ to Glutamic Acid-Rich Polypeptides from the Rod Outer Segment

    Science.gov (United States)

    Haber-Pohlmeier, S.; Abarca-Heidemann, K.; Körschen, H. G.; Dhiman, H. Kaur; Heberle, J.; Schwalbe, H.; Klein-Seetharaman, J.; Kaupp, U. B.; Pohlmeier, A.

    2007-01-01

    Rod photoreceptors contain three different glutamic acid-rich proteins (GARPs) that have been proposed to control the propagation of Ca2+ from the site of its entry at the cyclic nucleotide-gated channel to the cytosol of the outer segment. We tested this hypothesis by measuring the binding of Ca2+ to the following five constructs related to GARPs of rod photoreceptors: a 32-mer peptide containing 22 carboxylate groups, polyglutamic acid, a recombinant segment comprising 73 carboxylate groups (GLU), GARP1, and GARP2. Ca2+ binding was investigated by means of a Ca2+-sensitive electrode. In all cases, Ca2+ binds with low affinity; the half-maximum binding constant K1/2 ranges from 6 to 16 mM. The binding stoichiometry between Ca2+ ions and carboxylic groups is ∼1:1; an exception is GARP2, where a binding stoichiometry of ∼1:2 was found. Hydrodynamic radii of 1.6, 2.8, 3.3, 5.7, and 6.7 nm were determined by dynamic light scattering for the 32-mer, polyglutamic acid, GLU, GARP2, and GARP1 constructs, respectively. These results suggest that the peptides as well as GARP1 and GARP2 do not adopt compact globular structures. We conclude that the structures should be regarded as loose coils with low-affinity, high-capacity Ca2+ binding. PMID:17218469

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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 amino acid mixtures.

  7. The sialic acid binding activity of the S protein facilitates infection by porcine transmissible gastroenteritis coronavirus

    Directory of Open Access Journals (Sweden)

    Enjuanes Luis

    2011-09-01

    Full Text Available Abstract Background Transmissible gastroenteritis virus (TGEV has a sialic acid binding activity that is believed to be important for enteropathogenicity, but that has so far appeared to be dispensable for infection of cultured cells. The aims of this study were to determine the effect of sialic acid binding for the infection of cultured cells under unfavorable conditions, and comparison of TGEV strains and mutants, as well as the avian coronavirus IBV concerning their dependence on the sialic acid binding activity. Methods The infectivity of different viruses was analyzed by a plaque assay after adsorption times of 5, 20, and 60 min. Prior to infection, cultured cells were either treated with neuraminidase to deplete sialic acids from the cell surface, or mock-treated. In a second approach, pre-treatment of the virus with porcine intestinal mucin was performed, followed by the plaque assay after a 5 min adsorption time. A student's t-test was used to verify the significance of the results. Results Desialylation of cells only had a minor effect on the infection by TGEV strain Purdue 46 when an adsorption period of 60 min was allowed for initiation of infection. However, when the adsorption time was reduced to 5 min the infectivity on desialylated cells decreased by more than 60%. A TGEV PUR46 mutant (HAD3 deficient in sialic acid binding showed a 77% lower titer than the parental virus after a 5 min adsorption time. After an adsorption time of 60 min the titer of HAD3 was 58% lower than that of TGEV PUR46. Another TGEV strain, TGEV Miller, and IBV Beaudette showed a reduction in infectivity after neuraminidase treatment of the cultured cells irrespective of the virion adsorption time. Conclusions Our results suggest that the sialic acid binding activity facilitates the infection by TGEV under unfavorable environmental conditions. The dependence on the sialic acid binding activity for an efficient infection differs in the analyzed TGEV strains.

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

    Science.gov (United States)

    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.

  9. Binding, tuning and mechanical function of the 4-hydroxy-cinnamic acid chromophore in photoactive yellow protein

    NARCIS (Netherlands)

    Horst, M.A. van der; Arents, J.C.; Kort, R.; Hellingwerf, K.J.

    2007-01-01

    The bacterial photoreceptor protein photoactive yellow protein (PYP) covalently binds the chromophore 4-hydroxy coumaric acid, tuning (spectral) characteristics of this cofactor. Here, we study this binding and tuning using a combination of pointmutations and chromophore analogs. In all photosensor

  10. Sialic Acid Binding Properties of Soluble Coronavirus Spike (S1 Proteins: Differences between Infectious Bronchitis Virus and Transmissible Gastroenteritis Virus

    Directory of Open Access Journals (Sweden)

    Christine Winter

    2013-07-01

    Full Text Available The spike proteins of a number of coronaviruses are able to bind to sialic acids present on the cell surface. The importance of this sialic acid binding ability during infection is, however, quite different. We compared the spike protein of transmissible gastroenteritis virus (TGEV and the spike protein of infectious bronchitis virus (IBV. Whereas sialic acid is the only receptor determinant known so far for IBV, TGEV requires interaction with its receptor aminopeptidase N to initiate infection of cells. Binding tests with soluble spike proteins carrying an IgG Fc-tag revealed pronounced differences between these two viral proteins. Binding of the IBV spike protein to host cells was in all experiments sialic acid dependent, whereas the soluble TGEV spike showed binding to APN but had no detectable sialic acid binding activity. Our results underline the different ways in which binding to sialoglycoconjugates is mediated by coronavirus spike proteins.

  11. Effect of liver fatty acid binding protein on fatty acid movement between liposomes and rat liver microsomes.

    Science.gov (United States)

    McCormack, M; Brecher, P

    1987-01-01

    Although movement of fatty acids between bilayers can occur spontaneously, it has been postulated that intracellular movement is facilitated by a class of proteins named fatty acid binding proteins (FABP). In this study we have incorporated long chain fatty acids into multilamellar liposomes made of phosphatidylcholine, incubated them with rat liver microsomes containing an active acyl-CoA synthetase, and measured formation of acyl-CoA in the absence or presence of FABP purified from rat liver. FABP increased about 2-fold the accumulation of acyl-CoA when liposomes were the fatty acid donor. Using fatty acid incorporated into liposomes made either of egg yolk lecithin or of dipalmitoylphosphatidylcholine, it was found that the temperature dependence of acyl-CoA accumulation in the presence of FABP correlated with both the physical state of phospholipid molecules in the liposomes and the binding of fatty acid to FABP, suggesting that fatty acid must first desorb from the liposomes before FABP can have an effect. An FABP-fatty acid complex incubated with microsomes, in the absence of liposomes, resulted in greater acyl-CoA formation than when liposomes were present, suggesting that desorption of fatty acid from the membrane is rate-limiting in the accumulation of acyl-CoA by this system. Finally, an equilibrium dialysis cell separating liposomes from microsomes on opposite sides of a Nuclepore filter was used to show that liver FABP was required for the movement and activation of fatty acid between the compartments. These studies show that liver FABP interacts with fatty acid that desorbs from phospholipid bilayers, and promotes movement to a membrane-bound enzyme, suggesting that FABP may act intracellularly by increasing net desorption of fatty acid from cell membranes. PMID:3446187

  12. Binding of phenazinium dye safranin T to polyriboadenylic acid: spectroscopic and thermodynamic study.

    Science.gov (United States)

    Pradhan, Ankur Bikash; Haque, Lucy; Roy, Snigdha; Das, Suman

    2014-01-01

    Here, we report results from experiments designed to explore the association of the phenazinium dye safranin T (ST, 3,7-diamino-2,8-dimethyl-5-phenylphenazinium chloride) with single and double stranded form of polyriboadenylic acid (hereafter poly-A) using several spectroscopic techniques. We demonstrate that the dye binds to single stranded polyriboadenylic acid (hereafter ss poly-A) with high affinity while it does not interact at all with the double stranded (ds) form of the polynucleotide. Fluorescence and absorption spectral studies reveal the molecular aspects of binding of ST to single stranded form of the polynucleotide. This observation is also supported by the circular dichroism study. Thermodynamic data obtained from temperature dependence of binding constant reveals that association is driven by negative enthalpy change and opposed by negative entropy change. Ferrocyanide quenching studies have shown intercalative binding of ST to ss poly-A. Experiments on viscosity measurements confirm the binding mode of the dye to be intercalative. The effect of [Na⁺] ion concentration on the binding process suggests the role of electrostatic forces in the complexation. Present studies reveal the utility of the dye in probing nucleic acid structure.

  13. Binding of phenazinium dye safranin T to polyriboadenylic acid: spectroscopic and thermodynamic study.

    Directory of Open Access Journals (Sweden)

    Ankur Bikash Pradhan

    Full Text Available Here, we report results from experiments designed to explore the association of the phenazinium dye safranin T (ST, 3,7-diamino-2,8-dimethyl-5-phenylphenazinium chloride with single and double stranded form of polyriboadenylic acid (hereafter poly-A using several spectroscopic techniques. We demonstrate that the dye binds to single stranded polyriboadenylic acid (hereafter ss poly-A with high affinity while it does not interact at all with the double stranded (ds form of the polynucleotide. Fluorescence and absorption spectral studies reveal the molecular aspects of binding of ST to single stranded form of the polynucleotide. This observation is also supported by the circular dichroism study. Thermodynamic data obtained from temperature dependence of binding constant reveals that association is driven by negative enthalpy change and opposed by negative entropy change. Ferrocyanide quenching studies have shown intercalative binding of ST to ss poly-A. Experiments on viscosity measurements confirm the binding mode of the dye to be intercalative. The effect of [Na⁺] ion concentration on the binding process suggests the role of electrostatic forces in the complexation. Present studies reveal the utility of the dye in probing nucleic acid structure.

  14. A Large-Scale Assessment of Nucleic Acids Binding Site Prediction Programs.

    Directory of Open Access Journals (Sweden)

    Zhichao Miao

    2015-12-01

    Full Text Available Computational prediction of nucleic acid binding sites in proteins are necessary to disentangle functional mechanisms in most biological processes and to explore the binding mechanisms. Several strategies have been proposed, but the state-of-the-art approaches display a great diversity in i the definition of nucleic acid binding sites; ii the training and test datasets; iii the algorithmic methods for the prediction strategies; iv the performance measures and v the distribution and availability of the prediction programs. Here we report a large-scale assessment of 19 web servers and 3 stand-alone programs on 41 datasets including more than 5000 proteins derived from 3D structures of protein-nucleic acid complexes. Well-defined binary assessment criteria (specificity, sensitivity, precision, accuracy… are applied. We found that i the tools have been greatly improved over the years; ii some of the approaches suffer from theoretical defects and there is still room for sorting out the essential mechanisms of binding; iii RNA binding and DNA binding appear to follow similar driving forces and iv dataset bias may exist in some methods.

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

    Science.gov (United States)

    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.

  16. Evolutionarily conserved bias of amino-acid usage refines the definition of PDZ-binding motif.

    Science.gov (United States)

    Chimura, Takahiko; Launey, Thomas; Ito, Masao

    2011-06-08

    The interactions between PDZ (PSD-95, Dlg, ZO-1) domains and PDZ-binding motifs play central roles in signal transductions within cells. Proteins with PDZ domains bind to PDZ-binding motifs almost exclusively when the motifs are located at the carboxyl (C-) terminal ends of their binding partners. However, it remains little explored whether PDZ-binding motifs show any preferential location at the C-terminal ends of proteins, at genome-level. Here, we examined the distribution of the type-I (x-x-S/T-x-I/L/V) or type-II (x-x-V-x-I/V) PDZ-binding motifs in proteins encoded in the genomes of five different species (human, mouse, zebrafish, fruit fly and nematode). We first established that these PDZ-binding motifs are indeed preferentially present at their C-terminal ends. Moreover, we found specific amino acid (AA) bias for the 'x' positions in the motifs at the C-terminal ends. In general, hydrophilic AAs were favored. Our genomics-based findings confirm and largely extend the results of previous interaction-based studies, allowing us to propose refined consensus sequences for all of the examined PDZ-binding motifs. An ontological analysis revealed that the refined motifs are functionally relevant since a large fraction of the proteins bearing the motif appear to be involved in signal transduction. Furthermore, co-precipitation experiments confirmed two new protein interactions predicted by our genomics-based approach. Finally, we show that influenza virus pathogenicity can be correlated with PDZ-binding motif, with high-virulence viral proteins bearing a refined PDZ-binding motif. Our refined definition of PDZ-binding motifs should provide important clues for identifying functional PDZ-binding motifs and proteins involved in signal transduction.

  17. Evolutionarily conserved bias of amino-acid usage refines the definition of PDZ-binding motif

    Directory of Open Access Journals (Sweden)

    Launey Thomas

    2011-06-01

    Full Text Available Abstract Background The interactions between PDZ (PSD-95, Dlg, ZO-1 domains and PDZ-binding motifs play central roles in signal transductions within cells. Proteins with PDZ domains bind to PDZ-binding motifs almost exclusively when the motifs are located at the carboxyl (C- terminal ends of their binding partners. However, it remains little explored whether PDZ-binding motifs show any preferential location at the C-terminal ends of proteins, at genome-level. Results Here, we examined the distribution of the type-I (x-x-S/T-x-I/L/V or type-II (x-x-V-x-I/V PDZ-binding motifs in proteins encoded in the genomes of five different species (human, mouse, zebrafish, fruit fly and nematode. We first established that these PDZ-binding motifs are indeed preferentially present at their C-terminal ends. Moreover, we found specific amino acid (AA bias for the 'x' positions in the motifs at the C-terminal ends. In general, hydrophilic AAs were favored. Our genomics-based findings confirm and largely extend the results of previous interaction-based studies, allowing us to propose refined consensus sequences for all of the examined PDZ-binding motifs. An ontological analysis revealed that the refined motifs are functionally relevant since a large fraction of the proteins bearing the motif appear to be involved in signal transduction. Furthermore, co-precipitation experiments confirmed two new protein interactions predicted by our genomics-based approach. Finally, we show that influenza virus pathogenicity can be correlated with PDZ-binding motif, with high-virulence viral proteins bearing a refined PDZ-binding motif. Conclusions Our refined definition of PDZ-binding motifs should provide important clues for identifying functional PDZ-binding motifs and proteins involved in signal transduction.

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

    Science.gov (United States)

    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.

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

    OpenAIRE

    Zahid, Henna; Miah, Layeque; Lau, Andy; Brochard, Lea; Hati, Debolina; Bui, T. T.; Drake, A. 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 investigate...

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

    Science.gov (United States)

    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

  1. Development of a Novel Tetravalent Synthetic Peptide That Binds to Phosphatidic Acid.

    Directory of Open Access Journals (Sweden)

    Rina Ogawa

    Full Text Available We employed a multivalent peptide-library screening technique to identify a peptide motif that binds to phosphatidic acid (PA, but not to other phospholipids such as phosphatidylcholine (PC, phosphatidylethanolamine (PE, and phosphatidylserine (PS. A tetravalent peptide with the sequence motif of MARWHRHHH, designated as PAB-TP (phosphatidic acid-binding tetravalent peptide, was shown to bind as low as 1 mol% of PA in the bilayer membrane composed of PC and cholesterol. Kinetic analysis of the interaction between PAB-TP and the membranes containing 10 mol% of PA showed that PAB-TP associated with PA with a low dissociation constant of KD = 38 ± 5 nM. Coexistence of cholesterol or PE with PA in the membrane enhanced the PAB-TP binding to PA by increasing the ionization of the phosphomonoester head group as well as by changing the microenvironment of PA molecules in the membrane. Amino acid replacement analysis demonstrated that the tryptophan residue at position 4 of PAB-TP was involved in the interaction with PA. Furthermore, a series of amino acid substitutions at positions 5 to 9 of PAB-TP revealed the involvement of consecutive histidine and arginine residues in recognition of the phosphomonoester head group of PA. Our results demonstrate that the recognition of PA by PAB-TP is achieved by a combination of hydrophobic, electrostatic and hydrogen-bond interactions, and that the tetravalent structure of PAB-TP contributes to the high affinity binding to PA in the membrane. The novel PA-binding tetravalent peptide PAB-TP will provide insight into the molecular mechanism underlying the recognition of PA by PA-binding proteins that are involved in various cellular events.

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

    Science.gov (United States)

    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.

  3. Relationship between serum adiopocyte fatty acid binding protein and atherosclerosis in chronic kidney disease

    Institute of Scientific and Technical Information of China (English)

    吴晶

    2014-01-01

    Objective To investigate the expression of serum adiopocyte fatty acid binding protein(A-FABP)in chronic kidney disease(CKD)and the role that A-FABP plays in CKD with atherosclerosis.Methods A total of 138 patients with CKD and 20 health control volunteers(HC)were involved in this study.The levels of serum AFABP,free fatty acid(FFA),interleukin-6(IL-6),

  4. Determination of solvation and binding site profile within electropolymerised poly(pyrrole-N-propionic acid)

    DEFF Research Database (Denmark)

    Glidle, A.; Swann, M.J.; Gadegaard, Nikolaj

    2000-01-01

    Specular neutron reflectivity measurements were performed on electropolymerised films of poly(pyrrole-N-propionic acid) to determine the degree to which the solvents are capable of supporting biological macromolecules can penetrate the polymer film. The permeation profile of Ni2+ ions which chela...... to the polymer's carboxylic acid moieties for subsequent modified protein binding was also determined. (C) 2000 Elsevier Science B.V. All rights reserved....

  5. Hepatic phenotype of liver fatty acid binding protein gene-ablated mice

    OpenAIRE

    Martin, Gregory G.; Atshaves, Barbara P.; Huang, Huan; McIntosh, Avery L.; Williams, Brad J.; Pai, Pei-Jing; Russell, David H.; Kier, Ann B.; Schroeder, Friedhelm

    2009-01-01

    Although the function of liver fatty acid binding protein in hepatic fatty acid metabolism has been extensively studied, its potential role in hepatic cholesterol homeostasis is less clear. Although hepatic cholesterol accumulation was initially reported in L-FABP-null female mice, that study was performed with early N2 backcross generation mice. To resolve whether the hepatic cholesterol phenotype in these L-FABP−/− mice was attributable to genetic inhomogeneity, these L-FABP−/− mice were fu...

  6. CIN85 associates with endosomal membrane and binds phosphatidic acid

    Institute of Scientific and Technical Information of China (English)

    Jing Zhang; Xiudan Zheng; Xiao Yang; Kan Liao

    2009-01-01

    CIN85 (Cbl-interacting protein of 85 kDa) is an important molecule involved in receptor tyrosine kinase endocy-tosis. Here we report that through its positively charged C-terminus, CIN85 associates with a fusogenic lipid - phos-phatidic acid. Its coiled-coil domain plays an important role in mediating this protein-lipid interaction. Deletion of the coiled-coil domain results in loss of membrane association, and reduced interaction with c-cbl, finally causing the blockage of epidermal growth factor receptor downregulation, In addition, a significant portion of CIN85 is located on the endosomai compartment and is related to endocytic cargo sorting, characterized by CIN85's localization on the "E class" compartment and EGF degradation blockage in CIN85 knockdown cells. Taken together, our results suggest that CIN85 may function as a scaffold molecule in both the internalization and endocytic cargo sorting pro-cesses through its association with the endosomal membrane.

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

    Science.gov (United States)

    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)

  8. Selective binding of glutathione conjugates of fatty acid derivatives by plant glutathione transferases.

    Science.gov (United States)

    Dixon, David P; Edwards, Robert

    2009-08-07

    Proteomic studies with Arabidopsis thaliana have revealed that the plant-specific Tau (U) class glutathione transferases (GSTs) are selectively retained by S-hexylglutathione affinity supports. Overexpression of members of the Arabidopsis GST superfamily in Escherichia coli showed that 25 of the complement of 28 GSTUs caused the aberrant accumulation of acylated glutathione thioesters in vivo, a perturbation that was not observed with other GST classes. Each GSTU caused a specific group of fatty acyl derivatives to accumulate, which varied in chain length (C(6) to C(18)), additional oxygen content (0 or 1), and desaturation (0 or 1). Thioesters bound tightly to recombinant GSTs (K(d) approximately 1 microm), explaining their accumulation. Transient expression of GSTUs in Nicotiana benthamiana followed by recovery by Strep-tag affinity chromatography allowed the respective plant ligands to be extracted and characterized. Again, each GST showed a distinct profile of recovered metabolites, notably glutathionylated oxophytodienoic acid and related oxygenated fatty acids. Similarly, the expression of the major Tau protein GSTU19 in the endogenous host Arabidopsis led to the selective binding of the glutathionylated oxophytodienoic acid-glutathione conjugate, with the enzyme able to catalyze the conjugation reaction. Additional ligands identified in planta included other fatty acid derivatives including divinyl ethers and glutathionylated chlorogenic acid. The strong and specific retention of various oxygenated fatty acids by each GSTU and the conservation in binding observed in the different hosts suggest that these proteins have selective roles in binding and conjugating these unstable metabolites in vivo.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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. Compartmentation of hepatic fatty-acid-binding protein in liver cells and its effect on microsomal phosphatidic acid biosynthesis.

    Science.gov (United States)

    Bordewick, U; Heese, M; Börchers, T; Robenek, H; Spener, F

    1989-03-01

    Fatty-acid-binding proteins are known to occur in the cytosol of mammalian cells and to bind fatty acids and their CoA-esters. Application of the postembedding protein A-gold labeling method with antibody against the hepatic type fatty-acid-binding protein (hFABP) to cross-sections of liver cells and a newly developed gel-chromatographic immunofluorescence assay established qualitatively (1) that hFABP in mitochondria was confined to outer mitochondrial membranes, (2) the presence of this protein in microsomes and (3) that nuclei were also filled with hFABP. Quantitative data elaborated with a non-competitive ELISA confirmed these results. A significant difference to the distribution of cardiac FABP in heart muscle cells, where this type of protein was found in cytosol, matrix and nuclei, was observed (Börchers et al. (1989) Biochim. Biophys. Acta, in the press). hFABP-containing rat liver microsomes were incubated with long-chain acyl-CoAs in the presence of hFABP (isolated from rat liver cytosol) in a study on the acylation of sn-glycerol-3-phosphate and lysophosphatidic acid. Both acyltransferases were stimulated by addition of hFABP to the incubation medium. The morphological, immunochemical as well as kinetic data infer a direct interaction of hFABP with microsomal membranes in liver cells.

  12. Lack of upregulation of epidermal fatty acid binding protein in dithranol induced irritation.

    NARCIS (Netherlands)

    Kucharekova, M.; Vissers, W.H.P.M.; Schalkwijk, J.; Kerkhof, P.C.M. van de; Valk, P.G.M. van der

    2003-01-01

    The exact role of epidermal fatty acid binding protein (E-FABP) in skin is unknown. A restoration of the barrier function may be associated with an upregulation of E-FABP. Moreover, E-FABP is upregulated in a variety of cells in response to oxidative stress. A recent observation that dithranol induc

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

    Science.gov (United States)

    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.

  14. Urinary excretion of fatty acid-binding proteins in idiopathic membranous nephropathy.

    NARCIS (Netherlands)

    Hofstra, J.M.; Deegens, J.K.J.; Steenbergen, E.J.; Wetzels, J.F.M.

    2008-01-01

    BACKGROUND: It is suggested that proteinuria contributes to progressive renal failure by inducing tubular cell injury. The site of injury is unknown. Most studies have used markers of proximal tubular cell damage. Fatty acid-binding proteins (FABPs) are intracellular carrier proteins with different

  15. Early Diagnosis of Intestinal Ischemia Using Urinary and Plasma Fatty Acid Binding Proteins

    NARCIS (Netherlands)

    Thuijls, Geertje; van Wijck, Kim; Grootjans, Joep; Derikx, Joep P. M.; van Bijnen, Annemarie A.; Heineman, Erik; Dejong, Cornelis H. C.; Buurman, Wim A.; Poeze, Martijn

    2011-01-01

    Objective: This study aims at improving diagnosis of intestinal ischemia, by measuring plasma and urinary fatty acid binding protein (FABP) levels. Methods: Fifty consecutive patients suspected of intestinal ischemia were included and blood and urine were sampled at time of suspicion. Plasma and uri

  16. Heart-type fatty-acid-binding protein (FABP3 is a lysophosphatidic acid-binding protein in human coronary artery endothelial cells

    Directory of Open Access Journals (Sweden)

    Ryoko Tsukahara

    2014-01-01

    Full Text Available Fatty-acid-binding protein 3, muscle and heart (FABP3, also known as heart-type FABP, is a member of the family of intracellular lipid-binding proteins. It is a small cytoplasmic protein with a molecular mass of about 15 kDa. FABPs are known to be carrier proteins for transporting fatty acids and other lipophilic substances from the cytoplasm to the nucleus, where these lipids are released to a group of nuclear receptors such as peroxisome proliferator-activated receptors (PPARs. In this study, using lysophosphatidic acid (LPA-coated agarose beads, we have identified FABP3 as an LPA carrier protein in human coronary artery endothelial cells (HCAECs. Administration of LPA to HCAECs resulted in a dose-dependent increase in PPARγ activation. Furthermore, the LPA-induced PPARγ activation was abolished when the FABP3 expression was reduced using small interfering RNA (siRNA. We further show that the nuclear fraction of control HCAECs contained a significant amount of exogenously added LPA, whereas FABP3 siRNA-transfected HCAECs had a decreased level of LPA in the nucleus. Taken together, these results suggest that FABP3 governs the transcriptional activities of LPA by targeting them to cognate PPARγ in the nucleus.

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

    Directory of Open Access Journals (Sweden)

    Gabriela Alvite

    Full Text Available 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.

  18. Towards an Understanding of Mesocestoides vogae Fatty Acid Binding Proteins’ Roles

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  1. Proton binding by humic and fulvic acids from pig slurry and amended soils.

    Science.gov (United States)

    Plaza, César; García-Gil, Juan C; Polo, Alfredo; Senesi, Nicola; Brunetti, Gennaro

    2005-01-01

    The knowledge of acid-base characteristics of humic acid (HA) and fulvic acid (FA) fractions of organic amendments and amended soils is of considerable importance for assessing their agronomic efficacy and environmental impact. In this work, the acid-base properties of HAs and FAs isolated from pig slurry, soils amended with either 90 or 150 m(3) ha(-1) yr(-1) of pig slurry for 3 yr, and the corresponding nonamended control soil were investigated by using a current potentiometric titration method. The nonideal competitive adsorption (NICA) model that describes proton binding by two classes of binding sites (carboxylic- and phenolic-type groups) was successfully fit to titration data. With respect to the control soil HA and FA, pig-slurry HA and FA were generally characterized by smaller carboxylic-type group contents, slightly smaller phenolic-type group contents, larger affinities for proton binding by the carboxylic-type groups, and much smaller, in the case of the HA fraction, or similar, in the case of the FA fraction, affinities for proton binding by the phenolic-type groups. Amendment with pig slurry determined a number of modifications in soil HAs and FAs, including decrease of acidic functional group contents, and slight increase of the proton affinity of the carboxylic-type groups. Further, a slight decrease of the affinities for proton binding by the phenolic-type groups of HAs was observed. These effects can have a large impact on the biological availability, mobilization, and transport of macro- and micronutrients, toxic metal ions, and xenobiotic organic cations in pig slurry-amended soils.

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

    Science.gov (United States)

    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.

  3. Analysis of the binding interaction in uric acid - Human hemoglobin system by spectroscopic techniques

    Science.gov (United States)

    Makarska-Bialokoz, Magdalena

    2017-05-01

    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.

  4. Roles played by acidic lipids in HIV-1 Gag membrane binding.

    Science.gov (United States)

    Olety, Balaji; Ono, Akira

    2014-11-26

    The MA domain mediates plasma membrane (PM) targeting of HIV-1 Gag, leading to particle assembly at the PM. The interaction between MA and acidic phospholipids, in addition to N-terminal myristoyl moiety, promotes Gag binding to lipid membranes. Among acidic phospholipids, PI(4,5)P2, a PM-specific phosphoinositide, is essential for proper HIV-1 Gag localization to the PM and efficient virus particle production. Recent studies further revealed that MA-bound RNA negatively regulates HIV-1 Gag membrane binding and that PI(4,5)P2 is necessary to overcome this RNA-imposed block. In this review, we will summarize the current understanding of Gag-membrane interactions and discuss potential roles played by acidic phospholipids.

  5. Chemical functionalization of oligodeoxynucleotides with multiple boronic acids for the polyvalent binding of saccharides.

    Science.gov (United States)

    Hargrove, Amanda E; Ellington, Andrew D; Anslyn, Eric V; Sessler, Jonathan L

    2011-03-16

    A novel saccharide host containing four boronic acid recognition units on a single DNA duplex terminus was constructed. This construct allowed boronic acid sugar recognition in the context of double-stranded DNA to be established while highlighting the benefits of multivalency. Following the solid-phase synthesis of a bis-boronic acid tag, two end-functionalized oligonucleotides with complementary sequences were functionalized through amide ligation. By annealing the boronic acid-DNA conjugates, a tetra-boronic acid DNA duplex was assembled. The saccharide binding ability of this tetra-boronic acid host was revealed through cellulose paper chromatography in the absence and presence of various saccharides. While no appreciable saccharide binding was seen in the case of a bis-boronic DNA conjugate, the increased migration of the tetra-boronic acid host relative to the control sequences in the presence of selected monosaccharides underscored the importance of multivalent effects. We thus identified a requirement for multiple recognition sites in these conjugate systems and expect the results to facilitate future efforts toward applying synthetic recognition systems to the realm of macromolecules.

  6. In Vitro bile acid binding of kale, mustard greens, broccoli, cabbage and green bell pepper improves with microwave cooking

    Science.gov (United States)

    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...

  7. Tolerance to acetic acid is improved by mutations of the TATA-binding protein gene.

    Science.gov (United States)

    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.

  8. Interactions between Human Liver Fatty Acid Binding Protein and Peroxisome Proliferator Activated Receptor Selective Drugs

    Directory of Open Access Journals (Sweden)

    Tony Velkov

    2013-01-01

    Full Text Available Fatty acid binding proteins (FABPs act as intracellular shuttles for fatty acids as well as lipophilic xenobiotics to the nucleus, where these ligands are released to a group of nuclear receptors called the peroxisome proliferator activated receptors (PPARs. PPAR mediated gene activation is ultimately involved in maintenance of cellular homeostasis through the transcriptional regulation of metabolic enzymes and transporters that target the activating ligand. Here we show that liver- (L- FABP displays a high binding affinity for PPAR subtype selective drugs. NMR chemical shift perturbation mapping and proteolytic protection experiments show that the binding of the PPAR subtype selective drugs produces conformational changes that stabilize the portal region of L-FABP. NMR chemical shift perturbation studies also revealed that L-FABP can form a complex with the PPAR ligand binding domain (LBD of PPARα. This protein-protein interaction may represent a mechanism for facilitating the activation of PPAR transcriptional activity via the direct channeling of ligands between the binding pocket of L-FABP and the PPARαLBD. The role of L-FABP in the delivery of ligands directly to PPARα via this channeling mechanism has important implications for regulatory pathways that mediate xenobiotic responses and host protection in tissues such as the small intestine and the liver where L-FABP is highly expressed.

  9. SAAMBE: Webserver to Predict the Charge of Binding Free Energy Caused by Amino Acids Mutations.

    Science.gov (United States)

    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/.

  10. Glutamate-containing dipeptides do not modulate ligand binding at excitatory amino acid receptors.

    Science.gov (United States)

    Baud, J; Fagg, G E

    1986-10-08

    Dipeptides of the structure X-Glu (e.g. X = Phe, Leu) have been proposed as allosteric modulators of excitatory amino acid receptors in rat brain membranes. Here we report that these dipeptides reduce the binding of L-[3H]Glu (predominantly N-methyl-D-aspartate-sensitive sites) and of [3H]kainate to postsynaptic density preparations isolated from rat brain. However, several observations indicate that the effects of these dipeptides are mediated not by allosteric modulation, but by free L-Glu liberated by the actions of a membrane-associated aminopeptidase. The absolute and relative potencies of the dipeptides are similar at all acidic amino acid binding sites examined to date, suggesting the involvement of a factor with similar activity at each site (e.g. L-Glu). N-Acetyl-Met-Glu is a weak inhibitor of L-Glu and kainate binding, and N-blocked peptides are known to be poor substrates of aminopeptidases. Bestatin, an inhibitor of aminopeptidases, decreases or abolishes the effects of substrate dipeptides on L-Glu and kainate receptor binding, while having no effect itself.

  11. The lactococcal abortive infection protein AbiP is membrane-anchored and binds nucleic acids.

    Science.gov (United States)

    Domingues, Susana; McGovern, Stephen; Plochocka, Danuta; Santos, Mário A; Ehrlich, S Dusko; Polard, Patrice; Chopin, Marie-Christine

    2008-03-30

    AbiP, a lactococcal abortive phage infection system, has previously been shown to arrest phage bIL66M1 DNA replication around 10 min after infection and to inhibit the switch off of phage early transcripts. We report here the functional characterization and implication in the abortive infection phenotype of two domains identified in the AbiP sequence. We show that AbiP is a protein anchored to the membrane by an N-terminal membrane-spanning domain. Our results further suggest that membrane localization may be required for the anti-phage activity of AbiP. The remainder of the protein, which contains a putative nucleic acid binding domain, is shown to be located on the cytosolic side. Purified AbiP is shown to bind nucleic acids with an approximately 10-fold preference for RNA relative to ssDNA. AbiP interaction with both ssDNA and RNA molecules occurs in a sequence-independent manner. We have analyzed the effect of substitutions of aromatic and basic residues on the surface of the putative binding fold. In vitro and in vivo studies of these AbiP derivatives indicate that the previously reported effects on phage development might be dependent on the nucleic acid binding activity displayed by the membrane-bound protein.

  12. Recent insights into the biological functions of liver fatty acid binding protein 1.

    Science.gov (United States)

    Wang, GuQi; Bonkovsky, Herbert L; de Lemos, Andrew; Burczynski, Frank J

    2015-12-01

    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).

  13. Binding of acylated peptides and fatty acids to phospholipid vesicles: pertinence to myristoylated proteins.

    Science.gov (United States)

    Peitzsch, R M; McLaughlin, S

    1993-10-01

    We studied the binding of fatty acids and acylated peptides to phospholipid vesicles by making electrophoretic mobility and equilibrium dialysis measurements. The binding energies of the anionic form of the fatty acids and the corresponding acylated glycines were identical; the energies increased by 0.8 kcal/mol per number of carbons in the acyl chain (Ncarbon = 10, 12, 14, 16), a value identical to that for the classical entropy-driven hydrophobic effect discussed by Tanford [The Hydrophobic Effect (1980) Wiley, New York]. The unitary Gibbs free binding energy, delta Gou, of myristoylated glycine, 8 kcal/mol, is independent of the nature of the electrically neutral lipids used to form the vesicles. Similar binding energies were obtained with other myristoylated peptides (e.g., Gly-Ala, Gly-Ala-Ala). The 8 kcal/mol, which corresponds to an effective dissociation constant of 10(-4) M for myristoylated peptides with lipids, provides barely enough energy to attach a myristoylated protein in the cytoplasm to the plasma membrane. Thus, other factors that reduce (e.g., hydrophobic interaction of myristate with the covalently attached protein) or enhance (e.g., electrostatic interactions of basic residues with acidic lipids; protein-protein interactions with intrinsic receptor proteins) the interaction of myristoylated proteins with membranes are likely to be important and may cause reversible translocation of these proteins to the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

  14. The linoleic acid derivative DCP-LA selectively activates PKC-epsilon, possibly binding to the phosphatidylserine binding site.

    Science.gov (United States)

    Kanno, Takeshi; Yamamoto, Hideyuki; Yaguchi, Takahiro; Hi, Rika; Mukasa, Takeshi; Fujikawa, Hirokazu; Nagata, Tetsu; Yamamoto, Satoshi; Tanaka, Akito; Nishizaki, Tomoyuki

    2006-06-01

    This study examined the effect of 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA), a newly synthesized linoleic acid derivative with cyclopropane rings instead of cis-double bonds, on protein kinase C (PKC) activity. In the in situ PKC assay with reverse-phase high-performance liquid chromatography, DCP-LA significantly activated PKC in PC-12 cells in a concentration-dependent (10 nM-100 microM) manner, with the maximal effect at 100 nM, and the DCP-LA effect was blocked by GF109203X, a PKC inhibitor, or a selective inhibitor peptide of the novel PKC isozyme PKC-epsilon. Furthermore, DCP-LA activated PKC in HEK-293 cells that was inhibited by the small, interfering RNA against PKC-epsilon. In the cell-free PKC assay, of the nine isozymes examined here, DCP-LA most strongly activated PKC-epsilon, with >7-fold potency over other PKC isozymes, in the absence of dioleoyl-phosphatidylserine and 1,2-dioleoyl-sn-glycerol; instead, the DCP-LA action was inhibited by dioleoyl-phosphatidylserine. DCP-LA also activated PKC-gamma, a conventional PKC, but to a much lesser extent compared with that for PKC-epsilon, by a mechanism distinct from PKC-epsilon activation. Thus, DCP-LA serves as a selective activator of PKC-epsilon, possibly by binding to the phosphatidylserine binding site on PKC-epsilon. These results may provide fresh insight into lipid signaling in PKC activation.

  15. IgE binding to peanut allergens is inhibited by combined D-aspartic and D-glutamic acids

    Science.gov (United States)

    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...

  16. Synthesis of Glutamic Acid-based Cluster Galactosides and Their Binding Affinities with Liver Cells

    Institute of Scientific and Technical Information of China (English)

    ZHANG,Xiao-Ru(张晓茹); Ll,Ying-Xia(李英霞); CHU,Shi-Dong(褚世栋); DING,Ning(丁宁); Ll,Chun-Xia(李春霞); GUAN,Hua-Shi(管华诗)

    2004-01-01

    Structurally well defined di-,tri-and tetra-valent cluster galactosides were synthesized in a convenient way.Oligo-glutamic acids were assembled as scaffolds.The presence of amine groups in these three ligands is expected to couple with drugs or genes for delivery.The binding affinities of these cluster galactoses to liver cells were determined by in vitro binding studies.Among them,the tetravalent cluster galactose (19) showed the highest affinity to liver cell.It is therefore a promising targeting device for the specific delivery of drugs or genes to parenchymal liver cells.

  17. Liver-type fatty acid binding protein interacts with hepatocyte nuclear factor 4α

    OpenAIRE

    McIntosh, Avery L.; Petrescu, Anca D.; Hostetler, Heather A.; Kier, Ann B.; Schroeder, Friedhelm

    2013-01-01

    Hepatocyte nuclear factor 4α (HNF4α) regulates liver type fatty acid binding protein (L-FABP) gene expression. Conversely as shown herein, L-FABP structurally and functionally also interacts with HNF4α. Fluorescence resonance energy transfer (FRET) between Cy3-HNF4α (donor) and Cy5-L-FABP (acceptor) as well as FRET microscopy detected L-FABP in close proximity (~80 Å) to HNF4α, binding with high affinity Kd ~250–300 nM. Circular dichroism (CD) determined that the HNF4α/L-FABP interaction alte...

  18. The binding of phosphonic acids at aluminium oxide surfaces and correlation with passivation of aluminium flake.

    Science.gov (United States)

    Cooper, Rachel J; Camp, Philip J; Henderson, David K; Lovatt, Paul A; Nation, David A; Richards, Stuart; Tasker, Peter A

    2007-04-07

    Measurements of adsorption isotherms of a series of thirteen mono- and di-phosphonic acids have shown that these bind strongly to the surface of high surface area aluminium trihydroxide. The incorporation of such phosphonates into a suspension of aluminium flake in an aqueous medium, modelling the continuous phase of a water-based paint, greatly suppresses the evolution of hydrogen. Whilst strong binding of the phosphonate to aluminium oxides is an essential criterion for good passivation, other factors such as the hydrophobicity of the ligand are also important in suppressing hydrogen-evolution.

  19. Biochemical Roles for Conserved Residues in the Bacterial Fatty Acid-binding Protein Family.

    Science.gov (United States)

    Broussard, Tyler C; Miller, Darcie J; Jackson, Pamela; Nourse, Amanda; White, Stephen W; Rock, Charles O

    2016-03-18

    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.

  20. Recent Advances in Nucleic Acid Binding Aspects of Berberine Analogs and Implications for Drug Design.

    Science.gov (United States)

    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.

  1. Spectroscopic study of binding of chlorogenic acid with the surface of ZnO nanoparticles

    Science.gov (United States)

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

    2017-09-01

    Understanding the interaction properties of biological materials with ZnO NPs is fundamental interest in the field of biotechnological applications as well as in the formation of optoelectronic devices. In this research, the binding of ZnO NPs and chlorogenic acid (CGA) were investigated using fluorescence quenching, UV-Vis absorption spectroscopy, Fourier transform infrared (FTIR), Raman spectroscopy, scanning electron microscopy (TEM), and dynamic light scattering (DLS) techniques. The study results indicated the fluorescence quenching between ZnO NPs and CGA rationalized in terms of static quenching mechanism or the formation of nonfluorescent CGA-ZnO. From fluorescence quenching spectral analysis the binding constant ( K a ), number of binding sites ( n), and thermodynamic properties, were determined. The quenching constants ( K sv) and binding constant ( K a ), decrease with increasing the temperature and their binding sites n are 2. The thermodynamic parameters determined using Van't Hoff equation indicated binding occurs spontaneously involving the hydrogen bond and van der Walls forces played the major role in the reaction of ZnO NPs with CGA. The Raman, SEM, DLS, and Zeta potential measurements were also indicated the differences in the structure, morphology and sizes of CGA, ZnO NPs, and their corresponding CGA-ZnO due to adsorption of CGA on the surface of ZnO NPs

  2. Correlation study between sperm concentration, hyaluronic acid-binding capacity and sperm aneuploidy in Hungarian patients.

    Science.gov (United States)

    Mokánszki, Attila; Molnár, Zsuzsanna; Ujfalusi, Anikó; Balogh, Erzsébet; Bazsáné, Zsuzsa Kassai; Varga, Attila; Jakab, Attila; Oláh, Éva

    2012-12-01

    Infertile men with low sperm concentration and/or less motile spermatozoa have an increased risk of producing aneuploid spermatozoa. Selecting spermatozoa by hyaluronic acid (HA) binding may reduce genetic risks such as chromosomal rearrangements and numerical aberrations. Fluorescence in-situ hybridization (FISH) has been used to evaluate the presence of aneuploidies. This study examined spermatozoa of 10 oligozoospermic, 9 asthenozoospermic, 9 oligoasthenozoospermic and 17 normozoospermic men by HA binding and FISH. Mean percentage of HA-bound spermatozoa in the normozoospermic group was 81%, which was significantly higher than in the oligozoospermic (Psex chromosomes (P=0.014) and chromosome 17 (P=0.0019), diploidy (P=0.03) and estimated numerical chromosome aberrations (P=0.004) were significantly higher in the oligoasthenozoospermic group compared with the other groups. There were statistically significant relationships (Pchromosome aberrations (r=-0.668) and between HA binding and estimated numerical chromosome aberrations (r=-0.682). HA binding and aneuploidy studies of spermatozoa in individual cases allow prediction of reproductive prognosis and provision of appropriate genetic counselling. Infertile men with normal karyotypes and low sperm concentrations and/or less motile spermatozoa have significantly increased risks of producing aneuploid (diminished mature) spermatozoa. Selecting spermatozoa by hyaluronic acid (HA) binding, based on a binding between sperm receptors for zona pellucida and HA, may reduce the potential genetic risks such as chromosomal rearrangements and numerical aberrations. In the present study we examined sperm samples of 45 men with different sperm parameters by HA-binding assay and fluorescence in-situ hybridization (FISH). Mean percentage of HA-bound spermatozoa in the normozoospermic group was significantly higher than the oligozoospermic, the asthenozoospermic and the oligoasthenozoospermic groups. Using FISH, disomy of sex

  3. Combined spectroscopy and molecular modeling studies on the binding of galbanic acid and MMP9.

    Science.gov (United States)

    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.

  4. Amino Acid Derivatives as New Zinc Binding Groups for the Design of Selective Matrix Metalloproteinase Inhibitors

    Directory of Open Access Journals (Sweden)

    Mariateresa Giustiniano

    2013-01-01

    Full Text Available 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.

  5. Electrostatic Binding and Hydrophobic Collapse of Peptide-Nucleic Acid Aggregates Quantified Using Force Spectroscopy

    CERN Document Server

    Camunas-Soler, Joan; Bizarro, Cristiano V; de Loreno, Sara; Fuentes-Perez, Maria Eugenia; Ramsch, Roland; Vilchez, Susana; Solans, Conxita; Moreno-Herrero, Fernando; Albericio, Fernando; Eritja, Ramon; Giralt, Ernest; Dev, Sukhendu B; Ritort, Felix

    2014-01-01

    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 which 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 interact...

  6. Probing the orthosteric binding site of GABAA receptors with heterocyclic GABA carboxylic acid bioisosteres

    DEFF Research Database (Denmark)

    Petersen, Jette G; Bergmann, Rikke; Krogsgaard-Larsen, Povl;

    2013-01-01

    selective and potent GABAAR agonists. This review investigates the use of heterocyclic carboxylic acid bioisosteres within the GABAAR area. Several heterocycles including 3-hydroxyisoxazole, 3-hydroxyisoxazoline, 3-hydroxyisothiazole, and the 1- and 3-hydroxypyrazole rings have been employed in order to map...... the orthosteric binding site. The physicochemical properties of the heterocyclic moieties making them suitable for bioisosteric replacement of the carboxylic acid in the molecule of GABA are discussed. A variety of synthetic strategies for synthesis of the heterocyclic scaffolds are available. Likewise, methods...... for introduction of substituents into specific positions of the heterocyclic scaffolds facilitate the investigation of different regions in the orthosteric binding pocket in close vicinity of the core scaffolds of muscimol/GABA. The development of structural models, from pharmacophore models to receptor homology...

  7. Biological characterization of liver fatty acid binding gene from miniature pig liver cDNA library.

    Science.gov (United States)

    Gao, Y H; Wang, K F; Zhang, S; Fan, Y N; Guan, W J; Ma, Y H

    2015-01-01

    Liver fatty acid binding proteins (L-FABP) are a family of small, highly conserved, cytoplasmic proteins that bind to long-chain fatty acids and other hydrophobic ligands. In this study, a full-length enriched cDNA library was successfully constructed from Wuzhishan miniature pig, and then the L-FABP gene was cloned from this cDNA library and an expression vector (pEGFP-N3-L-FABP) was constructed in vitro. This vector was transfected into hepatocytes to test its function. The results of western blotting analysis demonstrated that the L-FABP gene from our full-length enriched cDNA library regulated downstream genes, including the peroxisome proliferator-activated receptor family in hepatocytes. This study provides a theoretical basis and experimental evidence for the application of L-FABP for the treatment of liver injury.

  8. Extracts of Edible Plants Inhibit Pancreatic Lipase, Cholesterol Esterase and Cholesterol Micellization, and Bind Bile Acids

    Directory of Open Access Journals (Sweden)

    Julnaryn Intrawangso

    2012-01-01

    Full Text Available The application of edible plants with more effective ability to inhibit fat digestion and absorption has recently been explored for possible treatment of hyperlipidaemia. The aim of the present study is to investigate the effect of nine edible plants on the inhibition of pancreatic lipase and pancreatic cholesterol esterase activities, as well as the inhibition of cholesterol micelle formation, and bile acid binding. Our findings have shown strong pancreatic lipase inhibitory activity and the inhibition of cholesterol micellization by mulberry leaf extract. Safflower extract was the most potent inhibitor of pancreatic cholesterol esterase. In addition, cat’s whiskers and safflower extracts had a potent bile acid binding activity. It is suggested that a daily intake of these edible plants may delay postprandial hypertriacylglycerolaemia and hypercholesterolaemia, and therefore may be applied for the prevention and treatment of hyperlipidaemia.

  9. Response of fatty acid synthesis genes to the binding of human salivary amylase by Streptococcus gordonii.

    Science.gov (United States)

    Nikitkova, Anna E; Haase, Elaine M; Vickerman, M Margaret; Gill, Steven R; Scannapieco, Frank A

    2012-03-01

    Streptococcus gordonii, an important primary colonizer of dental plaque biofilm, specifically binds to salivary amylase via the surface-associated amylase-binding protein A (AbpA). We hypothesized that a function of amylase binding to S. gordonii may be to modulate the expression of chromosomal genes, which could influence bacterial survival and persistence in the oral cavity. Gene expression profiling by microarray analysis was performed to detect genes in S. gordonii strain CH1 that were differentially expressed in response to the binding of purified human salivary amylase versus exposure to purified heat-denatured amylase. Selected genes found to be differentially expressed were validated by quantitative reverse transcription-PCR (qRT-PCR). Five genes from the fatty acid synthesis (FAS) cluster were highly (10- to 35-fold) upregulated in S. gordonii CH1 cells treated with native amylase relative to those treated with denatured amylase. An abpA-deficient strain of S. gordonii exposed to amylase failed to show a response in FAS gene expression similar to that observed in the parental strain. Predicted phenotypic effects of amylase binding to S. gordonii strain CH1 (associated with increased expression of FAS genes, leading to changes in fatty acid synthesis) were noted; these included increased bacterial growth, survival at low pH, and resistance to triclosan. These changes were not observed in the amylase-exposed abpA-deficient strain, suggesting a role for AbpA in the amylase-induced phenotype. These results provide evidence that the binding of salivary amylase elicits a differential gene response in S. gordonii, resulting in a phenotypic adjustment that is potentially advantageous for bacterial survival in the oral environment.

  10. Natural and synthetic sialic acid-containing inhibitors of influenza virus receptor binding.

    Science.gov (United States)

    Matrosovich, Mikhail; Klenk, Hans-Dieter

    2003-01-01

    Influenza viruses attach to susceptible cells via multivalent interactions of their haemagglutinins with sialyloligosaccharide moieties of cellular glycoconjugates. Soluble macromolecules containing sialic acid from animal sera and mucosal fluids can act as decoy receptors and competitively inhibit virus-mediated haemagglutination and infection. Although a role for these natural inhibitors in the innate anti-influenza immunity is still not clear, studies are in progress on the design of synthetic sialic acid-containing inhibitors of receptor binding which could be used as anti-influenza drugs.

  11. Protecting Cell Walls from Binding Aluminum by Organic Acids Contributes to Aluminum Resistance

    Institute of Scientific and Technical Information of China (English)

    Ya-Ying Li; Yue-Jiao Zhang; Yuan Zhou; Jian-Li Yang; Shao-Jian Zheng

    2009-01-01

    Aluminum-induced secretion of organic acids from the root apex has been demonstrated to be one major AI resistance mechanism in plants. However, whether the organic acid concentration is high enough to detoxify AI in the growth medium is frequently questioned. The genotypes of Al-resistant wheat, Cassia tora L. and buckwheat secrete malate, citrate and oxalate, respectively. In the present study we found that at a 35% inhibition of root elongation, the AI activities in the solution were 10, 20, and 50 μM with the corresponding malate, citrate, and oxalate exudation at the rates of 15, 20 and 21 nmol/cm2 per 12 h, respectively, for the above three plant species. When exogenous organic acids were added to ameliorate Al toxicity, twofold and eightfold higher oxalate and malate concentrations were required to produce the equal effect by citrate. After the root apical cell walls were isolated and preincubated in 1 mM malate, oxalate or citrate solution overnight, the total amount of AI adsorbed to the cell walls all decreased significantly to a similar level, implying that these organic acids own an equal ability to protect the cell walls from binding AI. These findings suggest that protection of cell walls from binding Al by organic acids may contribute significantly to AI resistance.

  12. Binding of single stranded nucleic acids to cationic ligand functionalized gold nanoparticles.

    Science.gov (United States)

    Nash, Jessica A; Tucker, Tasha L; Therriault, William; Yingling, Yaroslava G

    2016-11-11

    The interactions of nanoparticles (NPs) with single stranded nucleic acids (NAs) have important implications in gene delivery, and nanotechnological and biomedical applications. Here, the complexation of cationic ligand functionalized gold nanoparticles with single stranded deoxyribose nucleic acid (DNA) and ribonucleic acid (RNA) are examined using all atom molecular dynamics simulations. The results indicated that complexation depends mostly on charge of nanoparticle, and, to lesser extent, sequence and type of nucleic acid. For cationic nanoparticles, electrostatic interactions between charged ligands and the nucleic acid backbone dominate binding regardless of nanoparticle charge. Highly charged nanoparticles bind more tightly and cause compaction of the single-stranded NAs through disruption of intrastrand π-π stacking and hydrogen bonding. However, poly-purine strands (polyA-DNA, polyA-RNA) show less change in structure than poly-pyrimidine strands (polyT-DNA, polyU-RNA). Overall, the results show that control over ssNA structure may be achieved with cationic NPs with a charge of more than 30, but the extent of the structural changes depends on sequence.

  13. Zinc complexes of the antibacterial drug oxolinic acid: structure and DNA-binding properties.

    Science.gov (United States)

    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.

  14. Absolute binding free energies for octa-acids and guests in SAMPL5

    Science.gov (United States)

    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.

  15. Fatty Acid Binding Proteins—The Evolutionary Crossroads of Inflammatory and Metabolic Responses1

    OpenAIRE

    Makowski, Liza; Hotamisligil, Gökhan S.

    2004-01-01

    Fatty acid binding proteins (FABPs) are members of a highly conserved family of proteins with the task of protecting a cell’s delicate lipid balance. Yet they fail when faced with metabolic or inflammatory stress, turning the cytosol into an inhospitable environment with less than ideal outcomes. This review will focus on how FABPs direct lipid traffic and simultaneously control inflammatory and metabolic pathways under the pressures of the Metabolic Syndrome.

  16. 1-Aminocyclopropane-1-carboxylic acid oxidase: insight into cofactor binding from experimental and theoretical studies.

    Science.gov (United States)

    Brisson, Lydie; El Bakkali-Taheri, Nadia; Giorgi, Michel; Fadel, Antoine; Kaizer, József; Réglier, Marius; Tron, Thierry; Ajandouz, El Hassan; Simaan, A Jalila

    2012-08-01

    1-Aminocyclopropane-1-carboxylic acid oxidase (ACCO) is a nonheme Fe(II)-containing enzyme that is related to the 2-oxoglutarate-dependent dioxygenase family. The binding of substrates/cofactors to tomato ACCO was investigated through kinetics, tryptophan fluorescence quenching, and modeling studies. α-Aminophosphonate analogs of the substrate (1-aminocyclopropane-1-carboxylic acid, ACC), 1-aminocyclopropane-1-phosphonic acid (ACP) and (1-amino-1-methyl)ethylphosphonic acid (AMEP), were found to be competitive inhibitors versus both ACC and bicarbonate (HCO(3)(-)) ions. The measured dissociation constants for Fe(II) and ACC clearly indicate that bicarbonate ions improve both Fe(II) and ACC binding, strongly suggesting a stabilization role for this cofactor. A structural model of tomato ACCO was constructed and used for docking experiments, providing a model of possible interactions of ACC, HCO(3)(-), and ascorbate at the active site. In this model, the ACC and bicarbonate binding sites are located close together in the active pocket. HCO(3)(-) is found at hydrogen-bond distance from ACC and interacts (hydrogen bonds or electrostatic interactions) with residues K158, R244, Y162, S246, and R300 of the enzyme. The position of ascorbate is also predicted away from ACC. Individually docked at the active site, the inhibitors ACP and AMEP were found coordinating the metal ion in place of ACC with the phosphonate groups interacting with K158 and R300, thus interlocking with both ACC and bicarbonate binding sites. In conclusion, HCO(3)(-) and ACC together occupy positions similar to the position of 2-oxoglutarate in related enzymes, and through a hydrogen bond HCO(3)(-) likely plays a major role in the stabilization of the substrate in the active pocket.

  17. Urinary liver-type fatty acid-binding protein change in gestational diabetes mellitus.

    Science.gov (United States)

    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.

  18. Diverse roles of the nucleic acid binding protein KHSRP in cell differentiation and disease

    Science.gov (United States)

    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

  19. Backbone and sidechain 1H, 13C and 15N resonance assignments of the human brain-type fatty acid binding protein (FABP7) in its apo form and the holo forms binding to DHA, oleic acid, linoleic acid and elaidic acid

    DEFF Research Database (Denmark)

    Oeemig, Jesper S; Jørgensen, Mathilde L; Hansen, Mikka S

    2009-01-01

    In this manuscript, we present the backbone and side chain assignments of human brain-type fatty acid binding protein, also known as FABP7, in its apo form and in four different holo forms, bound to DHA, oleic acid, linoleic acid and elaidic acid.......In this manuscript, we present the backbone and side chain assignments of human brain-type fatty acid binding protein, also known as FABP7, in its apo form and in four different holo forms, bound to DHA, oleic acid, linoleic acid and elaidic acid....

  20. Binding of [alpha, alpha]-Disubstituted Amino Acids to Arginase Suggests New Avenues for Inhibitor Design

    Energy Technology Data Exchange (ETDEWEB)

    Ilies, Monica; Di Costanzo, Luigi; Dowling, Daniel P.; Thorn, Katherine J.; Christianson, David W. (MIT); (Episcopal U); (Rutgers); (Drexel); (Penn)

    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.

  1. Iron binding efficiency of polyphenols: Comparison of effect of ascorbic acid and ethylenediaminetetraacetic acid on catechol and galloyl groups.

    Science.gov (United States)

    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.

  2. Synthesis, saccharide-binding and anti-cancer cell proliferation properties of arylboronic acid derivatives of indoquinolines.

    Science.gov (United States)

    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.

  3. Accurate determination of the binding energy of the formic acid dimer: The importance of geometry relaxation

    Science.gov (United States)

    Kalescky, Robert; Kraka, Elfi; Cremer, Dieter

    2014-02-01

    The formic acid dimer in its C2h-symmetrical cyclic form is stabilized by two equivalent H-bonds. The currently accepted interaction energy is 18.75 kcal/mol whereas the experimental binding energy D0 value is only 14.22 ±0.12 kcal/mol [F. Kollipost, R. W. Larsen, A. V. Domanskaya, M. Nörenberg, and M. A. Suhm, J. Chem. Phys. 136, 151101 (2012)]. Calculation of the binding energies De and D0 at the CCSD(T) (Coupled Cluster with Single and Double excitations and perturbative Triple excitations)/CBS (Complete Basis Set) level of theory, utilizing CCSD(T)/CBS geometries and the frequencies of the dimer and monomer, reveals that there is a 3.2 kcal/mol difference between interaction energy and binding energy De, which results from (i) not relaxing the geometry of the monomers upon dissociation of the dimer and (ii) approximating CCSD(T) correlation effects with MP2. The most accurate CCSD(T)/CBS values obtained in this work are De = 15.55 and D0 = 14.32 kcal/mol where the latter binding energy differs from the experimental value by 0.1 kcal/mol. The necessity of employing augmented VQZ and VPZ calculations and relaxing monomer geometries of H-bonded complexes upon dissociation to obtain reliable binding energies is emphasized.

  4. Valproic acid: in vitro plasma protein binding and interaction with phenytoin.

    Science.gov (United States)

    Cramer, J A; Mattson, R H

    1979-01-01

    Because valproic acid (VPA) is highly bound to plasma protein, several variables affecting binding will significantly alter the quantity of free drug which is pharmacologically active. Therefore, total VPA plasma concentrations do not reflect the therapeutic strength of the drug in tissue. We have performed equilibrium dialysis and ultrafiltration studies of VPA binding to plasma protein. The converging data in these in vitro studies indicate a clinically significant alteration in the percent of free VPA when total drug concentration exceeds 80 micrograms/ml. Saturation of drug binding sites probably occurs in this range. At 20--60 micrograms/ml VPA there is 5% free drug, with a significant increase to 8% free at 80 micrograms/ml; free drug increases to over 20% at 145 micrograms/ml total VPA. Human plasma, which is low in albumin, has twice the quantity of free VPA as normal plasma (10 versus 5% free). The clinical evidence of interaction between VPA and phenytoin is confirmed in vitro by the increase in the free fraction of both drugs. VPA binding decreases by 3--6%, while phenytoin binding decreases 5--6% as both drugs reach high plasma concentrations. When appropriate, laboratory reports should be available defining concentration of free drug in plasma for optimal interpretation of drug concetrations relative to clinical effects.

  5. Hyaluronic acid binding, endocytosis and degradation by sinusoidal liver endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    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.

  6. Identification of gamma-aminobutyric acid and its binding sites in Caenorhabditis elegans

    Energy Technology Data Exchange (ETDEWEB)

    Schaeffer, J.M.; Bergstrom, A.R.

    1988-01-01

    Gamma-aminobutyric acid (GABA), glutamate decarboxylase and GABA-transaminase were identified in the nematode Caenorhabditis elegans. The concentration of GABA in C. elegans is approximately 10-fold lower than the concentration of GABA in rat brain. Glutamate decarboxylase and GABA-transaminase, the GABA anabolic and catabolic enzymes, are also present in C. elegans. Crude membrane fractions were prepared from C. elegans and used to study specific (/sup 3/H) GABA binding sites. GABA binds to C. elegans membranes with high affinity and low capacity. Muscimol is a competitive inhibitor of specific GABA binding with a K/sub I/ value of 120 nM. None of the other GABA agonists or antagonists inhibited greater than 40% of the specific GABA binding at concentrations up to 10/sup -4/M. Thirteen spider venoms were examined as possible GABA agonists or antagonists, the venom from Calilena agelenidae inhibits specific GABA binding with a K/sub I/ value of 6 nl/ml. These results suggest that GABA has a physiological role as a neurotransmitter in C. elegans.

  7. A human microRNA precursor binding to folic acid discovered by small RNA transcriptomic SELEX.

    Science.gov (United States)

    Terasaka, Naohiro; Futai, Kazuki; Katoh, Takayuki; Suga, Hiroaki

    2016-12-01

    RNA aptamers are structured motifs that bind to specific molecules. A growing number of RNAs bearing aptamer elements, whose functions are modulated by direct binding of metabolites, have been found in living cells. Recent studies have suggested that more small RNAs binding to metabolites likely exist and may be involved in diverse cellular processes. However, conventional methods are not necessarily suitable for the discovery of such RNA aptamer elements in small RNAs with lengths ranging from 50 to 200 nucleotides, due to the far more abundant tRNAs in this size range. Here, we describe a new in vitro selection method to uncover naturally occurring small RNAs capable of binding to a ligand of interest, referred to as small RNA transcriptomic SELEX (smaRt-SELEX). By means of this method, we identified a motif in human precursor microRNA 125a (hsa-pre-miR-125a) that interacts with folic acid. Mutation studies revealed that the terminal loop region of hsa-pre-miR-125a is important for this binding interaction. This method has potential for the discovery of new RNA aptamer elements or catalytic motifs in biological small RNA fractions. © 2016 Terasaka et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  8. Interactions of chrysotile and crocidolite asbestos with red blood cell membranes. Chrysotile binds to sialic acid.

    Science.gov (United States)

    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

  9. Sterol regulatory element binding transcription factor 1 (SREBF1) polymorphism and milk fatty acid composition.

    Science.gov (United States)

    Nafikov, R A; Schoonmaker, J P; Korn, K T; Noack, K; Garrick, D J; Koehler, K J; Minick-Bormann, J; Reecy, J M; Spurlock, D E; Beitz, D C

    2013-04-01

    Milk is known to contain high concentrations of saturated fatty acids-such as palmitic (16:0), myristic (14:0), and lauric (12:0) acids-that can raise plasma cholesterol in humans, making their presence in milk undesirable. The main objective of our candidate gene study was to develop genetic markers that can be used to improve the healthfulness of bovine milk. The sterol regulatory element binding transcription factor 1 (SREBF1) known to regulate the transcription of lipogenic genes together with SREBF chaperone and insulin induced gene 1 were the candidate genes. The results showed significant association of the overall SREBF1 haplotypes with milk production and variations in lauric (12:0) and myristic (14:0) acid concentrations in milk. Haplotype H1 of SREBF1 was the most desirable to improve milk healthfulness because it was significantly associated with lower lauric (12:0) and myristic (14:0) acid concentrations compared with haplotype H3 of SREBF1, and lower lauric acid (12:0) concentration compared with haplotype H2 of SREBF1. Haplotype H1 of SREBF1, however, was significantly associated with lower milk production compared with haplotype H3 of SREBF1. We did not detect any significant associations between genetic polymorphisms in insulin induced gene 1 (INSIG1) and SREBF chaperone and milk fatty acid composition. In conclusion, genetic polymorphisms in SREBF1 can be used to develop genetic tools for the selection of animals producing milk with healthier fatty acid composition.

  10. Structure of the human-heart fatty-acid-binding protein 3 in complex with the fluorescent probe 1-anilinonaphthalene-8-sulphonic acid

    Energy Technology Data Exchange (ETDEWEB)

    Hirose, Mika; Sugiyama, Shigeru, E-mail: sugiyama@chem.eng.osaka-u.ac.jp [Lipid Active Structure Project, 1-1 Machikaneyama-cho, Toyonaka 560-0043 (Japan); Osaka University, 1-1 Machikaneyama-cho, Toyonaka 560-0043 (Japan); Ishida, Hanako; Niiyama, Mayumi [Lipid Active Structure Project, 1-1 Machikaneyama-cho, Toyonaka 560-0043 (Japan); Osaka University, 2-1 Yamadaoka, Suita 565-0871 (Japan); Matsuoka, Daisuke; Hara, Toshiaki [Lipid Active Structure Project, 1-1 Machikaneyama-cho, Toyonaka 560-0043 (Japan); Osaka University, 1-1 Machikaneyama-cho, Toyonaka 560-0043 (Japan); Mizohata, Eiichi [Osaka University, 2-1 Yamadaoka, Suita 565-0871 (Japan); Murakami, Satoshi [Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama, Kanagaw 226-8501 (Japan); Inoue, Tsuyoshi [Osaka University, 2-1 Yamadaoka, Suita 565-0871 (Japan); Matsuoka, Shigeru; Murata, Michio [Lipid Active Structure Project, 1-1 Machikaneyama-cho, Toyonaka 560-0043 (Japan); Osaka University, 1-1 Machikaneyama-cho, Toyonaka 560-0043 (Japan)

    2013-11-01

    The crystal structure of human-heart-type fatty-acid-binding protein in complex with anilinonaphthalene-8-sulfonate was solved at 2.15 Å resolution revealing the detailed binding mechanism of the fluorescent probe 1-anilinonaphthalene-8-sulfonate. Heart-type fatty-acid-binding protein (FABP3), which is a cytosolic protein abundantly found in cardiomyocytes, plays a role in trafficking fatty acids throughout cellular compartments by reversibly binding intracellular fatty acids with relatively high affinity. The fluorescent probe 1-anilinonaphthalene-8-sulfonate (ANS) is extensively utilized for examining the interaction of ligands with fatty-acid-binding proteins. The X-ray structure of FABP3 was determined in the presence of ANS and revealed the detailed ANS-binding mechanism. Furthermore, four water molecules were clearly identified in the binding cavity. Through these water molecules, the bound ANS molecule forms indirect hydrogen-bond interactions with FABP3. The adipocyte-type fatty-acid-binding protein (FABP4) exhibits 67% sequence identity with FABP3 and its crystal structure is almost the same as that of FABP3. However, FABP4 can bind with a higher affinity to ANS than FABP3. To understand the difference in their ligand specificities, a structural comparison was performed between FABP3–ANS and FABP4–ANS complexes. The result revealed that the orientation of ANS binding to FABP3 is completely opposite to that of ANS binding to FABP4, and the substitution of valine in FABP4 to leucine in FABP3 may result in greater steric hindrance between the side-chain of Leu115 and the aniline ring of ANS.

  11. Binding of diazepam, salicylic acid and digitoxin to albumin isolated from fetal and adult serum.

    Science.gov (United States)

    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.

  12. Selectivity of substrate binding and ionization of 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase.

    Science.gov (United States)

    Luanloet, Thikumporn; Sucharitakul, Jeerus; Chaiyen, Pimchai

    2015-08-01

    2-Methyl-3-hydroxypyridine-5-carboxylic acid (MHPC) oxygenase (EC 1.14.12.4) from Pseudomonas sp. MA-1 is a flavin-dependent monooxygenase that catalyzes a hydroxylation and aromatic ring cleavage reaction. The functional roles of two residues, Tyr223 and Tyr82, located ~ 5 Å away from MHPC, were characterized using site-directed mutagenesis, along with ligand binding, product analysis and transient kinetic experiments. Mutation of Tyr223 resulted in enzyme variants that were impaired in their hydroxylation activity and had Kd values for substrate binding 5-10-fold greater than the wild-type enzyme. Because this residue is adjacent to the water molecule that is located next to the 3-hydroxy group of MHPC, the results indicate that the interaction between Tyr223, H2 O and the 3-hydroxyl group of MHPC are important for substrate binding and hydroxylation. By contrast, the Kd for substrate binding of Tyr82His and Tyr82Phe variants were similar to that of the wild-type enzyme. However, only ~ 40-50% of the substrate was hydroxylated in the reactions of both variants, whereas most of the substrate was hydroxylated in the wild-type enzyme reaction. In free solution, MHPC or 5-hydroxynicotinic acid exists in a mixture of monoanionic and tripolar ionic forms, whereas only the tripolar ionic form binds to the wild-type enzyme. The binding of tripolar ionic MHPC would allow efficient hydroxylation through an electrophilic aromatic substitution mechanism. For the Tyr82His and Tyr82Phe variants, both forms of substrates can bind to the enzymes, indicating that the mutation at Tyr82 abolished the selectivity of the enzyme towards the tripolar ionic form. Transient kinetic studies indicated that the hydroxylation rate constants of both Tyr82 variants are approximately two- to 2.5-fold higher than that of the wild-type enzyme. Altogether, our findings suggest that Tyr82 is important for the binding selectivity of MHPC oxygenase towards the tripolar ionic species, whereas the

  13. Fatty acids induce leukotriene C4 synthesis in macrophages in a fatty acid binding protein-dependent manner.

    Science.gov (United States)

    Long, Eric K a; Hellberg, Kristina; Foncea, Rocio; Hertzel, Ann V; Suttles, Jill; Bernlohr, David A

    2013-07-01

    Obesity results in increased macrophage recruitment to adipose tissue that promotes a chronic low-grade inflammatory state linked to increased fatty acid efflux from adipocytes. Activated macrophages produce a variety of pro-inflammatory lipids such as leukotriene C4 (LTC4) and 5-, 12-, and 15-hydroxyeicosatetraenoic acid (HETE) suggesting the hypothesis that fatty acids may stimulate eicosanoid synthesis. To assess if eicosanoid production increases with obesity, adipose tissue of leptin deficient ob/ob mice was analyzed. In ob/ob mice, LTC4 and 12-HETE levels increased in the visceral (but not subcutaneous) adipose depot while the 5-HETE levels decreased and 15-HETE abundance was unchanged. Since macrophages produce the majority of inflammatory molecules in adipose tissue, treatment of RAW264.7 or primary peritoneal macrophages with free fatty acids led to increased secretion of LTC4 and 5-HETE, but not 12- or 15-HETE. Fatty acid binding proteins (FABPs) facilitate the intracellular trafficking of fatty acids and other hydrophobic ligands and in vitro stabilize the LTC4 precursor leukotriene A4 (LTA4) from non-enzymatic hydrolysis. Consistent with a role for FABPs in LTC4 synthesis, treatment of macrophages with HTS01037, a specific FABP inhibitor, resulted in a marked decrease in both basal and fatty acid-stimulated LTC4 secretion but no change in 5-HETE production or 5-lipoxygenase expression. These results indicate that the products of adipocyte lipolysis may stimulate the 5-lipoxygenase pathway leading to FABP-dependent production of LTC4 and contribute to the insulin resistant state.

  14. Design, Synthesis, and in Vitro Pharmacology of New Radiolabeled γ-Hydroxybutyric Acid Analogues Including Photolabile Analogues with Irreversible Binding to the High-Affinity γ-Hydroxybutyric Acid Binding Sites

    DEFF Research Database (Denmark)

    Sabbatini, Paola; Wellendorph, Petrine; Høg, Signe;

    2010-01-01

    γ-Hydroxybutyric acid (GHB) is a psychotropic compound endogenous to the brain. Despite its potential physiological significance, the complete molecular mechanisms of action remain unexplained. To facilitate the isolation and identification of the high-affinity GHB binding site, we herein report...... the design and synthesis of the first 125I-labeled radioligands in the field, one of which contains a photoaffinity label which enables it to bind irreversibly to the high-affinity GHB binding sites....

  15. Sex Differences in Long Chain Fatty Acid Utilization and Fatty Acid Binding Protein Concentration in Rat Liver

    Science.gov (United States)

    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

  16. Cytotoxicity and comparative binding mechanism of piperine with human serum albumin and α-1-acid glycoprotein.

    Science.gov (United States)

    Yeggoni, Daniel Pushparaju; Rachamallu, Aparna; Kallubai, Monika; Subramanyam, Rajagopal

    2015-01-01

    Human serum albumin (HSA) and α-1-acid glycoprotein (AGP) (acute phase protein) are the plasma proteins in blood system which transports many drugs. To understand the pharmacological importance of piperine molecule, here, we studied the anti-inflammatory activity of piperine on mouse macrophages (RAW 264.7) cell lines, which reveals that piperine caused an increase in inhibition growth of inflammated macrophages. Further, the fluorescence maximum quenching of proteins were observed upon binding of piperine to HSA and AGP through a static quenching mechanism. The binding constants obtained from fluorescence emission were found to be K(piperine) = 5.7 ± .2 × 10(5) M(-1) and K(piperine) = 9.3± .25 × 10(4) M(-1) which correspond to the free energy of -7.8 and -6.71 kcal M(-1)at 25 °C for HSA and AGP, respectively. Further, circular dichrosim studies revealed that there is a marginal change in the secondary structural content of HSA due to partial destabilization of HSA-piperine complexes. Consequently, inference drawn from the site-specific markers (phenylbutazone, site I marker) studies to identify the binding site of HSA noticed that piperine binds at site I (IIA), which was further authenticated by molecular docking and molecular dynamic (MD) studies. The binding constants and free energy corresponding to experimental and computational analysis suggest that there are hydrophobic and hydrophilic interactions when piperine binds to HSA. Additionally, the MD studies have showed that HSA-piperine complex reaches equilibration state at around 3 ns, which prove that the HSA-piperine complex is stable in nature.

  17. Staphylococcus aureus immunodominant surface antigen B is a cell-surface associated nucleic acid binding protein

    Directory of Open Access Journals (Sweden)

    Cerca Nuno

    2009-03-01

    Full Text Available Abstract Background Staphylococcus aureus immunodominant surface antigen B (IsaB elicits an immune response during septicemia and is generally classified as a virulence factor, but its biological function remains completely undefined. In an attempt to identify staphylococcal RNA-binding proteins, we designed an RNA Affinity Chromatography assay and subsequently isolated IsaB. Results Western analysis indicated that IsaB was both secreted and cell-surface associated. Gel Shift analysis confirmed the RNA binding activity but revealed that IsaB bound to any nucleic acid without sequence specificity. IsaB exhibited the highest affinity for double-stranded DNA followed by single-stranded DNA and RNA. Because extracellular DNA has been shown to play a role in biofilm formation, we investigated the biofilm-forming capacity of an isogenic isaB deletion mutant but we found that IsaB did not contribute to biofilm formation under any conditions tested. Conclusion IsaB is an extracellular nucleic acid binding protein, with little to no sequence specificity, but its role in virulence remains unclear.

  18. Binding and detoxification of chlorpyrifos by lactic acid bacteria on rice straw silage fermentation.

    Science.gov (United States)

    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.

  19. Fatty acid binding protein 7 and n-3 poly unsaturated fatty acid supply in early rat brain development.

    Science.gov (United States)

    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.

  20. Assessment of coronary reperfusion in patients with myocardial infarction using fatty acid binding protein concentrations in plasma

    NARCIS (Netherlands)

    M.J.M. de Groot; A.M.M. Muijtjens; M.L. Simoons (Maarten); W.T. Hermens (Wim); J.F.C. Glatz

    2001-01-01

    textabstractOBJECTIVE: To examine whether successful coronary reperfusion after thrombolytic treatment in patients with confirmed acute myocardial infarction can be diagnosed from the plasma marker fatty acid binding protein (FABP), for either acute clinical decision making or retrospective purposes

  1. Model of β-Sheet of Muscle Fatty Acid Binding Protein of Locusta migratoria Displays Characteristic Topology.

    Science.gov (United States)

    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.

  2. Binding of ring-substituted indole-3-acetic acids to human serum albumin.

    Science.gov (United States)

    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.

  3. Retinoic acid receptors recognize the mouse genome through binding elements with diverse spacing and topology.

    Science.gov (United States)

    Moutier, Emmanuel; Ye, Tao; Choukrallah, Mohamed-Amin; Urban, Sylvia; Osz, Judit; Chatagnon, Amandine; Delacroix, Laurence; Langer, Diana; Rochel, Natacha; Moras, Dino; Benoit, Gerard; Davidson, Irwin

    2012-07-27

    Retinoic acid receptors (RARs) heterodimerize with retinoid X receptors (RXRs) and bind to RA response elements (RAREs) in the regulatory regions of their target genes. Although previous studies on limited sets of RA-regulated genes have defined canonical RAREs as direct repeats of the consensus RGKTCA separated by 1, 2, or 5 nucleotides (DR1, DR2, DR5), we show that in mouse embryoid bodies or F9 embryonal carcinoma cells, RARs occupy a large repertoire of sites with DR0, DR8, and IR0 (inverted repeat 0) elements. Recombinant RAR-RXR binds these non-canonical spacings in vitro with comparable affinities to DR2 and DR5. Most DR8 elements comprise three half-sites with DR2 and DR0 spacings. This specific half-site organization constitutes a previously unrecognized but frequent signature of RAR binding elements. In functional assays, DR8 and IR0 elements act as independent RAREs, whereas DR0 does not. Our results reveal an unexpected diversity in the spacing and topology of binding elements for the RAR-RXR heterodimer. The differential ability of RAR-RXR bound to DR0 compared to DR2, DR5, and DR8 to mediate RA-dependent transcriptional activation indicates that half-site spacing allosterically regulates RAR function.

  4. Liver fatty acid binding protein (L-Fabp) modifies intestinal fatty acid composition and adenoma formation in ApcMin/+ mice

    OpenAIRE

    Dharmarajan, Sekhar; Newberry, Elizabeth P.; Montenegro, Grace; Nalbantoglu, ILKe; Davis, Victoria R.; Clanahan, Michael J.; Blanc, Valerie; Xie, Yan; Luo, Jianyang; Fleshman, James W.; Kennedy, Susan; Davidson, Nicholas O.

    2013-01-01

    Evidence suggests a relationship between dietary fat intake, obesity and colorectal cancer, implying a role for fatty acid (FA) metabolism in intestinal tumorigenesis that is incompletely understood. Liver fatty acid binding protein (L-Fabp), a dominant intestinal FA binding protein, regulates intestinal FA trafficking and metabolism and L-Fabp deletion attenuates diet-induced obesity. Here we examined whether changes in intestinal FA metabolism following L-Fabp deletion modify adenoma develo...

  5. Structural basis for the ligand-binding specificity of fatty acid-binding proteins (pFABP4 and pFABP5) in gentoo penguin.

    Science.gov (United States)

    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.

  6. Synthesis, Characterization, and Saccharide Binding Studies of Bile Acid − Porphyrin Conjugates

    Directory of Open Access Journals (Sweden)

    Vladimír Král

    2007-01-01

    Full Text Available Synthesis and characterization of bile acid-porphyrin conjugates (BAPs are reported. Binding of saccharides with BAPs in aqueous methanol was studied by monitoring changes in the visible absorption spectral of the porphyrin-moieties. Although these studies clearly showed absorbance changes, suggesting quite high if non-selective binding, the mass spectral studies do not unambiguously support these results.

  7. Metal loading effect on rare earth element binding to humic acid: Experimental and modelling evidence

    Science.gov (United States)

    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

  8. Tertiary structure of human alpha1-acid glycoprotein (orosomucoid). Straightforward fluorescence experiments revealing the presence of a binding pocket.

    Science.gov (United States)

    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.

  9. Preferential lectin binding of cancer cells upon sialic acid treatment under nutrient deprivation.

    Science.gov (United States)

    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. A homology model of Xyloglucan Xylosyltransferase 2 reveals critical amino acids involved in substrate binding.

    Science.gov (United States)

    Culbertson, Alan T; Tietze, Alesia A; Tietze, Daniel; Chou, Yi-Hsiang; Smith, Adrienne L; Young, Zachary T; Zabotina, Olga A

    2016-09-01

    In dicotyledonous plants, xyloglucan (XyG) is the most abundant hemicellulose of the primary cell wall. The enzymes involved in XyG biosynthesis have been identified through reverse-genetics and activity was characterized by heterologous expression. Currently, there is no information on the atomic structures or amino acids involved in activity or substrate binding of any of the Golgi-localized XyG biosynthetic enzymes. A homology model of the xyloglucan xylosyltransferase 2 (XXT2) catalytic domain was built on the basis of the crystal structure of A64Rp. Molecular dynamics simulations revealed that the homology model retains the glycosyltransferase (GT)-A fold of the template structure used to build the homology model indicating that XXT2 likely has a GT-A fold. According to the XXT2 homology model, six amino acids (Phe204, Lys207, Asp228, Ser229, Asp230, His378) were selected and their contribution in catalytic activity was investigated. Site-directed mutagenesis studies show that Asp228, Asp230 and His378 are critical for XXT2 activity and are predicted to be involved in coordination of manganese ion. Lys207 was also found to be critical for protein activity and the homology model indicates a critical role in substrate binding. Additionally, Phe204 mutants have less of an impact on XXT2 activity with the largest effect when replaced with a polar residue. This is the first study that investigates the amino acids involved in substrate binding of the XyG-synthesizing xylosyltransferases and contributes to the understanding of the mechanisms of polysaccharide-synthesizing GTs and XyG biosynthesis. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  11. NR4A orphan nuclear receptors influence retinoic acid and docosahexaenoic acid signaling via up-regulation of fatty acid binding protein 5

    Energy Technology Data Exchange (ETDEWEB)

    Volakakis, Nikolaos; Joodmardi, Eliza [Ludwig Institute for Cancer Research Ltd., Box 240, S-17177 Stockholm (Sweden); Perlmann, Thomas, E-mail: thomas.perlmann@licr.ki.se [Ludwig Institute for Cancer Research Ltd., Box 240, S-17177 Stockholm (Sweden); The Department of Cell and Molecular Biology, Karolinska Institute, S-17177 Stockholm (Sweden)

    2009-12-25

    The orphan nuclear receptor (NR) Nurr1 is expressed in the developing and adult nervous system and is also induced as an immediate early gene in a variety of cell types. In silico analysis of human promoters identified fatty acid binding protein 5 (FABP5), a protein shown to enhance retinoic acid-mediated PPAR{beta}/{delta} signaling, as a potential Nurr1 target gene. Nurr1 has previously been implicated in retinoid signaling via its heterodimerization partner RXR. Since NRs are commonly involved in cross-regulatory control we decided to further investigate the regulatory relationship between Nurr1 and FABP5. FABP5 expression was up-regulated by Nurr1 and other NR4A NRs in HEK293 cells, and Nurr1 was shown to activate and bind to the FABP5 promoter, supporting that FABP5 is a direct downstream target of NR4A NRs. We also show that the RXR ligand docosahexaenoic acid (DHA) can induce nuclear translocation of FABP5. Moreover, via up-regulation of FABP5 Nurr1 can enhance retinoic acid-induced signaling of PPAR{beta}/{delta} and DHA-induced activation of RXR. We also found that other members of the NR4A orphan NRs can up-regulate FABP5. Thus, our findings suggest that NR4A orphan NRs can influence signaling events of other NRs via control of FABP5 expression levels.

  12. Characterization and functional analysis of fatty acid binding protein from the carcinogenic liver fluke, Opisthorchis viverrini.

    Science.gov (United States)

    Sripa, Jittiyawadee; Laha, Thewarach; Sripa, Banchob

    2017-08-01

    In the present study, the cDNA encoding FABP (Ov-FABP) was isolated from the adult stage of Opisthorchis viverrini and characterized. The Ov-FABP protein sequence (107 amino acids) was predicted to have a molecular mass of 12.26kDa and an isoelectric point (PI) of 6.82. This sequence had a high identity and similarity to Cs-FABP of the related opisthorchid Clonorchis sinensis. Multiple sequence alignment with FABPs from other parasitic flatworms and mammals showed a number of conserved amino acids including Phe(34), Gly(37), Glu(38), Glu(39),Val(50), Iso(62), Gly(81), Ile(84), Ser(87) and Arg(101). In addition, the structure of Ov-FABP was predicted to have eleven β-sheets and one α-helix based on the known structures for FABPs from human (hL-FABP), rat and a schistosome. Phylogenetic analysis of amino acid sequence data revealed a close relationship of Ov-FABP with Cs-FABP and hL-FABP. Reverse transcription-PCR revealed that Ov-FABP was transcribed in the egg, metacercaria, juvenile and adult stages. The soluble form of recombinant Ov-FABP (rOv-FABP) was shown to specifically bind fatty acids, including oleic acid, palmitic acid and linoleic acid, as shown for other animals. Anti-serum against rOv-FABP (produced in mice) located the protein to parenchyma, egg, sucker musculature, testes and tegument of adult O. viverrini. Taken together, the findings suggest key functional roles for Ov-FABP in development, reproduction and/or host-parasite interactions. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

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

    DEFF Research Database (Denmark)

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

    2017-01-01

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

  14. Visualization of Iron-Binding Micelles in Acidic Recombinant Biomineralization Protein, MamC

    Energy Technology Data Exchange (ETDEWEB)

    Kashyap, Sanjay [Ames Laboratory; Woehl, Taylor [Ames Laboratory; Valverde-Tercedor, Carmen [University of Granada; Sanchez-Quesada, Miguel [University of Granada; Lopez, Concepcion Jimenez [University of Granada; Prozorov, Tanya [Ames Laboratory

    2014-03-07

    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 formation of biotemplated inorganic nanoparticles. The self-organization of MamC is compared to the behavior of another acidic recombinant iron-binding protein, Mms6.

  15. Visualization of Iron-Binding Micelles in Acidic Recombinant Biomineralization Protein, MamC

    Directory of Open Access Journals (Sweden)

    Sanjay Kashyap

    2014-01-01

    Full Text Available 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 formation of biotemplated inorganic nanoparticles. The self-organization of MamC is compared to the behavior of another acidic recombinant iron-binding protein, Mms6.

  16. Synthesis and DNA-binding properties of novel DNA cyclo-intercalators containing purine-glucuronic acid hybrids.

    Science.gov (United States)

    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.

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

    NARCIS (Netherlands)

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

    2001-01-01

    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

  18. Steam Cooking Significantly Improves in Vitro Bile Acid Binding of Beets, Eggplant, Asparagus, Carrots, Green Beans and Cauliflower

    Science.gov (United States)

    The relative healthful potential of cooked beets, okra, eggplant, asparagus, carrots, green beans, cauliflower and turnips was evaluated by determining their in vitro bile acid binding using a mixture of bile acids secreted in human bile at a duodenal physiological pH of 6.3. Six treatments and two...

  19. [Application of aspartic acid as a non-specific binding inhibitor in the enrichment of phosphopeptides with titanium dioxide].

    Science.gov (United States)

    Chi, Ming; Bi, Wei; Lu, Zhuang; Song, Lina; Jia, Wei; Zhang, Yangjun; Qian, Xiaohong; Cai, Yun

    2010-02-01

    Titanium dioxide (TiO2) is one of metal oxides widely used for phosphopeptide enrichment in phosphoproteomic research nowadays. However it can bind to some non-phosphorylated peptides containing one or more aspartic acid residues and/or glutamic acid residues. These non-phosphorylated peptides can be eluted along with phosphorylated peptides and cause the reduction of the selectivity. Conventional inhibitors for the non-specific binding of non-phosphorylated peptides can often contaminate the ion source of mass spectrometry and therefore their applications are limited in liquid chromatography-mass spectrometry (LC-MS). In this study, aspartic acid was reported as a novel non-specific binding inhibitor for phosphopeptide enrichment by titanium dioxide. Firstly, the tryptic peptide mixtures of 3 and 9 standard proteins were used for the comparison of the enrichment efficiency of titanium dioxide. The effects with the presence of aspartic acid, glutamic acid and no-inhibitor in the enrichment systems were compared separately. The results showed that aspartic acid can greatly improve the selectivity of titanium dioxide for phosphopeptide enrichment. Then, aspartic acid was used for the enrichment of tryptic peptide mixture of C57BL/6J mouse liver lysate and good results were also obtained which demonstrated that aspartic acid was a promising non-specific binding inhibitor for complex biological samples. Besides, no contamination in the ion source occurred during the mass spectrometric analysis.

  20. Sequence and structural features of binding site residues in protein-protein complexes: comparison with protein-nucleic acid complexes

    Directory of Open Access Journals (Sweden)

    Selvaraj S

    2011-10-01

    Full Text Available Abstract Background Protein-protein interactions are important for several cellular processes. Understanding the mechanism of protein-protein recognition and predicting the binding sites in protein-protein complexes are long standing goals in molecular and computational biology. Methods We have developed an energy based approach for identifying the binding site residues in protein–protein complexes. The binding site residues have been analyzed with sequence and structure based parameters such as binding propensity, neighboring residues in the vicinity of binding sites, conservation score and conformational switching. Results We observed that the binding propensities of amino acid residues are specific for protein-protein complexes. Further, typical dipeptides and tripeptides showed high preference for binding, which is unique to protein-protein complexes. Most of the binding site residues are highly conserved among homologous sequences. Our analysis showed that 7% of residues changed their conformations upon protein-protein complex formation and it is 9.2% and 6.6% in the binding and non-binding sites, respectively. Specifically, the residues Glu, Lys, Leu and Ser changed their conformation from coil to helix/strand and from helix to coil/strand. Leu, Ser, Thr and Val prefer to change their conformation from strand to coil/helix. Conclusions The results obtained in this study will be helpful for understanding and predicting the binding sites in protein-protein complexes.

  1. Heart-type Fatty Acid-binding Protein Is Essential for Efficient Brown Adipose Tissue Fatty Acid Oxidation and Cold Tolerance*

    OpenAIRE

    Vergnes, Laurent; Chin, Robert; Young, Stephen G.; Reue, Karen

    2010-01-01

    Brown adipose tissue has a central role in thermogenesis to maintain body temperature through energy dissipation in small mammals and has recently been verified to function in adult humans as well. Here, we demonstrate that the heart-type fatty acid-binding protein, FABP3, is essential for cold tolerance and efficient fatty acid oxidation in mouse brown adipose tissue, despite the abundant expression of adipose-type fatty acid-binding protein, FABP4 (also known as aP2). Fabp3−/− mice exhibit ...

  2. DNA binding mode of novel tetradentate amino acid based 2-hydroxybenzylidene-4-aminoantipyrine complexes

    Science.gov (United States)

    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.

  3. Introduction of Ca(2+)-binding amino-acid sequence into the T4 lysozyme.

    Science.gov (United States)

    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.

  4. Relationship between binding activity of sup 67 Ga and low sulfated acid glycosaminoglycans

    Energy Technology Data Exchange (ETDEWEB)

    Ohkubo, Yasuhito; Tsukada, Fumitake; Kohno, Hiroyuki (Tohoku Coll. of Pharmacy, Sendai (Japan)); Kubodera, Akiko (Science Univ. of Tokyo (Japan). School of Pharmaceutical Sciences)

    1989-01-01

    Sulfate content of acid glycosaminoglycan (AGAG) extracted from granuloma which had been produced by turpentine oil was inversely proportional to the amount of {sub 67}Ga accumulation in the granuloma. Additionally, the lowest sulfation occurred in granuloma at a peak of inflammation when the uptake of {sub 67}Ga had reached a maximum. On the basis of electrophoretic pattern, sulfate content, and specific optical rotation, it was concluded that acid glycosaminoglycans obtained from granuloma are mainly composed of chondroitin sulfate-A, -B, and desulfated heparin, while haparan sulfate was a minor component. From in vitro assays, desulfated acid glycosaminoglycans, especially desulfated-heparin and desulfated-heparan sulfate, were found to have a high affinity to {sub 67}Ga. These results suggest that low- or de-sulfation of AGAG is related to the accumulation of {sub 67}Ga in inflammatory lesions such as granuloma. Moreover, these results suggest that {sub 67}Ga does not bind to glycosaminoglycans via sulfuric acid residues. (author).

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

    Science.gov (United States)

    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.

  6. Expression Pattern of Fatty Acid Binding Proteins in Celiac Disease Enteropathy

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    Natalia M. Bottasso Arias

    2015-01-01

    Full Text Available 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.

  7. Polymorphic Nucleic Acid Binding of Bioactive Isoquinoline Alkaloids and Their Role in Cancer

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    Motilal Maiti

    2010-01-01

    Full Text Available 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.

  8. Aflatoxin M1 in raw milk and binding of aflatoxin by lactic acid bacteria

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    Aflatoxin M1 in raw milk and binding of aflatoxin by lactic acid bacteria

    2010-12-01

    Full Text Available Aflatoxin M1 (AFM1 is potential human carcinogen. Its presence in milk and dairy products represents risk for human health. Therefore, this study was carried out in order to determine thedegree of microbiological contamination by mold, and the potential presence of aflatoxin M1 in 60 raw milk samples, randomly taken from individual producers from different regions of the continental Croatia. The most common genera isolated fungi were Geotrichum (78.3 %, Aspergillus (32.4 % and Penicillium (27.0 %. From total of 60 studied milk samples, 86.66 % were positive for the presence of aflatoxin M1, and 6.66 % of samples were above the prescribed limits. Lactic acid bacteria used in fermented dairy products as a starter culture may play a role in reduction of aflatoxin in foods and nutrients. In this paper the ability of lactic acid bacteria: Lactobacillus rhamnosus GG (ATCC 53103, Lactobacillus delbrueckii S1 and Lactobacillus plantarum A1 to bind aflatoxin M1 was investigated. Standard strain L. rhamnosus GG (ATCC 53103 and L. delbrueckii S1 can significantly (P50 % compared to L. plantarum A1, which binds AFM1 between 18.7 to 28.7 %.

  9. Expression and characterization of an enantioselective antigen-binding fragment directed against α-amino acids

    Science.gov (United States)

    Eleniste, Pierre P.; Hofstetter, Heike; Hofstetter, Oliver

    2013-01-01

    This work describes the design and expression of a stereoselective Fab that possesses binding properties comparable to those displayed by the parent monoclonal antibody. Utilizing mRNA from hybridoma clones that secrete a stereoselective anti-L-amino acid antibody, a corresponding biotechnologically produced Fab was generated. For that, appropriate primers were designed based on extensive literature and databank searches. Using these primers in PCR resulted in successful amplification of the VH, VL, CL and CH1 gene fragments. Overlap PCR was utilized to combine the VH and CH1 sequences and the VL and CL sequences, respectively, to obtain the genes encoding the HC and LC fragments. These sequences were separately cloned into the pEXP5-CT/TOPO expression vector and used for transfection of BL21(DE3) cells. Separate expression of the two chains, followed by assembly in a refolding buffer, yielded an Fab that was demonstrated to bind to L-amino acids but not to recognize the corresponding D-enantiomers. PMID:23827208

  10. Inhibition of fatty acid binding proteins elevates brain anandamide levels and produces analgesia.

    Directory of Open Access Journals (Sweden)

    Martin Kaczocha

    Full Text Available The endocannabinoid anandamide (AEA is an antinociceptive lipid that is inactivated through cellular uptake and subsequent catabolism by fatty acid amide hydrolase (FAAH. Fatty acid binding proteins (FABPs are intracellular carriers that deliver AEA and related N-acylethanolamines (NAEs to FAAH for hydrolysis. The mammalian brain expresses three FABP subtypes: FABP3, FABP5, and FABP7. Recent work from our group has revealed that pharmacological inhibition of FABPs reduces inflammatory pain in mice. The goal of the current work was to explore the effects of FABP inhibition upon nociception in diverse models of pain. We developed inhibitors with differential affinities for FABPs to elucidate the subtype(s that contributes to the antinociceptive effects of FABP inhibitors. Inhibition of FABPs reduced nociception associated with inflammatory, visceral, and neuropathic pain. The antinociceptive effects of FABP inhibitors mirrored their affinities for FABP5, while binding to FABP3 and FABP7 was not a predictor of in vivo efficacy. The antinociceptive effects of FABP inhibitors were mediated by cannabinoid receptor 1 (CB1 and peroxisome proliferator-activated receptor alpha (PPARα and FABP inhibition elevated brain levels of AEA, providing the first direct evidence that FABPs regulate brain endocannabinoid tone. These results highlight FABPs as novel targets for the development of analgesic and anti-inflammatory therapeutics.

  11. Interaction of aconitine with bovine serum albumin and effect of atropine sulphate and glycyrrhizic acid on the binding

    Energy Technology Data Exchange (ETDEWEB)

    Huang Yun, E-mail: hy9317536@126.com [Pharmaceutical College, Hebei Medical University, 361 Zhongshan East Road, Shijiazhuang City, Hebei Province 050017 (China); Cui Lijian [Traditional Chinese Medical College, Hebei Medical University, Shijiazhuang 050091 (China); Wang Jianming; Huo Kun; Chen Chen; Zhan Wenhong; Wang Yongli [Pharmaceutical College, Hebei Medical University, 361 Zhongshan East Road, Shijiazhuang City, Hebei Province 050017 (China)

    2012-02-15

    The interaction of aconitine with bovine serum albumin (BSA) and effect of atropine sulphate and glycyrrhizic acid on binding constant, binding sites, and conformation were studied in an aqueous buffer solution (pH 7.40) by ultraviolet absorption and fluorescence spectroscopy. The study results show that aconitine quenched the endogenous fluorescence of BSA via a dynamic quenching procedure. Predominant intermolecular forces between aconitine and BSA were hydrophobic interactions, which stabilized the complex of aconitine-BSA. The distance between the donor and acceptor was 2.62 nm. The conformation of BSA was investigated by synchronous fluorescence techniques, indicating that the microenvironment around tryptophan (Trp) residues was changed. Furthermore, with the addition of atropine sulphate or glycyrrhizic acid, binding constant and the number of binding sites of aconitine to BSA were decreased, and the conformation had no change, which provide an important theoretical support for aconitine detoxification by atropine sulphate and glycyrrhizic acid. - Highlights: Black-Right-Pointing-Pointer Effect of atropine or glycyrrhizic acid on aconitine-BSA binding. Black-Right-Pointing-Pointer UV-vis absorption and fluorescence spectroscopic techniques used. Black-Right-Pointing-Pointer Aconitine quenched BSA fluorescence via dynamic quenching with r=2.62 nm. Black-Right-Pointing-Pointer Atropine sulphate and glycyrrhizic acid decreased K{sub A} and n of aconitine-BSA. Black-Right-Pointing-Pointer Support for aconitine detoxification by atropine and glycyrrhizic acid.

  12. Molecular cloning and analysis of functional cDNA and genomic clones encoding bovine cellular retinoic acid-binding protein.

    OpenAIRE

    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-bindin...

  13. The antiviral drug acyclovir is a slow-binding inhibitor of (D)-amino acid oxidase.

    Science.gov (United States)

    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. SNBRFinder: A Sequence-Based Hybrid Algorithm for Enhanced Prediction of Nucleic Acid-Binding Residues.

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    Xiaoxia Yang

    Full Text Available Protein-nucleic acid interactions are central to various fundamental biological processes. Automated methods capable of reliably identifying DNA- and RNA-binding residues in protein sequence are assuming ever-increasing importance. The majority of current algorithms rely on feature-based prediction, but their accuracy remains to be further improved. Here we propose a sequence-based hybrid algorithm SNBRFinder (Sequence-based Nucleic acid-Binding Residue Finder by merging a feature predictor SNBRFinderF and a template predictor SNBRFinderT. SNBRFinderF was established using the support vector machine whose inputs include sequence profile and other complementary sequence descriptors, while SNBRFinderT was implemented with the sequence alignment algorithm based on profile hidden Markov models to capture the weakly homologous template of query sequence. Experimental results show that SNBRFinderF was clearly superior to the commonly used sequence profile-based predictor and SNBRFinderT can achieve comparable performance to the structure-based template methods. Leveraging the complementary relationship between these two predictors, SNBRFinder reasonably improved the performance of both DNA- and RNA-binding residue predictions. More importantly, the sequence-based hybrid prediction reached competitive performance relative to our previous structure-based counterpart. Our extensive and stringent comparisons show that SNBRFinder has obvious advantages over the existing sequence-based prediction algorithms. The value of our algorithm is highlighted by establishing an easy-to-use web server that is freely accessible at http://ibi.hzau.edu.cn/SNBRFinder.

  15. Salicylic acid-dependent and -independent impact of an RNA-binding protein on plant immunity.

    Science.gov (United States)

    Hackmann, Christian; Korneli, Christin; Kutyniok, Magdalene; Köster, Tino; Wiedenlübbert, Matthias; Müller, Caroline; Staiger, Dorothee

    2014-03-01

    Plants overexpressing the RNA-binding protein AtGRP7 (AtGRP7-ox plants) constitutively express the PR-1 (PATHOGENESIS-RELATED-1), PR-2 and PR-5 transcripts associated with salicylic acid (SA)-mediated immunity and show enhanced resistance against Pseudomonas syringae pv. tomato (Pto) DC3000. Here, we investigated whether the function of AtGRP7 in plant immunity depends on SA. Endogenous SA was elevated fivefold in AtGRP7-ox plants. The elevated PR-1, PR-2 and PR-5 levels were eliminated upon expression of the salicylate hydroxylase nahG in AtGRP7-ox plants and elevated PR-1 levels were suppressed by sid (salicylic acid deficient) 2-1 that is impaired in SA biosynthesis. RNA immunoprecipitation showed that AtGRP7 does not bind the PR-1 transcript in vivo, whereas it binds PDF1.2. Constitutive or inducible AtGRP7 overexpression increases PR-1 promoter activity, indicating that AtGRP7 affects PR-1 transcription. In line with this, the effect of AtGRP7 on PR-1 is suppressed by npr (non-expressor of PR genes) 1. Whereas AtGRP7-ox plants restricted growth of Pto DC3000 compared with wild type (wt), sid2-1 AtGRP7-ox plants allowed more growth than AtGRP7-ox plants. Furthermore, we show an enhanced hypersensitive response triggered by avirulent Pto DC3000 (AvrRpt2) in AtGRP7-ox compared with wt. In sid2-1 AtGRP7-ox, an intermediate phenotype was observed. Thus, AtGRP7 has both SA-dependent and SA-independent effects on plant immunity.

  16. Characterization of the binding strengths between boronic acids and cis-diol-containing biomolecules by affinity capillary electrophoresis.

    Science.gov (United States)

    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. Evolutionary diversification of retinoic acid receptor ligand-binding pocket structure by molecular tinkering

    Science.gov (United States)

    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

  18. Structural and functional interaction of fatty acids with human liver fatty acid-binding protein (L-FABP) T94A variant.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2011-04-18

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

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

    Directory of Open Access Journals (Sweden)

    Chung Chun-wa

    2011-04-01

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

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

    Science.gov (United States)

    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.

  2. Allosteric Regulation in the Ligand Binding Domain of Retinoic Acid Receptorγ

    Science.gov (United States)

    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

  3. Structural Insights Into Amino Acid Binding and Gene Control by a Lysine Riboswitch

    Energy Technology Data Exchange (ETDEWEB)

    Serganov, A.; Huang, L; Patel, D

    2008-01-01

    In bacteria, the intracellular concentration of several amino acids is controlled by riboswitches1, 2, 3, 4. One of the important regulatory circuits involves lysine-specific riboswitches, which direct the biosynthesis and transport of lysine and precursors common for lysine and other amino acids. To understand the molecular basis of amino acid recognition by riboswitches, here we present the crystal structure of the 174-nucleotide sensing domain of the Thermotoga maritima lysine riboswitch in the lysine-bound (1.9 A) and free (3.1 A) states. The riboswitch features an unusual and intricate architecture, involving three-helical and two-helical bundles connected by a compact five-helical junction and stabilized by various long-range tertiary interactions. Lysine interacts with the junctional core of the riboswitch and is specifically recognized through shape-complementarity within the elongated binding pocket and through several direct and K+-mediated hydrogen bonds to its charged ends. Our structural and biochemical studies indicate preformation of the riboswitch scaffold and identify conformational changes associated with the formation of a stable lysine-bound state, which prevents alternative folding of the riboswitch and facilitates formation of downstream regulatory elements. We have also determined several structures of the riboswitch bound to different lysine analogues5, including antibiotics, in an effort to understand the ligand-binding capabilities of the lysine riboswitch and understand the nature of antibiotic resistance. Our results provide insights into a mechanism of lysine-riboswitch-dependent gene control at the molecular level, thereby contributing to continuing efforts at exploration of the pharmaceutical and biotechnological potential of riboswitches.

  4. Clusters of basic amino acids contribute to RNA binding and nucleolar localization of ribosomal protein L22.

    Directory of Open Access Journals (Sweden)

    Jennifer L Houmani

    Full Text Available The ribosomal protein L22 is a component of the 60S eukaryotic ribosomal subunit. As an RNA-binding protein, it has been shown to interact with both cellular and viral RNAs including 28S rRNA and the Epstein-Barr virus encoded RNA, EBER-1. L22 is localized to the cell nucleus where it accumulates in nucleoli. Although previous studies demonstrated that a specific amino acid sequence is required for nucleolar localization, the RNA-binding domain has not been identified. Here, we investigated the hypothesis that the nucleolar accumulation of L22 is linked to its ability to bind RNA. To address this hypothesis, mutated L22 proteins were generated to assess the contribution of specific amino acids to RNA binding and protein localization. Using RNA-protein binding assays, we demonstrate that basic amino acids 80-93 are required for high affinity binding of 28S rRNA and EBER-1 by L22. Fluorescence localization studies using GFP-tagged mutated L22 proteins further reveal that basic amino acids 80-93 are critical for nucleolar accumulation and for incorporation into ribosomes. Our data support the growing consensus that the nucleolar accumulation of ribosomal proteins may not be mediated by a defined localization signal, but rather by specific interaction with established nucleolar components such as rRNA.

  5. Sheep erythrocyte membrane binding and transfer of long-chain fatty acids

    DEFF Research Database (Denmark)

    Bojesen, Inge Norby; Bojesen, Eigil

    1999-01-01

    Palmitic acid, oleic acid, linoleic acid, arachidonic acid, sheep erythrocyte ghosts, transporting elements, transport kinetics, fatty acid transport, transport rate constants......Palmitic acid, oleic acid, linoleic acid, arachidonic acid, sheep erythrocyte ghosts, transporting elements, transport kinetics, fatty acid transport, transport rate constants...

  6. High- and low-affinity binding of S-citalopram to the human serotonin transporter mutated at 20 putatively important amino acid positions

    DEFF Research Database (Denmark)

    Plenge, Per; Wiborg, Ove

    2005-01-01

    of presumed importance. Binding of S-citalopram, both to the high-affinity-binding site and to the allosteric binding site, was measured in these mutants with the purpose of investigating the connection between the two binding sites. The amino acid substitutions did not introduce large changes in the two...

  7. Plasma Free Fatty Acids, Fatty Acid-binding Protein 4, and Mortality in Older Adults (From the Cardiovascular Health Study)

    Science.gov (United States)

    Miedema, Michael D.; Maziarz, Marlena; Biggs, Mary L.; Zieman, Susan J.; Kizer, Jorge R.; Ix, Joachim H.; Mozaffarian, Dariush; Tracy, Russell P.; Psaty, Bruce M.; Siscovick, David S.; Mukamal, Kenneth J.; Djousse, Luc

    2014-01-01

    Plasma free fatty acids (FFA) are largely derived from adipose tissue. Elevated levels of FFA and fatty acid-binding protein 4 (FABP4), a key cytoplasmic chaperone of fatty acids, have been associated with adverse cardiovascular outcomes but limited data are available on the relation of these biomarkers with cardiovascular and total mortality. We studied 4,707 participants with a mean age of 75 years who had plasma FFA and FABP4 measured in 1992–1993 as part of the Cardiovascular Health Study, an observational cohort of community dwelling older adults. Over a median follow-up of 11.8 years, 3,555 participants died. Cox proportional hazard regression was used to determine the association between FFA, FABP4, and mortality. In fully adjusted models, FFA were associated with dose-dependent significantly higher total mortality (hazard ratio (HR) per standard deviation (SD): 1.14, 95% confidence interval (CI) 1.09–1.18), but FABP4 levels were not (HR 1.04, 95% CI 0.98–1.09). In a cause-specific mortality analysis, higher concentrations of FFA were associated with significantly higher risk of death due to cardiovascular disease, dementia, infection, and respiratory causes, but not cancer or trauma. We did not find evidence of an interaction between FFA and FABP4 (p=0.45), but FABP4 appeared to be associated with total mortality differentially among men and women (HR 1.17 (1.08–1.26) for men, HR 1.02 (0.96–1.07) for women, interaction p-value <0.001). In conclusion, in a cohort of community-dwelling older individuals, elevated plasma concentrations of FFA, but not FABP4, were associated with cardiovascular and non-cardiovascular mortality. PMID:25073566

  8. Molecular cloning and analysis of functional cDNA and genomic clones encoding bovine cellular retinoic acid-binding protein.

    Science.gov (United States)

    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.

  9. Seasonal dynamics of flight muscle fatty acid binding protein and catabolic enzymes in a migratory shorebird.

    Science.gov (United States)

    Guglielmo, Christopher G; Haunerland, Norbert H; Hochachka, Peter W; Williams, Tony D

    2002-05-01

    We developed an ELISA to measure heart-type fatty acid binding protein (H-FABP) in muscles of the western sandpiper (Calidris mauri), a long-distance migrant shorebird. H-FABP accounted for almost 11% of cytosolic protein in the heart. Pectoralis H-FABP levels were highest during migration (10%) and declined to 6% in tropically wintering female sandpipers. Premigratory birds increased body fat, but not pectoralis H-FABP, indicating that endurance flight training may be required to stimulate H-FABP expression. Juveniles making their first migration had lower pectoralis H-FABP than adults, further supporting a role for flight training. Aerobic capacity, measured by citrate synthase activity, and fatty acid oxidation capacity, measured by 3-hydroxyacyl-CoA-dehydrogenase and carnitine palmitoyl transferase activities, did not change during premigration but increased during migration by 6, 12, and 13%, respectively. The greater relative induction of H-FABP (+70%) with migration than of catabolic enzymes suggests that elevated H-FABP is related to the enhancement of uptake of fatty acids from the circulation. Citrate synthase, 3-hydroxyacyl-CoA-dehydrogenase, and carnitine palmitoyl transferase were positively correlated within individuals, suggesting coexpression, but enzyme activities were unrelated to H-FABP levels.

  10. Differential gene expression of fatty acid binding proteins during porcine adipogenesis.

    Science.gov (United States)

    Samulin, Johanna; Berget, Ingunn; Lien, Sigbjørn; Sundvold, Hilde

    2008-10-01

    Four different subtypes of fatty acid binding proteins i.e. liver-type FABP1, heart/muscle-type FABP3, adipocyte-type FABP4 and epithelial/epidermal-type FABP5 are expressed in adipose tissue. However, only the regulatory role of FABP4 in adipogenesis has been thoroughly investigated. To increase the knowledge on possible roles of these FABP subtypes in preadipocyte differentiation, gene expression patterns were examined during adipogenesis in pig (Sus scrofa). FABP1 expression was induced in proliferating cells, whereas FABP3, FABP4 and FABP5 expression increased throughout preadipocyte differentiation. Interestingly, the FABP4 and FABP5 expression increased early in the differentiation, followed by FABP3 later in the differentiation process. This indicates a role of FABP4 and FABP5 in intracellular fatty acid transport during initiation of differentiation, whereas, FABP3 likely is involved in the transport of fatty acids during intermediate stages of adipogenesis. In this study we demonstrate that FABP3, FABP4 and FABP5 expression is correlated with that of the peroxisome proliferator-activated receptors alpha and gamma (PPARA and PPARG). Altogether, this suggests a role of FABP1 during cell proliferation, whereas a coordinated expression of FABP3, FABP4 and FABP5 together with that of PPARA, PPARG1 and PPARG2 might be critical for the metabolic regulation during porcine adipogenesis.

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

    Science.gov (United States)

    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).

  12. Study of the Binding Energies between Unnatural Amino Acids and Engineered Orthogonal Tyrosyl-tRNA Synthetases

    Science.gov (United States)

    Ren, Wei; Truong, Tan M.; Ai, Hui-wang

    2015-01-01

    We utilized several computational approaches to evaluate the binding energies of tyrosine (Tyr) and several unnatural Tyr analogs, to several orthogonal aaRSes derived from Methanocaldococcus jannaschii and Escherichia coli tyrosyl-tRNA synthetases. The present study reveals the following: (1) AutoDock Vina and ROSETTA were able to distinguish binding energy differences for individual pairs of favorable and unfavorable aaRS-amino acid complexes, but were unable to cluster together all experimentally verified favorable complexes from unfavorable aaRS-Tyr complexes; (2) MD-MM/PBSA provided the best prediction accuracy in terms of clustering favorable and unfavorable enzyme-substrate complexes, but also required the highest computational cost; and (3) MM/PBSA based on single energy-minimized structures has a significantly lower computational cost compared to MD-MM/PBSA, but still produced sufficiently accurate predictions to cluster aaRS-amino acid interactions. Although amino acid-aaRS binding is just the first step in a complex series of processes to acylate a tRNA with its corresponding amino acid, the difference in binding energy, as shown by MD-MM/PBSA, is important for a mutant orthogonal aaRS to distinguish between a favorable unnatural amino acid (unAA) substrate from unfavorable natural amino acid substrates. Our computational study should assist further designing and engineering of orthogonal aaRSes for the genetic encoding of novel unAAs. PMID:26220470

  13. Derivation of an amino acid similarity matrix for peptide:MHC binding and its application as a Bayesian prior

    Directory of Open Access Journals (Sweden)

    Sette Alessandro

    2009-11-01

    Full Text Available Abstract Background Experts in peptide:MHC binding studies are often able to estimate the impact of a single residue substitution based on a heuristic understanding of amino acid similarity in an experimental context. Our aim is to quantify this measure of similarity to improve peptide:MHC binding prediction methods. This should help compensate for holes and bias in the sequence space coverage of existing peptide binding datasets. Results Here, a novel amino acid similarity matrix (PMBEC is directly derived from the binding affinity data of combinatorial peptide mixtures. Like BLOSUM62, this matrix captures well-known physicochemical properties of amino acid residues. However, PMBEC differs markedly from existing matrices in cases where residue substitution involves a reversal of electrostatic charge. To demonstrate its usefulness, we have developed a new peptide:MHC class I binding prediction method, using the matrix as a Bayesian prior. We show that the new method can compensate for missing information on specific residues in the training data. We also carried out a large-scale benchmark, and its results indicate that prediction performance of the new method is comparable to that of the best neural network based approaches for peptide:MHC class I binding. Conclusion A novel amino acid similarity matrix has been derived for peptide:MHC binding interactions. One prominent feature of the matrix is that it disfavors substitution of residues with opposite charges. Given that the matrix was derived from experimentally determined peptide:MHC binding affinity measurements, this feature is likely shared by all peptide:protein interactions. In addition, we have demonstrated the usefulness of the matrix as a Bayesian prior in an improved scoring-matrix based peptide:MHC class I prediction method. A software implementation of the method is available at: http://www.mhc-pathway.net/smmpmbec.

  14. Role of a liver fatty acid-binding protein gene in lipid metabolism in chicken hepatocytes.

    Science.gov (United States)

    Gao, G L; Na, W; Wang, Y X; Zhang, H F; Li, H; Wang, Q G

    2015-01-01

    This study investigated the role of the chicken liver fatty acid-binding protein (L-FABP) gene in lipid metabolism in hepatocytes, and the regulatory relationships between L-FABP and genes related to lipid metabolism. The short hairpin RNA (shRNA) interference vector with L-FABP and an eukaryotic expression vector were used. Chicken hepatocytes were subjected to shRNA-mediated knockdown or L-FABP cDNA overexpression. Expression levels of lipid metabolism-related genes and biochemical parameters were detected 24, 36, 48, 60, and 72 h after transfection with the interference or overexpression plasmids for L-FABP, PPARα and L-BABP expression levels, and the total amount of cholesterol, were significantly affected by L-FABP expression. L-FABP may affect lipid metabolism by regulating PPARα and L-BABP in chicken hepatocytes.

  15. Mechanistic studies on the binding of Acid Yellow 99 on coir pith.

    Science.gov (United States)

    Khan, Md Motiar R; Ray, Manju; Guha, Arun K

    2011-02-01

    The interaction of Acid Yellow 99 (AY 99) with coir pith has been investigated in aqueous medium to understand the mechanism of adsorption and explore the potentiality of this biomass towards controlling pollution resulting from textile dyes. The obtained results establish that one gram of coir pith can adsorb 442.13 mg of AY 99. The adsorption process is found to be a function of pH of the solution, the optimum pH value being 2.0. The process follows Langmuir-Freundlich dual isotherm model. Scanning electron microscopic analysis demonstrates that on dye adsorption the biomass develops uneven and irregular surface. X-ray diffraction study indicates incorporation of the dye into the micropores and macropores of the adsorbent and thereby enhancing its degree of crystallinity. The results of Fourier transform infrared (FTIR) spectroscopy and chemical modification of the functional groups establish that binding of AY 99 on coir pith occurs through electrostatic and complexation reaction.

  16. Iron-binding characterization and polysaccharide production by Klebsiella oxytoca strain isolated from mine acid drainage

    Science.gov (United States)

    Baldi, F; Marchetto, D; Battistel, D; Daniele, S; Faleri, C; De Castro, C; Lanzetta, R

    2009-01-01

    Aims: To investigate Klebsiella oxytoca strain BAS-10 growth on ferric citrate under anaerobic conditions for exopolysaccharide (EPS) production and localization on cell followed by the purification and the EPS determination of the iron-binding stability constant to EPS or biotechnological applications. Methods and Results: Klebsiella oxytoca ferments ferric citrate under anaerobic conditions and produces a ferric hydrogel, whereas ferrous ions were formed in solution. During growth, cells precipitate and a hydrogel formation was observed: the organic material was constituted of an EPS bound to Fe(III) ions, this was found by chemical analyses of the iron species and transmission electron microscopy of the cell cultures. Iron binding to EPS was studied by cyclic voltammetric measurements, either directly on the hydrogel or in an aqueous solutions containing Fe(III)-citrate and purified Fe(III)-EPS. From the voltammetric data, the stability constant for the Fe(III)-EPS complex can be assumed to have values of approx. 1012–1013. It was estimated that this is higher than for the Fe(III)-citrate complex. Conclusions: The production of Fe(III)-EPS under anaerobic conditions is a strategy for the strain to survive in mine drainages and other acidic conditions. This physiological feature can be used to produce large amounts of valuable Fe(III)-EPS, starting from a low cost substrate such as Fe(III)-citrate. Significant and Impact of the Study: The data herein demonstrates that an interesting metal-binding molecule can be produced as a novel catalyst for a variety of potential applications and the EPS itself is a valuable source for rhamnose purification. PMID:19508299

  17. Bovine lactoferrin binds oleic acid to form an anti-tumor complex similar to HAMLET.

    Science.gov (United States)

    Fang, Bing; Zhang, Ming; Tian, Mai; Jiang, Lu; Guo, Hui Yuan; Ren, Fa Zheng

    2014-04-04

    α-Lactalbumin (α-LA) can bind oleic acid (OA) to form HAMLET-like complexes, which exhibited highly selective anti-tumor activity in vitro and in vivo. Considering the structural similarity to α-LA, we conjectured that lactoferrin (LF) could also bind OA to obtain a complex with anti-tumor activity. In this study, LF-OA was prepared and its activity and structural changes were compared with α-LA-OA. The anti-tumor activity was evaluated by methylene blue assay, while the apoptosis mechanism was analyzed using flow cytometry and Western blot. Structural changes of LF-OA were measured by fluorescence spectroscopy and circular dichroism. The interactions of OA with LF and α-LA were evaluated by isothermal titration calorimetry (ITC). LF-OA was obtained by heat-treatment at pH8.0 with LD50 of 4.88, 4.95 and 4.62μM for HepG2, HT29, and MCF-7 cells, respectively, all of which were 10 times higher than those of α-LA-OA. Similar to HAMLET, LF-OA induced apoptosis in tumor cells through both death receptor- and mitochondrial-mediated pathways. Exposure of tryptophan residues and the hydrophobic regions as well as the loss of tertiary structure were observed in LF-OA. Besides these similarities, LF showed different secondary structure changes when compared with α-LA, with a decrease of α-helix and β-turn and an increase of β-sheet and random coil. ITC results showed that there was a higher binding number of OA to LF than to α-LA, while both of the proteins interacted with OA through van der Waals forces and hydrogen bonds. This study provides a theoretical basis for further exploration of protein-OA complexes. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

  18. Identification and analysis of hepatitis C virus NS3 helicase inhibitors using nucleic acid binding assays.

    Science.gov (United States)

    Mukherjee, Sourav; Hanson, Alicia M; Shadrick, William R; Ndjomou, Jean; Sweeney, Noreena L; Hernandez, John J; Bartczak, Diana; Li, Kelin; Frankowski, Kevin J; Heck, Julie A; Arnold, Leggy A; Schoenen, Frank J; Frick, David N

    2012-09-01

    Typical assays used to discover and analyze small molecules that inhibit the hepatitis C virus (HCV) NS3 helicase yield few hits and are often confounded by compound interference. Oligonucleotide binding assays are examined here as an alternative. After comparing fluorescence polarization (FP), homogeneous time-resolved fluorescence (HTRF®; Cisbio) and AlphaScreen® (Perkin Elmer) assays, an FP-based assay was chosen to screen Sigma's Library of Pharmacologically Active Compounds (LOPAC) for compounds that inhibit NS3-DNA complex formation. Four LOPAC compounds inhibited the FP-based assay: aurintricarboxylic acid (ATA) (IC50=1.4 μM), suramin sodium salt (IC50=3.6 μM), NF 023 hydrate (IC50=6.2 μM) and tyrphostin AG 538 (IC50=3.6 μM). All but AG 538 inhibited helicase-catalyzed strand separation, and all but NF 023 inhibited replication of subgenomic HCV replicons. A counterscreen using Escherichia coli single-stranded DNA binding protein (SSB) revealed that none of the new HCV helicase inhibitors were specific for NS3h. However, when the SSB-based assay was used to analyze derivatives of another non-specific helicase inhibitor, the main component of the dye primuline, it revealed that some primuline derivatives (e.g. PubChem CID50930730) are up to 30-fold more specific for HCV NS3h than similarly potent HCV helicase inhibitors.

  19. Liposomal nanotransporter for targeted binding based on nucleic acid anchor system.

    Science.gov (United States)

    Nejdl, Lukas; Merlos Rodrigo, Miguel Angel; Kudr, Jiri; Ruttkay-Nedecky, Branislav; Konecna, Marie; Kopel, Pavel; Zitka, Ondrej; Hubalek, Jaromir; Kizek, Rene; Adam, Vojtech

    2014-02-01

    Microfluidic techniques have been developed intensively in recent years due to lower reagent consumption, faster analysis, and possibility of the integration of several analytical detectors into one chip. Electrochemical detectors are preferred in microfluidic systems, whereas liposomes can be used for amplification of the electrochemical signals. The aim of this study was to design a nanodevice for targeted anchoring of liposome as transport device. In this study, liposome with encapsulated Zn(II) was prepared. Further, gold nanoparticles were anchored onto the liposome surface allowing binding of thiol moiety-modified molecules (DNA). For targeted capturing of the transport device, DNA loops were used. DNA loops were represented by paramagnetic microparticles with oligo(DT)25 chain, on which a connecting DNA was bound. Capturing of transport device was subsequently done by hybridization to the loop. The individual steps were analyzed by electrochemistry and UV/Vis spectrometry. For detection of Zn(II) encapsulated in liposome, a microfluidic system was used. The study succeeded in demonstrating that liposome is suitable for the transport of Zn(II) and nucleic acids. Such transporter may be used for targeted binding using DNA anchor system.

  20. Fatty-acid binding proteins modulate sleep and enhance long-term memory consolidation in Drosophila.

    Directory of Open Access Journals (Sweden)

    Jason R Gerstner

    Full Text Available Sleep is thought to be important for memory consolidation, since sleep deprivation has been shown to interfere with memory processing. However, the effects of augmenting sleep on memory formation are not well known, and testing the role of sleep in memory enhancement has been limited to pharmacological and behavioral approaches. Here we test the effect of overexpressing the brain-type fatty acid binding protein (Fabp7 on sleep and long-term memory (LTM formation in Drosophila melanogaster. Transgenic flies carrying the murine Fabp7 or the Drosophila homologue dFabp had reduced baseline sleep but normal LTM, while Fabp induction produced increases in both net sleep and LTM. We also define a post-training consolidation "window" that is sufficient for the observed Fabp-mediated memory enhancement. Since Fabp overexpression increases consolidated daytime sleep bouts, these data support a role for longer naps in improving memory and provide a novel role for lipid-binding proteins in regulating memory consolidation concurrently with changes in behavioral state.

  1. Plasma Protein Binding of Anisomelic Acid: Spectroscopy and Molecular Dynamic Simulations.

    Science.gov (United States)

    Senthilkumar, Rajendran; Marimuthu, Parthiban; Paul, Preethy; Manojkumar, Yesaiyan; Arunachalam, Sankaralingam; Eriksson, John E; Johnson, Mark S

    2016-12-27

    Anisomelic acid (AA) is a macrocyclic cembranolide compound extracted from Anisomeles herbal species. Recently, we have shown that AA possesses both anticancer and antiviral activity. However, to date, the plasma protein binding properties of AA are unknown. Here, we describe the molecular interactions of AA with two serum proteins, human serum albumin (HSA) and bovine serum albumin (BSA), adopting multiple physicochemical methods. Besides, molecular docking and dynamics simulations were performed to predict the interaction mode and the dynamic behavior of AA with HSA and BSA. The experimental results revealed that hydrophobic forces play a significant part in the interaction of AA to HSA and BSA. The outcomes of the principal components analysis (PCA) of the poses based on root-mean-squared distances showed less variation in AA-HSA, opposed to what is seen for BSA-AA. Furthermore, binding free energies estimated for AA-HSA and AA-BSA complexes at different temperatures (298, 303, 308, and 313 K) based on molecular mechanics-generalized Born surface area (MMGBSA) approaches were well correlated with our experimental results.

  2. Binding equilibrium study of phosphotungstic acid and HSA or BSA with UV spectrum, fluorescence spectrum and equilibrium dialysis

    Institute of Scientific and Technical Information of China (English)

    HUANG; Jin(黄瑾); YUAN; Yuzhou(袁余洲); LIANG; Hong(梁宏)

    2002-01-01

    The binding equilibrium between phosphotungstic acid (H7[P(W2O7)6]@XH2O;PTA) and human serum albumin (HSA) or bovine serum albumin (BSA) has been studied by UV-Vis, fluorescence spectroscopies and equilibrium dialysis. It has been observed that UV absorption enhanced and the fluorescence quenched as the PTA binding to HSA or BSA at physiological pH 7.43( ± 0.02). The Scatchard analysis indicated that there exists a strong binding site of PTA in both HSA and BSA, and the successive stability constants of these two systems are obtained by nonlinear least-squares methods fitting Bjerrum formula.

  3. IgE binding to peanut allergens is inhibited by combined D-aspartic and D-glutamic acids.

    Science.gov (United States)

    Chung, Si-Yin; Reed, Shawndrika

    2015-01-01

    The objective of this study was to determine if D-amino acids (D-aas) bind and inhibit immunoglobulin E (IgE) binding to peanut allergens. D-aas such as D-Asp (aspartic acid), D-Glu (glutamic acid), combined D-[Asp/Glu] and others were each prepared in a cocktail of 9 other D-aas, along with L-amino acids (L-aas) and controls. Each sample was mixed with a pooled plasma from peanut-allergic donors, and tested by ELISA (enzyme-linked immunosorbent assay) and Western blots for IgE binding to peanut allergens. Results showed that D-[Asp/Glu] (4 mg/ml) inhibited IgE binding (75%) while D-Glu, D-Asp and other D-aas had no inhibitory effect. A higher inhibition was seen with D-[Asp/Glu] than with L-[Asp/Glu]. We concluded that IgE was specific for D-[Asp/Glu], not D-Asp or D-Glu, and that D-[Asp/Glu] was more reactive than was L-[Asp/Glu] in IgE inhibition. The finding indicates that D-[Asp/Glu] may have the potential for removing IgE or reducing IgE binding to peanut allergens in vitro.

  4. Inositol phosphates compete with nucleic acids for binding to bovine leukemia virus matrix protein: implications for deltaretroviral assembly.

    Science.gov (United States)

    Qualley, Dominic F; Lackey, Crystal M; Paterson, Justin P

    2013-08-01

    The matrix (MA) domain of retroviral Gag proteins plays a crucial role in virion assembly. In human immunodeficiency virus type 1 (HIV-1), a lentivirus, the presence of phosphatidylinositol-(4,5)-bisphosphate triggers a conformational change allowing the MA domain to bind the plasma membrane (PM). In this study, the MA protein from bovine leukemia virus (BLV) was used to investigate the mechanism of viral Gag binding to the membrane during replication of a deltaretrovirus. Fluorescence spectroscopy was used to measure the binding affinity of MA for two RNA constructs derived from the BLV genome as well as for single-stranded DNA (ssDNA). The importance of electrostatic interactions and the ability of inositol hexakisphosphate (IP6) to compete with nucleic acids for binding to MA were also investigated. Our data show that IP6 effectively competes with RNA and DNA for BLV MA binding, while [NaCl] of greater than 100 mM is required to produce any observable effect on DNA-MA binding. These results suggest that BLV assembly may be highly dependent on the specific interaction of the MA domain with components of the PM, as observed previously with HIV-1. The mode of MA binding to nucleic acids and the implications for BLV assembly are discussed.

  5. Renal liver-type fatty acid binding protein (L-FABP) attenuates acute kidney injury in aristolochic acid nephrotoxicity.

    Science.gov (United States)

    Matsui, Katsuomi; Kamijo-Ikemorif, Atsuko; Sugaya, Takeshi; Yasuda, Takashi; Kimura, Kenjiro

    2011-03-01

    Injection of aristolochic acid (AA) in mice causes AA-induced nephrotoxicity, in which oxidative stress contributes to development of tubulointerstitial damage (TID). Liver-type fatty acid binding protein (L-FABP) is expressed in human proximal tubules and has an endogenous antioxidative function. The renoprotection of renal L-FABP was examined in a model of AA-induced nephrotoxicity. Established human L-FABP (hL-FABP) transgenic (Tg) mice and wild-type (WT) mice were treated with AA for up to 5 days. Mice were sacrificed on days 1, 3, and 5 after the start of AA injection. Although mouse L-FABP was not expressed in proximal tubules of WT mice, hL-FABP was expressed in proximal tubules of Tg mice. The expression of renal hL-FABP was significantly increased in Tg mice administered AA (Tg-AA), compared with the control (saline-treated Tg mice). In WT-AA mice, there was high urinary excretion of N(ε)-(hexanoyl)-lysine, the production of heme oxygenase-1 and receptor for advanced glycation end products increased, and TID was provoked. In contrast, renal hL-FABP in Tg-AA mice suppressed production of N(ε)-(hexanoyl)lysine, heme oxygenase-1, and receptor for advanced glycation end products. Renal dysfunction was significantly milder in Tg-AA mice than in WT-AA mice. The degree of TID was significantly attenuated in Tg-AA mice, compared with WT-AA. In conclusion, renal hL-FABP reduced the oxidative stress in AA-induced nephrotoxicity and attenuated TID.

  6. Stimulation of Chromobacterium lipase activity and prevention of its adsorption to palmitoyl cellulose by hydrophobic binding of fatty acids.

    Science.gov (United States)

    Horiuti, Y; Imamura, S

    1978-05-01

    Fatty acids prevented adsorption of purified Chromobacterium lipase [triacylglycerol acylhydrolase, EC 3.1.1.3] onto palmitoyl cellulose (Pal-C) and also increased the activity of the purified lipase. These effects increased with increase in the concentration and chainlength (up to 16 carbon atoms) of the fatty acids, and long-chain unsaturated fatty acids, such as oleic acid, linoleic acid and erucic acid, were most effective. When the lipase was adsorbed (immobilized) on Pal-C, its activity was elevated to 20 times that of the free lipase in detergent-free reaction mixture (olive oil-buffer system). Thus lipase was adsorbed to Pal-C through a hydrophobic site distinct from its catalytic site and the binding of fatty acids to the hydrophobic site seems to result in stimulation of the lipase activity.

  7. Mth10b, a unique member of the Sac10b family, does not bind nucleic acid.

    Directory of Open Access Journals (Sweden)

    Yan-Feng Liu

    Full Text Available The Sac10b protein family is regarded as a group of nucleic acid-binding proteins that are highly conserved and widely distributed within archaea. All reported members of this family are basic proteins that exist as homodimers in solution and bind to DNA and/or RNA without apparent sequence specificity in vitro. Here, we reported a unique member of the family, Mth10b from Methanobacterium thermoautotrophicum ΔH, whose amino acid sequence shares high homology with other Sac10b family proteins. However, unlike those proteins, Mth10b is an acidic protein; its potential isoelectric point is only 4.56, which is inconsistent with the characteristics of a nucleic acid-binding protein. In this study, Mth10b was expressed in Escherichia coli and purified using a three-column chromatography purification procedure. Biochemical characterization indicated that Mth10b should be similar to typical Sac10b family proteins with respect to its secondary and tertiary structure and in its preferred oligomeric forms. However, an electrophoretic mobility shift analysis (EMSA showed that neither DNA nor RNA bound to Mth10b in vitro, indicating that either Mth10b likely has a physiological function that is distinct from those of other Sac10b family members or nucleic acid-binding ability may not be a fundamental factor to the actual function of the Sac10b family.

  8. Purification of Two Novel Sugar Acid-binding Lectins from Haplomitrium Mnioides (bryophyte, Plantae) and their Preliminary Characterization.

    Science.gov (United States)

    Masuzaki, Hiroaki; Hosono, Masahiro; Nitta, Kazuo

    2017-01-01

    Two novel sugar acid-binding lectins were purified from Haplomitrium mnioides (Lindb.) Schust. using a procedure consisting of ammonium sulfate precipitation, G-50 gel filtration, hydroxyapatite chromatography, and HW-50 gel filtration. We reported their partial physicochemical properties: molecular weight, affinity for carbohydrates and organic acids, pH stability, and dependence of their hemagglutination activity on metal ions. We also determined their N-terminal amino acid sequences. H. mnioides lectins (HMLs) were monomers (one with a molecular weight of approximately 27 kDa, and the other with a molecular weight of approximately 105 kDa) under both nonreducing and reducing conditions. They were named HML27 and HML105, respectively. Both HMLs had an affinity for N-acetylneuraminic acid, D-glucuronic acid, D-glucaric acid, bovine submaxillary mucin, heparin, and organic acids, such as citrate, 2-oxoglutaric acid, and D-2-hydroxyglutarate. Furthermore, HML27 had an affinity for α-D-galacturonic acid, D-malate, L-malate, and pyruvate, while HML105 had an affinity for D-gluconic acid. HML27 and HML105 are novel plant lectins: they have an affinity for sugar acids and organic acids and specifically recognize the carboxyl group, and there is no homology between their N-terminal amino acid sequences and those of the previously described lectins and agglutinins.

  9. StAR-related lipid transfer domain protein 5 binds primary bile acids[S

    Science.gov (United States)

    Létourneau, Danny; Lorin, Aurélien; Lefebvre, Andrée; Frappier, Vincent; Gaudreault, Francis; Najmanovich, Rafael; Lavigne, Pierre; LeHoux, Jean-Guy

    2012-01-01

    Steroidogenic acute regulatory-related lipid transfer (START) domain proteins are involved in the nonvesicular intracellular transport of lipids and sterols. The STARD1 (STARD1 and STARD3) and STARD4 subfamilies (STARD4–6) have an internal cavity large enough to accommodate sterols. To provide a deeper understanding on the structural biology of this domain, the binding of sterols to STARD5, a member of the STARD4 subfamily, was monitored. The SAR by NMR [1H-15N heteronuclear single-quantum coherence (HSQC)] approach, complemented by circular dichroism (CD) and isothermal titration calorimetry (ITC), was used. Titration of STARD5 with cholic (CA) and chenodeoxycholic acid (CDCA), ligands of the farnesoid X receptor (FXR), leads to drastic perturbation of the 1H-15N HSQC spectra and the identification of the residues in contact with those ligands. The most perturbed residues in presence of ligands are lining the internal cavity of the protein. Ka values of 1.8·10−4 M−1 and 6.3·104 M−1 were measured for CA and CDCA, respectively. This is the first report of a START domain protein in complex with a sterol ligand. Our original findings indicate that STARD5 may be involved in the transport of bile acids rather than cholesterol. PMID:23018617

  10. Selectivity and affinity determinants for ligand binding to the aromatic amino acid hydroxylases.

    Science.gov (United States)

    Teigen, Knut; McKinney, Jeffrey Alan; Haavik, Jan; Martínez, Aurora

    2007-01-01

    Hydroxylation of the aromatic amino acids phenylalanine, tyrosine and tryptophan is carried out by a family of non-heme iron and tetrahydrobiopterin (BH4) dependent enzymes, i.e. the aromatic amino acid hydroxylases (AAHs). The reactions catalyzed by these enzymes are important for biomedicine and their mutant forms in humans are associated with phenylketonuria (phenylalanine hydroxylase), Parkinson's disease and DOPA-responsive dystonia (tyrosine hydroxylase), and possibly neuropsychiatric and gastrointestinal disorders (tryptophan hydroxylase 1 and 2). We attempt to rationalize current knowledge about substrate and inhibitor specificity based on the three-dimensional structures of the enzymes and their complexes with substrates, cofactors and inhibitors. In addition, further insights on the selectivity and affinity determinants for ligand binding in the AAHs were obtained from molecular interaction field (MIF) analysis. We applied this computational structural approach to a rational analysis of structural differences at the active sites of the enzymes, a strategy that can help in the design of novel selective ligands for each AAH.

  11. Structural and functional characterization of solute binding proteins for aromatic compounds derived from lignin: p-coumaric acid and related aromatic acids.

    Science.gov (United States)

    Tan, Kemin; Chang, Changsoo; Cuff, Marianne; Osipiuk, Jerzy; Landorf, Elizabeth; Mack, Jamey C; Zerbs, Sarah; Joachimiak, Andrzej; Collart, Frank R

    2013-10-01

    Lignin comprises 15-25% of plant biomass and represents a major environmental carbon source for utilization by soil microorganisms. Access to this energy resource requires the action of fungal and bacterial enzymes to break down the lignin polymer into a complex assortment of aromatic compounds that can be transported into the cells. To improve our understanding of the utilization of lignin by microorganisms, we characterized the molecular properties of solute binding proteins of ATP-binding cassette transporter proteins that interact with these compounds. A combination of functional screens and structural studies characterized the binding specificity of the solute binding proteins for aromatic compounds derived from lignin such as p-coumarate, 3-phenylpropionic acid and compounds with more complex ring substitutions. A ligand screen based on thermal stabilization identified several binding protein clusters that exhibit preferences based on the size or number of aromatic ring substituents. Multiple X-ray crystal structures of protein-ligand complexes for these clusters identified the molecular basis of the binding specificity for the lignin-derived aromatic compounds. The screens and structural data provide new functional assignments for these solute-binding proteins which can be used to infer their transport specificity. This knowledge of the functional roles and molecular binding specificity of these proteins will support the identification of the specific enzymes and regulatory proteins of peripheral pathways that funnel these compounds to central metabolic pathways and will improve the predictive power of sequence-based functional annotation methods for this family of proteins.

  12. Development of a radioiodinated triazolopyrimidine probe for nuclear medical imaging of fatty acid binding protein 4.

    Directory of Open Access Journals (Sweden)

    Kantaro Nishigori

    Full Text Available Fatty acid binding protein 4 (FABP4 is the most well-characterized FABP isoform. FABP4 regulates inflammatory pathways in adipocytes and macrophages and is involved in both inflammatory diseases and tumor formation. FABP4 expression was recently reported for glioblastoma, where it may participate in disease malignancy. While FABP4 is a potential molecular imaging target, with the exception of a tritium labeled probe there are no reports of other nuclear imaging probes that target this protein. Here we designed and synthesized a nuclear imaging probe, [123I]TAP1, and evaluated its potential as a FABP4 targeting probe in in vitro and in vivo assays. We focused on the unique structure of a triazolopyrimidine scaffold that lacks a carboxylic acid to design the TAP1 probe that can undergo facilitated delivery across cell membranes. The affinity of synthesized TAP1 was measured using FABP4 and 8-anilino-1-naphthalene sulfonic acid. [125I]TAP1 was synthesized by iododestannylation of a precursor, followed by affinity and selectivity measurements using immobilized FABPs. Biodistributions in normal and C6 glioblastoma-bearing mice were evaluated, and excised tumors were subjected to autoradiography and immunohistochemistry. TAP1 and [125I]TAP1 showed high affinity for FABP4 (Ki = 44.5±9.8 nM, Kd = 69.1±12.3 nM. The FABP4 binding affinity of [125I]TAP1 was 11.5- and 35.5-fold higher than for FABP3 and FABP5, respectively. In an in vivo study [125I]TAP1 displayed high stability against deiodination and degradation, and moderate radioactivity accumulation in C6 tumors (1.37±0.24% dose/g 3 hr after injection. The radioactivity distribution profile in tumors partially corresponded to the FABP4 positive area and was also affected by perfusion. The results indicate that [125I]TAP1 could detect FABP4 in vitro and partly in vivo. As such, [125I]TAP1 is a promising lead compound for further refinement for use in in vivo FABP4 imaging.

  13. Residues in the Nucleosome Acidic Patch Regulate Histone Occupancy and Are Important for FACT Binding in Saccharomyces cerevisiae.

    Science.gov (United States)

    Hodges, Amelia J; Gloss, Lisa M; Wyrick, John J

    2017-07-01

    The essential histone chaperone FACT plays a critical role in DNA replication, repair, and transcription, primarily by binding to histone H2A-H2B dimers and regulating their assembly into nucleosomes. While FACT histone chaperone activity has been extensively studied, the exact nature of the H2A and H2B residues important for FACT binding remains controversial. In this study, we characterized the functions of residues in the histone H2A and H2B acidic patch, which is important for binding many chromatin-associated factors. We found that mutations in essential acidic patch residues cause a defect in histone occupancy in yeast, even though most of these histone mutants are expressed normally in yeast and form stable nucleosomes in vitro Instead, we show that two acidic patch residues, H2B L109 and H2A E57, are important for histone binding to FACT in vivo We systematically screened mutants in other H2A and H2B residues previously suspected to be important for FACT binding and confirmed the importance of H2B M62 using an in-vivo FACT-binding assay. Furthermore, we show that, like deletion mutants in FACT subunits, an H2A E57 and H2B M62 double mutant is lethal in yeast. In summary, we show that residues in the nucleosome acidic patch promote histone occupancy and are important for FACT binding to H2A-H2B dimers in yeast. Copyright © 2017 by the Genetics Society of America.

  14. Position-specific incorporation of fluorescent non-natural amino acids into maltose-binding protein for detection of ligand binding by FRET and fluorescence quenching.

    Science.gov (United States)

    Iijima, Issei; Hohsaka, Takahiro

    2009-04-17

    Position-specific incorporation of fluorescent groups is a useful method for analysis of the functions and structures of proteins. We have developed a method for the incorporation of visible-wavelength-fluorescent non-natural amino acids into proteins in a cell-free translation system. Using this technique, we introduced one or two BODIPY-linked amino acids into maltose-binding protein (MBP) to obtain MBP derivatives showing ligand-dependent changes in fluorescence intensity or intensity ratio. BODIPY-FL-aminophenylalanine was incorporated in place of 15 tyrosines, as well as the N-terminal Lys1, and the C-terminal Lys370 of MBP. Fluorescence measurements revealed that MBP containing a BODIPY-FL moiety in place of Tyr210 showed a 13-fold increase in fluorescence upon binding of maltose. Tryptophan-to-phenylalanine substitutions suggest that the increase in fluorescence was the result of a decrease in the quenching of BODIPY-FL by tryptophan located around the binding site. MBP containing a BODIPY-558 moiety also showed a maltose-dependent increase in fluorescence. BODIPY-FL was then additionally incorporated in place of Lys1 of the BODIPY-558-containing MBP as a response to the amber codon. Fluorescence measurements with excitation of BODIPY-FL showed a large change in fluorescence intensity ratio (0.13 to 1.25) upon binding of maltose; this change can be attributed to fluorescence resonance energy transfer (FRET) and maltose-dependent quenching of BODIPY-558. These results demonstrate the usefulness of the position-specific incorporation of fluorescent amino acids in the fluorescence-based detection of protein functions.

  15. Three amino acid residues bind corn odorants to McinOBP1 in the polyembryonic endoparasitoid of Macrocentrus cingulum Brischke.

    Directory of Open Access Journals (Sweden)

    Tofael Ahmed

    Full Text Available Odorant binding proteins (OBPs play a central role in transporting odorant molecules from the sensillum lymph to olfactory receptors to initiate behavioral responses. In this study, the OBP of Macrocentrus cingulum McinOBP1 was expressed in Escherichia coli and purified by Ni ion affinity chromatography. Real-time PCR experiments indicate that the McinOBP1 is expressed mainly in adult antennae, with expression levels differing by sex. Ligand-binding experiments using N-phenyl-naphthylamine (1-NPN as a fluorescent probe demonstrated that the McinOBP1 can bind green-leaf volatiles, including aldehydes and terpenoids, but also can bind aliphatic alcohols with good affinity, in the order trans-2-nonenal>cis-3-hexen-1-ol>trans-caryophelle, suggesting a role of McinOBP1 in general odorant chemoreception. We chose those three odorants for further homology modeling and ligand docking based on their binding affinity. The Val58, Leu62 and Glu130 are the key amino acids in the binding pockets that bind with these three odorants. The three mutants, Val58, Leu62 and Glu130, where the valine, leucine and glutamic residues were replaced by alanine, proline and alanine, respectively; showed reduced affinity to these odorants. This information suggests, Val58, Leu62 and Glu130 are involved in the binding of these compounds, possibly through the specific recognition of ligands that forms hydrogen bonds with the ligands functional groups.

  16. Determination of human serum alpha1-acid glycoprotein and albumin binding of various marketed and preclinical kinase inhibitors.

    Science.gov (United States)

    Zsila, Ferenc; Fitos, Ilona; Bencze, Gyula; Kéri, György; Orfi, László

    2009-01-01

    There are about 380 protein kinase inhibitors in drug development as of today and 15 drugs have been marketed already for the treatment of cancer. This time 139 validated kinase targets are in the focus of drug research of pharmaceutical companies and big efforts are made for the development of new, druglike kinase inhibitors. Plasma protein binding is an important factor of the ADME profiling of a drug compound. Human serum albumin (HSA) and alpha(1)-acid glycoprotein (AAG) are the most relevant drug carriers in blood plasma. Since previous literature data indicated that AAG is the principal plasma binding component of some kinase inhibitors the present work focuses on the comprehensive evaluation of AAG binding of a series of marketed and experimental kinase inhibitors by using circular dichroism (CD) spectroscopy approach. HSA binding was also evaluated by affinity chromatography. Protein binding interactions of twenty-six kinase inhibitors are characterized. The contribution of AAG and HSA binding data to the pharmacokinetic profiles of the investigated therapeutic agents is discussed. Structural, biological and drug binding properties of AAG as well as the applicability of the CD method in studying drug-protein binding interactions are also briefly reviewed.

  17. Synthesis and evaluation of boronic acids as inhibitors of Penicillin Binding Proteins of classes A, B and C.

    OpenAIRE

    Zervosen, Astrid; Bouillez, André; Herman, Alexandre; Amoroso, Ana Maria; Joris, Bernard; Sauvage, Eric; Charlier, Paulette; Luxen, André

    2012-01-01

    In response to the widespread use of beta-lactam antibiotics bacteria have evolved drug resistance mechanisms that include the production of resistant Penicillin Binding Proteins (PBPs). Boronic acids are potent beta-lactamase inhibitors and have been shown to display some specificity for soluble transpeptidases and PBPs, but their potential as inhibitors of the latter enzymes is yet to be widely explored. Recently, a (2,6-dimethoxybenzamido)methylboronic acid was identified as being a potent...

  18. Selective determination of 3,4-dihydroxyphenylacetic acid in the presence of ascorbic and uric acids using polymer film modified electrode

    Indian Academy of Sciences (India)

    Palraj Kalimuthu; S Abraham John

    2011-05-01

    We report here the highly sensitive and selective electrochemical determination of 3,4-dihydroxyphenylacetic acid (DOPAC), one of the dopamine metabolites in the presence of important interferents ascorbic acid (AA) and uric acid (UA) using an ultrathin electropolymerized film of 5-amino-1,3,4-thiadiazole-2-thiol (p-ATT) modified glassy carbon (GC) electrode in 0.20M phosphate buffer solution (pH 5.0). The bare GC electrode fails to resolve the oxidation peaks of AA, DOPAC and UA in a mixture. Further, the oxidation peak potentials of them were shifted to more positive potential with decreased peak currents in the subsequent cycles. On the other hand, the p-ATT modified electrode not only separated the voltammetric signals of AA, DOPAC and UA but also enhanced their peak currents. The amperometric current response was increased linearly with increasing DOPAC concentration in the range of 4.0 × 10−8 to 1.0 × 10−5 M and the detection limit was found to be 150pM ( =3).

  19. Study on the binding interaction of chromium(VI) with humic acid using UV-vis, fluorescence spectroscopy and molecular modeling

    Science.gov (United States)

    Gu, Yun-Lan; Yin, Ming-Xing; Zhang, Hong-Mei; Wang, Yan-Qing; Shi, Jing-hua

    2015-02-01

    In this report, the binding interaction of chromium(VI), as Cr2O72-, with humic acid was studied by using UV-visible absorption, fluorescence spectroscopy, and molecular modeling method. The fluorescence spectral data indicated that the binding interaction existed between Cr2O72- and humic acid and the order of magnitude of binding constants were 103. The rise in temperature caused a decrease in the values of the binding constant of humic acid with Cr2O72-. Thermodynamic analysis presented that multi-intermolecular forces including hydrogen bonding, hydrophobic, and electrostatic forces were involved in the binding process at pH 6.5. The spectral data also indicated that Cr2O72- affected the aromatic ring structures in humic acid. Furthermore, the molecular modeling analysis indicated that a lot of reactive groups and binding cavities in HA played a key role in its binding with Cr2O72-.

  20. Novel radioiodinated {gamma}-hydroxybutyric acid analogues for radiolabeling and Photolinking of high-affinity {gamma}-hydroxybutyric acid binding sites

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Høg, Signe; Sabbatini, Paola;

    2010-01-01

    ¿-Hydroxybutyric acid (GHB) is a therapeutic drug, a drug of abuse, and an endogenous substance that binds to low- and high-affinity sites in the mammalian brain. To target the specific GHB binding sites, we have developed a (125)I-labeled GHB analog and characterized its binding in rat brain...... homogenate and slices. Our data show that [(125)I]4-hydroxy-4-[4-(2-iodobenzyloxy)phenyl]butanoate ([(125)I]BnOPh-GHB) binds to one site in rat brain cortical membranes with low nanomolar affinity (K(d), 7 nM; B(max), 61 pmol/mg protein). The binding is inhibited by GHB and selected analogs......, but not by ¿-aminobutyric acid. Autoradiography using horizontal slices from rat brain demonstrates the highest density of binding in hippocampus and cortical regions and the lowest density in the cerebellum. Altogether, the findings correlate with the labeling and brain regional distribution of high-affinity GHB sites...

  1. Novel Radioiodinated γ-Hydroxybutyric Acid Analogues for Radiolabeling and Photolinking of High-Affinity γ-Hydroxybutyric Acid Binding Sites

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Høg, Signe; Sabbatini, Paola;

    2010-01-01

    γ-Hydroxybutyric acid (GHB) is a therapeutic drug, a drug of abuse, and an endogenous substance that binds to low- and high-affinity sites in the mammalian brain. To target the specific GHB binding sites, we have developed a 125I-labeled GHB analog and characterized its binding in rat brain...... homogenate and slices. Our data show that [125I]4-hydroxy-4-[4-(2-iodobenzyloxy)phenyl]butanoate ([125I]BnOPh-GHB) binds to one site in rat brain cortical membranes with low nanomolar affinity (Kd, 7 nM; Bmax, 61 pmol/mg protein). The binding is inhibited by GHB and selected analogs, but not by γ......-aminobutyric acid. Autoradiography using horizontal slices from rat brain demonstrates the highest density of binding in hippocampus and cortical regions and the lowest density in the cerebellum. Altogether, the findings correlate with the labeling and brain regional distribution of high-affinity GHB sites or [3H...

  2. Investigation of the binding of Salvianolic acid B to human serum albumin and the effect of metal ions on the binding

    Science.gov (United States)

    Chen, Tingting; Cao, Hui; Zhu, Shajun; Lu, Yapeng; Shang, Yanfang; Wang, Miao; Tang, Yanfeng; Zhu, Li

    2011-10-01

    The studies on the interaction between HSA and drugs have been an interesting research field in life science, chemistry and clinical medicine. There are also many metal ions present in blood plasma, thus the research about the effect of metal ions on the interaction between drugs and plasma proteins is crucial. In this study, the interaction of Salvianolic acid B (Sal B) with human serum albumin (HSA) was investigated by the steady-state, synchronous fluorescence and circular dichroism (CD) spectroscopies. The results showed that Sal B had a strong ability to quench the intrinsic fluorescence of HSA through a static quenching mechanism. Binding parameters calculated showed that Sal B was bound to HSA with the binding affinities of 10 5 L mol -1. The thermodynamic parameters studies revealed that the binding was characterized by positive enthalpy and positive entropy changes, and hydrophobic interactions were the predominant intermolecular forces to stabilize the complex. The specific binding distance r (2.93 nm) between donor (HSA) and acceptor (Sal B) was obtained according to Förster non-radiative resonance energy transfer theory. The synchronous fluorescence experiment revealed that Sal B cannot lead to the microenvironmental changes around the Tyr and Trp residues of HSA, and the binding site of Sal B on HSA is located in hydrophobic cavity of subdomain IIA. The CD spectroscopy indicated the secondary structure of HSA is not changed in the presence of Sal B. Furthermore, The effect of metal ions (e.g. Zn 2+, Cu 2+, Co 2+, Ni 2+, Fe 3+) on the binding constant of Sal B-HSA complex was also discussed.

  3. Molecular docking study investigating the possible mode of binding of C.I. Acid Red 73 with DNA.

    Science.gov (United States)

    Guo, Yumei; Yue, Qinyan; Gao, Baoyu

    2011-07-01

    C.I. Acid Red 73 is a reactive azo dye with a variable potential carcinogenicity. The mechanism mediating interactions that occur between the dye and DNA have not been completely understood thus far. In this study, molecular docking techniques were applied to describe the most probable mode of DNA binding as well as the sequence selectivity of the C.I. Acid Red 73 dye. These docking experiments revealed that the dye is capable of interacting with the minor groove of the DNA on the basis of its curved shape, which fits well with the topology of double-stranded DNA. In addition, the dye can bind selectively to the minor groove of the DNA by applying CGT sequence selectivity. Further, the minor groove can be recognized although DNA targets present intercalation gaps. However, intercalative binding can also occur when the DNA target possesses an appropriate intercalation gap. Compared with the other eight DNA sequences that were studied, the DNA dodecamer d(CGCGATATCGCG)(2) (PDB ID: 1DNE) presents a very favorable target for the binding of C.I. Acid Red 73 to the minor groove, with the lowest binding free energy -9.19 kcal/mol. Results reported from this study are expected to provide useful information for research involving further simulations of molecular dynamics and toxicology investigations of the dye.

  4. H7N9 influenza viruses interact preferentially with α2,3-linked sialic acids and bind weakly to α2,6-linked sialic acids.

    Science.gov (United States)

    Ramos, Irene; Krammer, Florian; Hai, Rong; Aguilera, Domingo; Bernal-Rubio, Dabeiba; Steel, John; García-Sastre, Adolfo; Fernandez-Sesma, Ana

    2013-11-01

    The recent human outbreak of H7N9 avian influenza A virus has caused worldwide concerns. Receptor binding specificity is critical for viral pathogenicity, and still not thoroughly studied for this emerging virus. Here, we evaluated the receptor specificity of the haemagglutinin (HA) of two human H7N9 isolates (A/Shanghai/1/13 and A/Anhui/1/13) through a solid-phase binding assay and a flow cytometry-based assay. In addition, we compared it with those from several HAs from human and avian influenza viruses. We observed that the HAs from the novel H7 isolates strongly interacted with α2,3-linked sialic acids. Importantly, they also showed low levels of binding to α2,6-linked sialic acids, but significantly higher than other avian H7s.

  5. Human adenovirus 52 uses sialic acid-containing glycoproteins and the coxsackie and adenovirus receptor for binding to target cells.

    Directory of Open Access Journals (Sweden)

    Annasara Lenman

    2015-02-01

    Full Text Available Most adenoviruses attach to host cells by means of the protruding fiber protein that binds to host cells via the coxsackievirus and adenovirus receptor (CAR protein. Human adenovirus type 52 (HAdV-52 is one of only three gastroenteritis-causing HAdVs that are equipped with two different fiber proteins, one long and one short. Here we show, by means of virion-cell binding and infection experiments, that HAdV-52 can also attach to host cells via CAR, but most of the binding depends on sialylated glycoproteins. Glycan microarray, flow cytometry, surface plasmon resonance and ELISA analyses reveal that the terminal knob domain of the long fiber (52LFK binds to CAR, and the knob domain of the short fiber (52SFK binds to sialylated glycoproteins. X-ray crystallographic analysis of 52SFK in complex with 2-O-methylated sialic acid combined with functional studies of knob mutants revealed a new sialic acid binding site compared to other, known adenovirus:glycan interactions. Our findings shed light on adenovirus biology and may help to improve targeting of adenovirus-based vectors for gene therapy.

  6. Proposed binding mechanism of galbanic acid extracted from Ferula assa-foetida to DNA.

    Science.gov (United States)

    Ahmadi, F; Shokoohinia, Y; Javaheri, Sh; Azizian, H

    2017-01-01

    Recently, galbanic acid (GA), a sesquiterpenoid coumarin, has been introduced as an apoptotic and geno/cytotoxicity agent. In the present study, GA has been extracted from Ferula assa-foetida, a native medicinal plant in Iran, and characterized by (1)H NMR, mass spectroscopy. Additionally, spectroscopic studies have been performed in order to investigate its DNA-interaction mode. The electrochemical behavior of GA has been studied by cyclic voltammetry (CV) in various scan rates. In neutral media (pH=7.3) one irreversible cathodic peak was obtained at -1.46 V, while in higher scan rates an irreversible one was determined at -1.67 V. According to the voltametric data GA can be easily reduced by 2e(-)/2H(+) mechanism at hanging mercury drop electrode (HMDE). The interaction of GA with ct-DNA was evaluated by CV, differential pulse voltammetry (DPV), enhancement fluorescence, UV-Vis, FT-IR spectroscopy and molecular docking. The molecular docking study shows that the GA interacts to DNA on partial intercalation mode via DNA groove binding and forms a complex by van der Waals and electroastatic interactions. In addition, the thermodynamic parameters of GA-DNA complex were investigated with ΔH°, ΔS° and ΔG° values of 15.81KJmol(-1), 133.95Jmol(-1) and -23.10KJmol(-1), respectively. All data revealed that the GA is binding to DNA by van der Waals and electrostatic interactions through the partial intercalations from the DNA's grooves.

  7. Intestinal fatty acid-binding protein as a diagnostic marker for complicated and uncomplicated necrotizing enterocolitis: a prospective cohort study

    NARCIS (Netherlands)

    Schurink, M.; Kooi, E.M.; Hulzebos, C.V.; Kox, R.G.; Groen, H.; Heineman, E.; Bos, A.F; Hulscher, J.B.

    2015-01-01

    BACKGROUND: Early NEC symptoms are non-specific and diagnostic tests lack discriminative power. Intestinal fatty acid-binding protein (I-FABP), mainly located in small bowel enterocytes, is released into the blood following NEC-associated enterocyte disruption. Aim of this prospective cohort trial w

  8. Fatty Acid binding protein 4 is associated with carotid atherosclerosis and outcome in patients with acute ischemic stroke

    DEFF Research Database (Denmark)

    Holm, Sverre; Ueland, Thor; Dahl, Tuva B

    2011-01-01

    Fatty acid binding protein 4 (FABP4) has been shown to play an important role in macrophage cholesterol trafficking and associated inflammation. To further elucidate the role of FABP4 in atherogenesis in humans, we examined the regulation of FABP4 in carotid atherosclerosis and ischemic stroke....

  9. TdaA Regulates Tropodithietic Acid Synthesis by Binding to the tdaC Promoter Region ▿ †

    OpenAIRE

    Geng, Haifeng; Belas, Robert

    2011-01-01

    Silicibacter sp. TM1040, a member of the marine Roseobacter clade, produces the antibiotic and quorum signaling molecule tropodithietic acid (TDA), encoded by tdaABCDEF. Here, we showed that an LysR-type transcriptional regulator, TdaA, is a positive regulator of tdaCDE gene expression and binds to the tdaC promoter region.

  10. Urinary liver-type fatty acid-binding protein predicts progression to nephropathy in type 1 diabetic patients

    DEFF Research Database (Denmark)

    Nielsen, Stine Elkjaer; Sugaya, Takeshi; Hovind, Peter;

    2010-01-01

    Urinary liver-type fatty acid-binding protein (u-LFABP) is a marker of tubulointerstitial inflammation and has been shown to be increased in patients with type 1 diabetes and is further increased in patients who progress to micro- and macroalbuminuria. Our aim was to evaluate u-LFABP as a predict...

  11. The effects of methylene blue infusion on gastric tonometry and intestinal fatty acid binding protein levels in septic shock patients.

    NARCIS (Netherlands)

    Haren, F.M.P. van; Pickkers, P.; Foudraine, N.; Heemskerk, S.; Sleigh, J.; Hoeven, J.G. van der

    2010-01-01

    OBJECTIVE: We prospectively studied the effect of methylene blue (MB) infusion on gastric mucosal metabolism perfusion ratio, assessed by gastric tonometry, and on mucosal cell damage, assessed by urinary levels of intestinal fatty acid binding protein, in septic shock patients. METHODS: Methylene

  12. Noninvasive measurement of fecal calprotectin and serum amyloid A combined with intestinal fatty acid-binding protein in necrotizing enterocolitis.

    NARCIS (Netherlands)

    Reisinger, K.W.; Zee, D.C. van der; Brouwers, H.A.A.; Kramer, B.W.; Heurn, L.W.E. van; Buurman, W.A.; Derikx, J.P.

    2012-01-01

    BACKGROUND: Diagnosis of necrotizing enterocolitis (NEC), prevalent in premature infants, remains challenging. Enterocyte damage in NEC can be assessed by intestinal fatty acid-binding protein (I-FABP), with a sensitivity of 93% and a specificity of 90%. Numerous markers of inflammation are known, s

  13. Noninvasive measurement of fecal calprotectin and serum amyloid A combined with intestinal fatty acid-binding protein in necrotizing enterocolitis

    NARCIS (Netherlands)

    Reisinger, Kostan W.; Van der Zee, David C.; Brouwers, Hens A. A.; Kramer, Boris W.; van Heurn, L. W. Ernest; Buurman, Wim A.; Derikx, Joep P. M.

    2012-01-01

    Background: Diagnosis of necrotizing enterocolitis (NEC), prevalent in premature infants, remains challenging. Enterocyte damage in NEC can be assessed by intestinal fatty acid-binding protein (I-FABP), with a sensitivity of 93% and a specificity of 90%. Numerous markers of inflammation are known, s

  14. Enantioselective binding of amino acids and amino alcohols by self-assembled chiral basket-shaped receptors

    NARCIS (Netherlands)

    Escuder, B.; Rowan, A.E.; Feiters, M.C.; Nolte, R.J.M.

    2004-01-01

    Amino acid appended diphenylglycoluril-based chiral molecular receptors 2 and 3 have been prepared and their aggregation has been studied in water at various pH's and in chloroform. The binding of several biologically relevant guests with aromatic moieties to these aggregates has been studied with U

  15. Enterocyte fatty acid-binding proteins (FABPs): different functions of liver and intestinal FABPs in the intestine.

    Science.gov (United States)

    Gajda, Angela M; Storch, Judith

    2015-02-01

    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 FABPs (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 different 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.

  16. Direct protein-protein interactions and substrate channeling between cellular retinoic acid binding proteins and CYP26B1.

    Science.gov (United States)

    Nelson, Cara H; Peng, Chi-Chi; Lutz, Justin D; Yeung, Catherine K; Zelter, Alex; Isoherranen, Nina

    2016-08-01

    Cellular retinoic acid binding proteins (CRABPs) bind all-trans-retinoic acid (atRA) tightly. This study aimed to determine whether atRA is channeled directly to cytochrome P450 (CYP) CYP26B1 by CRABPs, and whether CRABPs interact directly with CYP26B1. atRA bound to CRABPs (holo-CRABP) was efficiently metabolized by CYP26B1. Isotope dilution experiments showed that delivery of atRA to CYP26B1 in solution was similar with or without CRABP. Holo-CRABPs had higher affinity for CYP26B1 than free atRA, but both apo-CRABPs inhibited the formation of 4-OH-RA by CYP26B1. Similar protein-protein interactions between soluble binding proteins and CYPs may be important for other lipophilic CYP substrates.

  17. Targeting folded RNA: A branched peptide boronic acid that binds to a large surface area of HIV-1 RRE RNA†

    Science.gov (United States)

    Zhang, Wenyu; Bryson, David I.; Crumpton, Jason B.; Wynn, Jessica; Santos, Webster L.

    2013-01-01

    On-bead high throughput screening of a medium sized (1000–2000 Da) branched peptide boronic acid (BPBA) library consisting of 46,656 unique sequences against HIV-1 RRE RNA generated peptides with binding affinities in the low micromolar range. In particular, BPBA1 had a Kd of 1.4 µM with RRE IIB, preference for RNA over DNA (27 fold), and selectivity of up to >75 fold against a panel of RRE IIB variants. Structure-activity studies suggest that the boronic acid moiety and “branching” in peptides are key structural features for efficient binding and selectivity for the folded RNA target. BPBA1 was efficiently taken up by HeLa and A2780 cells. RNA-footprinting studies revealed that the BPBA1 binding site encompasses a large surface area that spans both the upper stem as well as the internal loop regions of RRE IIB. PMID:23925474

  18. Homology modeling of human γ-butyric acid transporters and the binding of pro-drugs 5-aminolevulinic acid and methyl aminolevulinic acid used in photodynamic therapy.

    Directory of Open Access Journals (Sweden)

    Yan Baglo

    Full Text Available Photodynamic therapy (PDT is a safe and effective method currently used in the treatment of skin cancer. In ALA-based PDT, 5-aminolevulinic acid (ALA, or ALA esters, are used as pro-drugs to induce the formation of the potent photosensitizer protoporphyrin IX (PpIX. Activation of PpIX by light causes the formation of reactive oxygen species (ROS and toxic responses. Studies have indicated that ALA and its methyl ester (MAL are taken up into the cells via γ-butyric acid (GABA transporters (GATs. Uptake via GATs into peripheral sensory nerve endings may also account for one of the few adverse side effects of ALA-based PDT, namely pain. In the present study, homology models of the four human GAT subtypes were constructed using three x-ray crystal structures of the homologous leucine transporter (LeuT as templates. Binding of the native substrate GABA and the possible substrates ALA and MAL was investigated by molecular docking of the ligands into the central putative substrate binding sites in the outward-occluded GAT models. Electrostatic potentials (ESPs of the putative substrate translocation pathway of each subtype were calculated using the outward-open and inward-open homology models. Our results suggested that ALA is a substrate of all four GATs and that MAL is a substrate of GAT-2, GAT-3 and BGT-1. The ESP calculations indicated that differences likely exist in the entry pathway of the transporters (i.e. in outward-open conformations. Such differences may be exploited for development of inhibitors that selectively target specific GAT subtypes and the homology models may hence provide tools for design of therapeutic inhibitors that can be used to reduce ALA-induced pain.

  19. Complex Binding of the FabR Repressor of Bacterial Unsaturated Fatty Acid Biosynthesis to its Cognate Promoters

    OpenAIRE

    Feng, Youjun; Cronan, John E.

    2011-01-01

    Two transcriptional regulators, the FadR activator and the FabR repressor control biosynthesis of unsaturated fatty acids in Escherichia coli. FabR represses expression of the two genes, fabA and fabB, required for unsaturated fatty acid synthesis and has been reported to require the presence of an unsaturated thioester (of either acyl carrier protein or CoA) in order to bind the fabA and fabB promoters in vitro. We report in vivo experiments in which unsaturated fatty acid synthesis was bloc...

  20. Diversity in the structures and ligand-binding sites of nematode fatty acid and retinol-binding proteins revealed by Na-FAR-1 from Necator americanus

    Science.gov (United States)

    Rey-Burusco, M. Florencia; Ibáñez-Shimabukuro, Marina; Gabrielsen, Mads; Franchini, Gisela R.; Roe, Andrew J.; Griffiths, Kate; Zhan, Bin; Cooper, Alan; Kennedy, Malcolm W.; Córsico, Betina; Smith, Brian O.

    2015-01-01

    Fatty acid and retinol-binding proteins (FARs) comprise a family of unusual α-helix rich lipid-binding proteins found exclusively in nematodes. They are secreted into host tissues by parasites of plants, animals and humans. The structure of a FAR protein from the free-living nematode Caenorhabditis elegans is available, but this protein [C. elegans FAR-7 (Ce-FAR-7)] is from a subfamily of FARs that does not appear to be important at the host/parasite interface. We have therefore examined [Necator americanus FAR-1 (Na-FAR-1)] from the blood-feeding intestinal parasite of humans, N. americanus. The 3D structure of Na-FAR-1 in its ligand-free and ligand-bound forms, determined by NMR (nuclear magnetic resonance) spectroscopy and X-ray crystallography respectively, reveals an α-helical fold similar to Ce-FAR-7, but Na-FAR-1 possesses a larger and more complex internal ligand-binding cavity and an additional C-terminal α-helix. Titration of apo-Na-FAR-1 with oleic acid, analysed by NMR chemical shift perturbation, reveals that at least four distinct protein–ligand complexes can be formed. Na-FAR-1 and possibly other FARs may have a wider repertoire for hydrophobic ligand binding, as confirmed in the present study by our finding that a range of neutral and polar lipids co-purify with the bacterially expressed recombinant protein. Finally, we show by immunohistochemistry that Na-FAR-1 is present in adult worms with a tissue distribution indicative of possible roles in nutrient acquisition by the parasite and in reproduction in the male. PMID:26318523

  1. Diversity in the structures and ligand-binding sites of nematode fatty acid and retinol-binding proteins revealed by Na-FAR-1 from Necator americanus.

    Science.gov (United States)

    Rey-Burusco, M Florencia; Ibáñez-Shimabukuro, Marina; Gabrielsen, Mads; Franchini, Gisela R; Roe, Andrew J; Griffiths, Kate; Zhan, Bin; Cooper, Alan; Kennedy, Malcolm W; Córsico, Betina; Smith, Brian O

    2015-11-01

    Fatty acid and retinol-binding proteins (FARs) comprise a family of unusual α-helix rich lipid-binding proteins found exclusively in nematodes. They are secreted into host tissues by parasites of plants, animals and humans. The structure of a FAR protein from the free-living nematode Caenorhabditis elegans is available, but this protein [C. elegans FAR-7 (Ce-FAR-7)] is from a subfamily of FARs that does not appear to be important at the host/parasite interface. We have therefore examined [Necator americanus FAR-1 (Na-FAR-1)] from the blood-feeding intestinal parasite of humans, N. americanus. The 3D structure of Na-FAR-1 in its ligand-free and ligand-bound forms, determined by NMR (nuclear magnetic resonance) spectroscopy and X-ray crystallography respectively, reveals an α-helical fold similar to Ce-FAR-7, but Na-FAR-1 possesses a larger and more complex internal ligand-binding cavity and an additional C-terminal α-helix. Titration of apo-Na-FAR-1 with oleic acid, analysed by NMR chemical shift perturbation, reveals that at least four distinct protein-ligand complexes can be formed. Na-FAR-1 and possibly other FARs may have a wider repertoire for hydrophobic ligand binding, as confirmed in the present study by our finding that a range of neutral and polar lipids co-purify with the bacterially expressed recombinant protein. Finally, we show by immunohistochemistry that Na-FAR-1 is present in adult worms with a tissue distribution indicative of possible roles in nutrient acquisition by the parasite and in reproduction in the male.

  2. Molecular characterization, functional expression, tissue localization and protective potential of a Taenia solium fatty acid-binding protein.

    Science.gov (United States)

    Illescas, Oscar; Carrero, Julio C; Bobes, Raúl J; Flisser, Ana; Rosas, Gabriela; Laclette, Juan P

    2012-12-01

    The fatty acid-binding proteins (FABPs) comprise a family of proteins that are widely expressed in animal cells and perform a variety of vital functions. Here, we report the identification, characterization, recombinant expression, tissue localization and protective potential of a Taenia solium FABP (TsFABP1). The TsFABP1 primary structure showed all the conserved residues characteristic of the subfamily iv of the intracellular Lipid-Binding Proteins (iLBPs), including those involved in the binding stabilization of the fatty acid molecule. Through a competitive binding assay we found that TsFABP1 is able to bind at least six different fatty acids with preference toward palmitic and stearic acid, suggesting that TsFABP1 is a member of the iLBP subfamily iv. Immunolocalization assays carried out on larval and adult tissues of four species of taeniids using anti-TsFABP1 hyperimmune sera produced in mice and rabbit, showed intense labeling in the tegument of the spiral canal and in subtegumental cytons of the larvae. These findings suggest that the spiral canal might be a major place for FA uptake in the developing scolex. In contrast, only subtegumental cytons in the adult worms stained positive. We propose that TsFABP1 is involved in the mechanism to mobilize fatty acids between compartments in the extensive syncytial tissue of taeniids. Protection assays carried out in a murine model of cysticercosis showed that subcutaneous immunization with TsFABP1 resulted in about 45% reduction of parasite load against an intraperitoneal challenge with Taenia crassiceps cysts. This reduction in parasite load correlated with the level of cellular and humoral immune responses against TsFABP1, as determined in spleen lymphocyte proliferation and ELISA testing.

  3. Drug-binding energetics of human α-1-acid glycoprotein assessed by isothermal titration calorimetry and molecular docking simulations

    Science.gov (United States)

    Huang, Johnny X.; Cooper, Matthew A.; Baker, Mark A.; Azad, Mohammad A.K.; Nation, Roger L.; Li, Jian; Velkov, Tony

    2012-01-01

    This study utilizes sensitive, modern isothermal titration calorimetric (ITC) methods to characterize the microscopic thermodynamic parameters that drive the binding of basic drugs to α-1-acid glycoprotein (AGP) and thereby rationalize the thermodynamic data in relation to docking models and crystallographic structures of the drug-AGP complexes. The binding of basic compounds from the tricyclic antidepressant series, together with miaserine, chlorpromazine, disopyramide and cimetidine all displayed an exothermically driven binding interaction with AGP. The impact of protonation/deprotonation events, ionic strength, temperature and the individual selectivity of the A and F1*S AGP variants on drug-binding thermodynamics were characterized. A correlation plot of the thermodynamic parameters for all of the test compounds revealed enthalpy-entropy compensation is in effect. The exothermic binding energetics of the test compounds were driven by a combination of favorable (negative) enthalpic (ΔH°) and favorable (positive) entropic (ΔS°) contributions to the Gibbs free energy (ΔG°). Collectively, the data imply that the free energies that drive drug binding to AGP and its relationship to drug-serum residency evolve from the complex interplay of enthalpic and entropic forces from interactions with explicit combinations of hydrophobic and polar side-chain sub-domains within the multi-lobed AGP ligand binding cavity. PMID:23192962

  4. Retinoic acid receptor agonists regulate expression of ATP-binding cassette transporter G1 in macrophages.

    Science.gov (United States)

    Ayaori, Makoto; Yakushiji, Emi; Ogura, Masatsune; Nakaya, Kazuhiro; Hisada, Tetsuya; Uto-Kondo, Harumi; Takiguchi, Shunichi; Terao, Yoshio; Sasaki, Makoto; Komatsu, Tomohiro; Iizuka, Maki; Yogo, Makiko; Uehara, Yoshinari; Kagechika, Hiroyuki; Nakanishi, Tsuyoshi; Ikewaki, Katsunori

    2012-04-01

    ABC transporter G1 (ABCG1) plays a pivotal role in HDL-mediated cholesterol efflux and atherogenesis. We investigated whether, and how, retinoic acid receptors (RARs) regulate ABCG1 expression in macrophages. All-trans retinoic acid (ATRA), an RAR ligand, increased ABCG1 protein levels and apoA-I/HDL-mediated cholesterol efflux from the macrophages. Both ATRA and other RAR agonists, TTNPB and Am580, increased major transcripts driven by promoter B upstream of exon 5, though minor transcripts driven by promoter A upstream of exon 1 were only increased by ATRA. The stimulatory effects of ATRA on ABCG1 expression were completely abolished in the presence of RAR/RXR antagonists but were only partially canceled in the presence of an LXR antagonist. Adenovirus with overexpressed oxysterol sulfotransferase abolished the LXR pathway, as previously reported, and ATRA-responsiveness in ABCA1/ABCG1 expressions were respectively attenuated by 38 and 22% compared to the control virus. Promoter assays revealed that ABCG1 levels were regulated more by promoter B than promoter A, and ATRA activated promoter B in a liver X receptor-responsive element (LXRE)-dependent manner. Further, LXRE-B in intron 7, but not LXRE-A in intron 5, enhanced ATRA responsiveness under overexpression of all RAR isoforms-RARα/β/γ. In contrast, the activation of promoter B by TTNPB depended on LXRE-B and RARα, but not on RARβ/γ. Finally, chromatin immunoprecipitation and gel-shift assays revealed a specific and direct repeat 4-dependent binding of RARα to LXRE-B. In conclusion, RAR ligands increase ABCA1/G1 expression and apoA-I/HDL-mediated cholesterol efflux from macrophages, and modulate ABCG1 promoter activity via LXRE-dependent mechanisms.

  5. Examination of the Addictive and Behavioral Properties of Fatty Acid Binding Protein Inhibitor SBFI26

    Directory of Open Access Journals (Sweden)

    Panayotis eThanos

    2016-04-01

    Full Text Available Abstract:The therapeutic properties of cannabinoids have been well demonstrated but are overshadowed by such adverse effects as cognitive and motor dysfunction, as well as their potential for addiction. Recent research on the natural lipid ligands of cannabinoid receptors, also known as endocannabinoids, have shed light on the mechanisms of intracellular transport of the endocannabinoid anandamide by fatty acid binding proteins (FABPs and subsequent catabolism by fatty acid amide hydrolase (FAAH. These findings facilitated the recent development of SBFI26, a pharmacological inhibitor of epidermal- and brain-specific FABP5 and FABP7, which effectively increases anandamide signaling. The goal of this study was to examine this compound for any possible rewarding and addictive properties as well as effects on locomotor activity, working / recognition memory, and propensity for sociability and preference for social novelty given its recently reported anti-inflammatory and analgesic properties. Male C57BL mice were split into four treatment groups and conditioned with 5.0 mg/kg, 20.0 mg/kg, 40.0 mg/kg SBFI26 or vehicle during a conditioned placed preference (CPP paradigm. Following CPP, mice underwent a battery of behavioral tests (open field, novel object recognition (NOR, and social interaction (SI and novelty (SN paired with acute SBFI26 administration. Results showed that SBFI26 did not produce conditioned placed preference or conditioned place aversion regardless of dose, and did not induce any differences in locomotor and exploratory activity during CPP or SBFI26-paired open field activity. We also observed no differences between treatment groups in NOR, SI, and SN. In conclusion, as SBFI26 was shown previously by our group to have significant analgesic and anti-inflammatory properties, here we show that it does not pose a risk of dependence or motor and cognitive impairment under the conditions tested.

  6. Gamma-aminobutyric acid-modulated benzodiazepine binding sites in bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Lummis, S.C.R.; Johnston, G.A.R. (Univ. of Sydney, New South Wales (Australia)); Nicoletti, G. (Royal Melbourne Inst. of Tech. (Australia)); Holan, G. (CSIRO, Melbourne (Australia))

    1991-01-01

    Benzodiazepine binding sites, which were once considered to exist only in higher vertebrates, are here demonstrated in the bacteria E. coli. The bacterial ({sup 3}H)diazepam binding sites are modulated by GABA; the modulation is dose dependent and is reduced at high concentrations. The most potent competitors of E.Coli ({sup 3}H)diazepam binding are those that are active in displacing ({sup 3}H)benzodiazepines from vertebrate peripheral benzodiazepine binding sites. These vertebrate sites are not modulated by GABA, in contrast to vertebrate neuronal benzodiazepine binding sites. The E.coli benzodiazepine binding sites therefore differ from both classes of vertebrate benzodiazepine binding sites; however the ligand spectrum and GABA-modulatory properties of the E.coli sites are similar to those found in insects. This intermediate type of receptor in lower species suggests a precursor for at least one class of vertebrate benzodiazepine binding sites may have existed.

  7. 15N NMR investigation of the covalent binding of reduced TNT amines to soil humic acid, model compounds, and lignocellulose.

    Science.gov (United States)

    Thorn, K A; Kennedy, K R

    2002-09-01

    The five major reductive degradation products of TNT-4ADNT (4-amino-2,6-dinitrotoluene), 2ADNT (2-amino-4,6-dinitrotoluene), 2,4DANT (2,4-diamino-6-nitrotoluene), 2,6DANT (2,6-diamino-4-nitrotoluene), and TAT (2,4,6-triaminotoluene)-labeled with 15N in the amine positions, were reacted with the IHSS soil humic acid and analyzed by 15N NMR spectrometry. In the absence of catalysts, all five amines underwent nucleophilic addition reactions with quinone and other carbonyl groups in the soil humic acid to form both heterocyclic and nonheterocyclic condensation products. Imine formation via 1,2-addition of the amines to quinone groups in the soil humic acid was significant with the diamines and TAT but not the monoamines. Horseradish peroxidase (HRP) catalyzed an increase in the incorporation of all five amines into the humic acid. In the case of the diamines and TAT, HRP also shifted the binding away from heterocyclic condensation product toward imine formation. A comparison of quantitative liquid phase with solid-state CP/MAS 15N NMR indicated that the CP experiment underestimated imine and heterocyclic nitrogens in humic acid, even with contact times optimal for observation of these nitrogens. Covalent binding of the mono- and diamines to 4-methylcatechol, the HRP catalyzed condensation of 4ADNT and 2,4DANT to coniferyl alcohol, and the binding of 2,4DANT to lignocellulose with and without birnessite were also examined.

  8. L-amino acid oxidase from Naja atra venom activates and binds to human platelets

    Institute of Scientific and Technical Information of China (English)

    Rui Li; Shaowen Zhu; Jianbo Wu; Wanyu Wang; Qiumin Lu; Kenneth J.Clemetson

    2008-01-01

    An L-amino acid oxidase (LAAO),NA-LAAO,was purified from the venom of Naja atra.Its N-terminal sequence shows great similarity with LAAOs from other snake venoms.NALAAO dose-dependently induced aggregation of washed human platelets.However,it had no activity on platelets in platelet-rich plasma.A low concentration of NA-LAAO greatly promoted the effect of hydrogen peroxide,whereas hydrogen peroxide itself had little activation effect on platelets.NA-LAAO induced tyrosine phosphorylation of a number of platelet proteins including Src kinase,spleen tyrosine kinase,and phospholipase C γ2.Unlike convulxin,Fc receptor γ chain and T lymphocyte adapter protein are not phosphorylated in NA-LAAO activated platelets,suggesting an activation mechanism different from the glycoprotein VI pathway.Catalase inhibited the platelet aggregation and platelet protein phosphorylation induced by NA-LAAO.NA-LAAO bound to fixed platelets as well as to platelet lysates of Western blots.Furthermore,affinity chromatography of platelet proteins on an NA-LAAO Sepharose 4B column isolated a few platelet membrane proteins,suggesting that binding of NA-LAAO to the platelet membrane might play a role in its action on platelets.

  9. Branched-chain Amino Acid Biosensing Using Fluorescent Modified Engineered Leucine/Isoleucine/Valine Binding Protein

    Directory of Open Access Journals (Sweden)

    Koji Sode

    2007-06-01

    Full Text Available A novel fluorescence sensing system for branched-chain amino acids (BCAAswas developed based on engineered leucine/isoleucine/valine-binding proteins (LIVBPsconjugated with environmentally sensitive fluorescence probes. LIVBP was cloned fromEscherichia coli and Gln149Cys, Gly227Cys, and Gln254Cys mutants were generated bygenetic engineering. The mutant LIVBPs were then modified with environmentallysensitive fluorophores. Based on the fluorescence intensity change observed upon thebinding of the ligands, the MIANS-conjugated Gln149Cys mutant (Gln149Cys-M showedthe highest and most sensitive response. The BCAAs Leu, Ile, and Val can each bemonitored at the sub-micromolar level using Gln149Cys-M. Measurements were alsocarried out on a mixture of BCAFAs and revealed that Gln149Cys-M-based measurementis not significantly affected by the change in the molar ratio of Leu, Ile and Val in thesample. Its high sensitivity and group-specific molecular recognition ability make the newsensing system ideally suited for the measurement of BCAAs and the determination of theFischer ratio, an indicator of hepatic disease involving metabolic dysfunction.

  10. Changes in dynamics upon oligomerization regulate substrate binding and allostery in amino acid kinase family members.

    Directory of Open Access Journals (Sweden)

    Enrique Marcos

    2011-09-01

    Full Text Available Oligomerization is a functional requirement for many proteins. The interfacial interactions and the overall packing geometry of the individual monomers are viewed as important determinants of the thermodynamic stability and allosteric regulation of oligomers. The present study focuses on the role of the interfacial interactions and overall contact topology in the dynamic features acquired in the oligomeric state. To this aim, the collective dynamics of enzymes belonging to the amino acid kinase family both in dimeric and hexameric forms are examined by means of an elastic network model, and the softest collective motions (i.e., lowest frequency or global modes of motions favored by the overall architecture are analyzed. Notably, the lowest-frequency modes accessible to the individual subunits in the absence of multimerization are conserved to a large extent in the oligomer, suggesting that the oligomer takes advantage of the intrinsic dynamics of the individual monomers. At the same time, oligomerization stiffens the interfacial regions of the monomers and confers new cooperative modes that exploit the rigid-body translational and rotational degrees of freedom of the intact monomers. The present study sheds light on the mechanism of cooperative inhibition of hexameric N-acetyl-L-glutamate kinase by arginine and on the allosteric regulation of UMP kinases. It also highlights the significance of the particular quaternary design in selectively determining the oligomer dynamics congruent with required ligand-binding and allosteric activities.

  11. [L-type fatty acid binding protein (L-FABP) and kidney disease].

    Science.gov (United States)

    Kamijo-Ikemori, Atsuko; Sugaya, Takeshi; Kimura, Kenjiro

    2014-02-01

    Liver-type fatty acid binding protein (L-FABP) is expressed in the cytoplasm of human renal proximal tubules. Renal L-FABP expression is up-regulated and urinary excretion of renal L-FABP is increased by various stressors, such as urinary protein, hyperglycemia, tubular ischemia, toxins, and salt-sensitive hypertension, which lead to the progression of kidney disease. Urinary L-FABP levels accurately reflect the degree of tubulointerstitial damage and are strongly correlated with the prognosis of chronic kidney disease (CKD) patients in clinical studies. In patients with type I or type II diabetes, urinary L-FABP levels were reported to be significantly higher in patients with normal levels of urinary albumin than in those with microalbuminuria. Urinary L-FABP may be useful for the early detection of diabetic nephropathy. Furthermore, in a longitudinal study, a higher level of urinary L-FABP was found to be a risk factor for the progression of diabetic nephropathy. With respect to acute kidney disease (AKI), urinary L-FABP facilitates the early detection of AKI before an increase in serum creatinine. Therefore, urinary L-FABP was approved as a new tubular biomarker by the Ministry of Health, Labour and Welfare of Japan.

  12. Current approach to male infertility treatment: sperm selection procedure based on hyaluronic acid binding ability

    Directory of Open Access Journals (Sweden)

    A. V. Zobova

    2015-01-01

    Full Text Available Intracytoplasmic sperm injection into an oocyte is widely used throughout the world in assisted reproductive technologies programs in the presence of male infertility factor. However, this approach can allow selection of a single sperm, which is carrying different types of pathologies. Minimizing of any potential risks, entailing the occurrence of abnormalities in the embryos development (apoptosis, fragmentation of embryos, alterations in gene expression, aneuploidies is a very important condition for reducing the potential negative consequences resulting the manipulation with gametes. Processes that could be influenced by the embryologist must be fulfilled in safe and physiological way as much as it is possible. Data of numerous publications reporting about the positive effects of using the technology of sperm selection by hyaluronic acid binding, let make a conclusion about the high prospects of this approach in the treatment of male infertility by methods of in vitro fertilization. The selection of sperm with improved characteristics, which determine the maturity and genetic integrity, provides an opportunity to improve the parameters of pre-implantation embryogenesis, having thus a positive effect on clinical outcomes of assisted reproductive technologies programs.

  13. DNA Methylation of Cellular Retinoic Acid-Binding Proteins in Cervical Cancer

    Science.gov (United States)

    Arellano-Ortiz, Ana L.; Salcedo-Vargas, Mauricio; Vargas-Requena, Claudia L.; López-Díaz, José A.; De la Mora-Covarrubias, Antonio; Silva-Espinoza, Juan C.; Jiménez-Vega, Florinda

    2016-01-01

    This study determined the methylation status of cellular retinoic acid-binding protein (CRABP) gene promoters and associated them with demographic characteristics, habits, and the presence of human papilloma virus (HPV) in patients with cervical cancer (CC), low and high squamous intraepithelial lesions, and no intraepithelial lesion. Women (n = 158) were selected from the Colposcopy Clinic of Sanitary Jurisdiction II in Ciudad Juarez, Chihuahua, Mexico. Demographic characteristics and habit information were collected. Cervical biopsy and endocervical scraping were used to determine methylation in promoter regions by methylation-specific polymerase chain reaction technique. We found hemi-methylation patterns in the promoter regions of CRABP1 and CRABP2; there was 28.5% hemi-methylation in CRABP1 and 7.0% in that of CRABP2. Methylation in CRABP1 was associated with age (≥35 years, P = 0.002), family history of cancer (P = 0.032), the presence of HPV-16 (P = 0.013), and no alcohol intake (P = 0.035). These epigenetic changes could be involved in the CC process, and CRABP1 has the potential to be a predictive molecular marker of retinoid therapy response. PMID:27867303

  14. Ability of Intestinal Lactic Acid Bacteria to Bind and/or Metabolise Indole

    Directory of Open Access Journals (Sweden)

    Zdzisława Libudzisz

    2008-01-01

    Full Text Available Intestinal microbiota can contribute to the development of colon cancer by the production of many substances playing a role in carcinogenesis. Metabolites of protein fermentation in the colon, such as ammonia, amines, indole, phenol, skatole and their derivatives are toxic. On the other hand, lactic acid bacteria (LAB existing in the colon may exert an anticarcinogenic action, but the mechanism is still poorly understood. In the present study, the aim is to determine the ability of intestinal lactobacilli to adsorb or metabolise indole in vitro. Lactobacillus strains were cultivated in MRS and a modified MRS broth with reduced concentrations of nitrogen and carbon. Indole concentration in the media was from 2 to 20 μg/mL. The decrease in indole concentration was from 1 to 10 μg/mL after cultivation in MRS broth, and from 4.3 to 6.7 μg/mL after cultivation in a modified MRS broth. It was shown that the higher concentration of indole in the medium, the higher reduction of its level. Killed bacteria displayed slight binding capacity. After the interaction of alive lactobacilli with 10 μg/mL of indole, it displayed a lower genotoxicity, as evaluated by the alkaline comet assay. The phenomenon did not depend on the decrease of indole concentration, but on the culture medium and the strain of LAB and ranged from 7 to 96 %.

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

    Science.gov (United States)

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

    2017-01-18

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

  16. Liver fatty acid-binding protein (L-Fabp) modifies intestinal fatty acid composition and adenoma formation in ApcMin/+ mice.

    Science.gov (United States)

    Dharmarajan, Sekhar; Newberry, Elizabeth P; Montenegro, Grace; Nalbantoglu, Ilke; Davis, Victoria R; Clanahan, Michael J; Blanc, Valerie; Xie, Yan; Luo, Jianyang; Fleshman, James W; Kennedy, Susan; Davidson, Nicholas O

    2013-10-01

    Evidence suggests a relationship between dietary fat intake, obesity, and colorectal cancer, implying a role for fatty acid metabolism in intestinal tumorigenesis that is incompletely understood. Liver fatty acid-binding protein (L-Fabp), a dominant intestinal fatty acid-binding protein, regulates intestinal fatty acid trafficking and metabolism, and L-Fabp deletion attenuates diet-induced obesity. Here, we examined whether changes in intestinal fatty acid metabolism following L-Fabp deletion modify adenoma development in Apc(Min)(/+) mice. Compound L-Fabp(-/-)Apc(Min)(/+) mice were generated and fed a 10% fat diet balanced equally between saturated, monounsaturated, and polyunsaturated fat. L-Fabp(-/-)Apc(Min)(/+) mice displayed significant reductions in adenoma number and total polyp area compared with Apc(Min)(/+)controls, reflecting a significant shift in distribution toward smaller polyps. Adenomas from L-Fabp(-/-)Apc(Min)(/+) mice exhibited reductions in cellular proliferation, high-grade dysplasia, and nuclear β-catenin translocation. Intestinal fatty acid content was increased in L-Fabp(-/-)Apc(Min)(/+) mice, and lipidomic profiling of intestinal mucosa revealed significant shifts to polyunsaturated fatty acid species with reduced saturated fatty acid species. L-Fabp(-/-)Apc(Min)(/+) mice also showed corresponding changes in mRNA expression of enzymes involved in fatty acid elongation and desaturation. Furthermore, adenomas from L-Fabp(-/-)Apc(Min)(/+) mice displayed significant reductions in mRNA abundance of nuclear hormone receptors involved in cellular proliferation and in enzymes involved in lipogenesis. These findings collectively implicate L-Fabp as an important genetic modifier of intestinal tumorigenesis, and identify fatty acid trafficking and metabolic compartmentalization as an important pathway linking dietary fat intake, obesity, and intestinal tumor formation.

  17. Heart-type fatty acid-binding protein is essential for efficient brown adipose tissue fatty acid oxidation and cold tolerance.

    Science.gov (United States)

    Vergnes, Laurent; Chin, Robert; Young, Stephen G; Reue, Karen

    2011-01-07

    Brown adipose tissue has a central role in thermogenesis to maintain body temperature through energy dissipation in small mammals and has recently been verified to function in adult humans as well. Here, we demonstrate that the heart-type fatty acid-binding protein, FABP3, is essential for cold tolerance and efficient fatty acid oxidation in mouse brown adipose tissue, despite the abundant expression of adipose-type fatty acid-binding protein, FABP4 (also known as aP2). Fabp3(-/-) mice exhibit extreme cold sensitivity despite induction of uncoupling and oxidative genes and hydrolysis of brown adipose tissue lipid stores. However, using FABP3 gain- and loss-of-function approaches in brown adipocytes, we detected a correlation between FABP3 levels and the utilization of exogenous fatty acids. Thus, Fabp3(-/-) brown adipocytes fail to oxidize exogenously supplied fatty acids, whereas enhanced Fabp3 expression promotes more efficient oxidation. These results suggest that FABP3 levels are a determinant of fatty acid oxidation efficiency by brown adipose tissue and that FABP3 represents a potential target for modulation of energy dissipation.

  18. Receptor binding proteins of Listeria monocytogenes bacteriophages A118 and P35 recognize serovar-specific teichoic acids

    Energy Technology Data Exchange (ETDEWEB)

    Bielmann, Regula; Habann, Matthias; Eugster, Marcel R. [Institute of Food, Nutrition and Health, ETH Zurich, Schmelzbergstrasse 7, 8092 Zurich (Switzerland); Lurz, Rudi [Max-Planck Institute for Molecular Genetics, 14195 Berlin (Germany); Calendar, Richard [Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3202 (United States); Klumpp, Jochen, E-mail: jochen.klumpp@hest.ethz.ch [Institute of Food, Nutrition and Health, ETH Zurich, Schmelzbergstrasse 7, 8092 Zurich (Switzerland); Loessner, Martin J. [Institute of Food, Nutrition and Health, ETH Zurich, Schmelzbergstrasse 7, 8092 Zurich (Switzerland)

    2015-03-15

    Adsorption of a bacteriophage to the host requires recognition of a cell wall-associated receptor by a receptor binding protein (RBP). This recognition is specific, and high affinity binding is essential for efficient virus attachment. The molecular details of phage adsorption to the Gram-positive cell are poorly understood. We present the first description of receptor binding proteins and a tail tip structure for the siphovirus group infecting Listeria monocytogenes. The host-range determining factors in two phages, A118 and P35 specific for L. monocytogenes serovar 1/2 have been determined. Two proteins were identified as RBPs in phage A118. Rhamnose residues in wall teichoic acids represent the binding ligands for both proteins. In phage P35, protein gp16 could be identified as RBP and the role of both rhamnose and N-acetylglucosamine in phage adsorption was confirmed. Immunogold-labeling and transmission electron microscopy allowed the creation of a topological model of the A118 phage tail. - Highlights: • We present the first description of receptor binding proteins and a tail tip structure for the Siphovirus group infecting Listeria monocytogenes. • The host-range determining factors in two phages, A118 and P35 specific for L. monocytogenes serovar 1/2 have been determined. • Rhamnose residues in wall teichoic acids represent the binding ligands for both receptor binding proteins in phage A118. • Rhamnose and N-acetylglucosamine are required for adsorption of phage P35. • We preset a topological model of the A118 phage tail.

  19. Effect of repeat unit structure and molecular mass of lactic acid bacteria hetero-exopolysaccharides on binding to milk proteins

    DEFF Research Database (Denmark)

    Birch, Johnny; HarÐarson, HörÐur Kári; Khan, Sanaullah

    2017-01-01

    -exopolysaccharides (HePSs) of 0.14–4.9 MDa from lactic acid bacteria to different milk proteins (β-casein, κ-casein, native and heat-treated β-lactoglobulin) at pH 4.0–5.0. Maximum binding capacity (RUmax) and apparent affinity (KA,app) were HePS- and protein-dependent and varied for example 10- and 600-fold......Interactions of exopolysaccharides and proteins are of great importance in food science, but complicated to analyze and quantify at the molecular level. A surface plasmon resonance procedure was established to characterize binding of seven structure-determined, branched hetero...

  20. Short Peptide Nucleic Acids Bind Strongly to Homopurine Tract of Double Helical RNA at pH 5.5

    OpenAIRE

    Li, Ming; Zengeya, Thomas; Rozners, Eriks

    2010-01-01

    The important role that non-coding RNA plays in cell biology makes it an attractive target for molecular recognition. However, the discovery of small molecules that bind double helical RNA selectively and may serve as biochemical probes and potential drug leads has been relatively slow. Herein, we show that peptide nucleic acids, as short as six nucleobases, bind very strongly (Ka > 107) and sequence selectively to a homopurine tract of double helical RNA at pH 5.5. The isothermal titration c...

  1. Structural basis of nucleic acid recognition by FK506-binding protein 25 (FKBP25), a nuclear immunophilin.

    Science.gov (United States)

    Prakash, Ajit; Shin, Joon; Rajan, Sreekanth; Yoon, Ho Sup

    2016-04-07

    The nuclear immunophilin FKBP25 interacts with chromatin-related proteins and transcription factors and is suggested to interact with nucleic acids. Currently the structural basis of nucleic acid binding by FKBP25 is unknown. Here we determined the nuclear magnetic resonance (NMR) solution structure of full-length human FKBP25 and studied its interaction with DNA. The FKBP25 structure revealed that the N-terminal helix-loop-helix (HLH) domain and C-terminal FK506-binding domain (FKBD) interact with each other and that both of the domains are involved in DNA binding. The HLH domain forms major-groove interactions and the basic FKBD loop cooperates to form interactions with an adjacent minor-groove of DNA. The FKBP25-DNA complex model, supported by NMR and mutational studies, provides structural and mechanistic insights into the nuclear immunophilin-mediated nucleic acid recognition. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  2. In vitro and in vivo evidence for actin association of the naphthylphthalamic acid-binding protein from zucchini hypocotyls

    Science.gov (United States)

    Butler, J. H.; Hu, S.; Brady, S. R.; Dixon, M. W.; Muday, G. K.

    1998-01-01

    The N-1-naphthylphthalamic acid (NPA)-binding protein is part of the auxin efflux carrier, the protein complex that controls polar auxin transport in plant tissues. This study tested the hypothesis that the NPA-binding protein (NBP) is associated with the actin cytoskeleton in vitro and that an intact actin cytoskeleton is required for polar auxin transport in vivo. Cytoskeletal polymerization was altered in extracts of zucchini hypocotyls with reagents that stabilized either the polymeric or monomeric forms of actin or tubulin. Phalloidin treatment altered actin polymerization, as demonstrated by immunoblot analyses following native and denaturing electrophoresis. Phalloidin increased both filamentous actin (F-actin) and NPA-binding activity, while cytochalasin D and Tris decreased both F-actin and NPA-binding activity in cytoskeletal pellets. The microtubule stabilizing drug taxol increased pelletable tubulin, but did not alter either the amount of pelletable actin or NPA-binding activity. Treatment of etiolated zucchini hypocotyls with cytochalasin D decreased the amount of auxin transport and its regulation by NPA. These experimental results are consistent with an in vitro actin cytoskeletal association of the NPA-binding protein and with the requirement of an intact actin cytoskeleton for maximal polar auxin transport in vivo.

  3. Fatty Acid Amide Hydrolase Binding in Brain of Cannabis Users: Imaging With the Novel Radiotracer [(11)C]CURB.

    Science.gov (United States)

    Boileau, Isabelle; Mansouri, Esmaeil; Williams, Belinda; Le Foll, Bernard; Rusjan, Pablo; Mizrahi, Romina; Tyndale, Rachel F; Huestis, Marilyn A; Payer, Doris E; Wilson, Alan A; Houle, Sylvain; Kish, Stephen J; Tong, Junchao

    2016-11-01

    One of the major mechanisms for terminating the actions of the endocannabinoid anandamide is hydrolysis by fatty acid amide hydrolase (FAAH), and inhibitors of the enzyme were suggested as potential treatment for human cannabis dependence. However, the status of brain FAAH in cannabis use disorder is unknown. Brain FAAH binding was measured with positron emission tomography and [(11)C]CURB in 22 healthy control subjects and ten chronic cannabis users during early abstinence. The FAAH genetic polymorphism (rs324420) and blood, urine, and hair levels of cannabinoids and metabolites were determined. In cannabis users, FAAH binding was significantly lower by 14%-20% across the brain regions examined than in matched control subjects (overall Cohen's d = 0.96). Lower binding was negatively correlated with cannabinoid concentrations in blood and urine and was associated with higher trait impulsiveness. Lower FAAH binding levels in the brain may be a consequence of chronic and recent cannabis exposure and could contribute to cannabis withdrawal. This effect should be considered in the development of novel treatment strategies for cannabis use disorder that target FAAH and endocannabinoids. Further studies are needed to examine possible changes in FAAH binding during prolonged cannabis abstinence and whether lower FAAH binding predates drug use. Copyright © 2016 Society of Biological Psychiatry. All rights reserved.

  4. The pathogen-related yeast protein Pry1, a member of the CAP protein superfamily, is a fatty acid-binding protein.

    Science.gov (United States)

    Darwiche, Rabih; Mène-Saffrané, Laurent; Gfeller, David; Asojo, Oluwatoyin A; Schneiter, Roger

    2017-05-19

    Members of the CAP superfamily (cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins), also known as SCP superfamily (sperm-coating proteins), have been implicated in many physiological processes, including immune defenses, venom toxicity, and sperm maturation. Their mode of action, however, remains poorly understood. Three proteins of the CAP superfamily, Pry1, -2, and -3 (pathogen related in yeast), are encoded in the Saccharomyces cerevisiae genome. We have shown previously that Pry1 binds cholesterol in vitro and that Pry function is required for sterol secretion in yeast cells, indicating that members of this superfamily may generally bind sterols or related small hydrophobic compounds. On the other hand, tablysin-15, a CAP protein from the horsefly Tabanus yao, has been shown to bind leukotrienes and free fatty acids in vitro Therefore, here we assessed whether the yeast Pry1 protein binds fatty acids. Computational modeling and site-directed mutagenesis indicated that the mode of fatty acid binding is conserved between tablysin-15 and Pry1. Pry1 bound fatty acids with micromolar affinity in vitro, and its function was essential for fatty acid export in cells lacking the acyl-CoA synthetases Faa1 and Faa4. Fatty acid binding of Pry1 is independent of its capacity to bind sterols, and the two sterol- and fatty acid-binding sites are nonoverlapping. These results indicate that some CAP family members, such as Pry1, can bind different lipids, particularly sterols and fatty acids, at distinct binding sites, suggesting that the CAP domain may serve as a stable, secreted protein domain that can accommodate multiple ligand-binding sites. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Response of Fatty Acid Synthesis Genes to the Binding of Human Salivary Amylase by Streptococcus gordonii

    OpenAIRE

    Nikitkova, Anna E.; Haase, Elaine M.; Vickerman, M. Margaret; Gill, Steven R.; Scannapieco, Frank A.

    2012-01-01

    Streptococcus gordonii, an important primary colonizer of dental plaque biofilm, specifically binds to salivary amylase via the surface-associated amylase-binding protein A (AbpA). We hypothesized that a function of amylase binding to S. gordonii may be to modulate the expression of chromosomal genes, which could influence bacterial survival and persistence in the oral cavity. Gene expression profiling by microarray analysis was performed to detect genes in S. gordonii strain CH1 that were di...

  6. Molecular dynamics simulations of the auxin-binding protein 1 in complex with indole-3-acetic acid and naphthalen-1-acetic acid.

    Science.gov (United States)

    Grandits, Melanie; Oostenbrink, Chris

    2014-10-01

    Auxin-binding protein 1 (ABP1) is suggested to be an auxin receptor which plays an important role in several processes in green plants. Maize ABP1 was simulated with the natural auxin indole-3-acetic acid (IAA) and the synthetic analog naphthalen-1-acetic acid (NAA), to elucidate the role of the KDEL sequence and the helix at the C-terminus. The KDEL sequence weakens the intermolecular interactions between the monomers but stabilizes the C-terminal helix. Conformational changes at the C-terminus occur within the KDEL sequence and are influenced by the binding of the simulated ligands. This observation helps to explain experimental findings on ABP1 interactions with antibodies that are modulated by the presence of auxin, and supports the hypothesis that ABP1 acts as an auxin receptor. Stable hydrogen bonds between the monomers are formed between Glu40 and Glu62, Arg10 and Thr97, Lys39, and Glu62 in all simulations. The amino acids Ile22, Leu25, Trp44, Pro55, Ile130, and Phe149 are located in the binding pocket and are involved in hydrophobic interactions with the ring system of the ligand. Trp151 is stably involved in a face to end interaction with the ligand. The calculated free energy of binding using the linear interaction energy approach showed a higher binding affinity for NAA as compared to IAA. Our simulations confirm the asymmetric behavior of the two monomers, the stronger interaction of NAA than IAA and offers insight into the possible mechanism of ABP1 as an auxin receptor.

  7. Intestinal fatty acid-binding protein and gut permeability responses to exercise.

    Science.gov (United States)

    March, Daniel S; Marchbank, Tania; Playford, Raymond J; Jones, Arwel W; Thatcher, Rhys; Davison, Glen

    2017-05-01

    Intestinal cell damage due to physiological stressors (e.g. heat, oxidative, hypoperfusion/ischaemic) may contribute to increased intestinal permeability. The aim of this study was to assess changes in plasma intestinal fatty acid-binding protein (I-FABP) in response to exercise (with bovine colostrum supplementation, Col, positive control) and compare this to intestinal barrier integrity/permeability (5 h urinary lactulose/rhamnose ratio, L/R). In a double-blind, placebo-controlled, crossover design, 18 males completed two experimental arms (14 days of 20 g/day supplementation with Col or placebo, Plac). For each arm participants performed two baseline (resting) intestinal permeability assessments (L/R) pre-supplementation and one post-exercise following supplementation. Blood samples were collected pre- and post-exercise to determine I-FABP concentration. Two-way repeated measures ANOVA revealed an arm × time interaction for L/R and I-FABP (P exercise in Plac (273% of pre, P exercise values significantly lower with Col (P exercise in Plac (191% of pre-exercise, P = 0.002) but not in the Col arm (107%, P = 0.862) with post-exercise values significantly lower with Col (P = 0.013). Correlations between the increase in I-FABP and L/R were evident for visit one (P = 0.044) but not visit two (P = 0.200) although overall plots/patterns do appear similar for each. These findings suggest that exercise-induced intestinal cellular damage/injury is partly implicated in changes in permeability but other factors must also contribute.

  8. Heart fatty acid binding protein and myoglobin after reperfusion of acute myocardial infarction.

    Science.gov (United States)

    Ozdemir, Murat; Durakoğlugil, Emre; Gülbahar, Ozlem; Turkoglu, Sedat; Sancak, Banu; Paşaoğlu, Hatice; Cengel, Atiye

    2007-10-01

    The aim of this study was to disclose the release kinetics of heart fatty acid binding protein (HFABP) and myoglobin in acute myocardial infarction (AMI) reperfused by primary percutaneous coronary intervention (PPCI) and to determine the influence of the quality of coronary flow post PPCI on the release properties of these markers. Twenty-four patients with AMI who underwent successful PPCI and had no evidence of reocclusion within the first 120 minutes were studied. Serum myoglobin and HFABP levels at baseline and at 15, 30, 45, 60, 90 and 120 minutes after reperfusion were measured. Corrected TIMI frame count (CTFC) in the relevant vessel post PPCI was used to categorize patients in group I (CTFC > 21) and group 2 (CTFC < or = 21). Biomarker ratios at each sampling point were calculated by dividing the serum level of the biomarker at the specific sampling time by its baseline level. Baseline myoglobin and HFABP levels rose significantly at 15 minutes (153 +/- 251.5 microg/L vs. 904.3 +/- 542.6 microg/L, 10.9 +/- 8 microg/L vs. 17.8 +/- 9.1 microg/L, both P < 0.0001) after successful PPCI. Group 2 patients tended to have higher biomarker ratios at each time point as compared to group I. Successful PPCI for AMI results in a significant increase of both HFABP and myoglobin levels within 15 minutes of vessel opening and the quality of flow in the infarction-related artery post PCI as evaluated by CTFC does not influence the release kinetics of these biomarkers.

  9. Serum Heart-Type Fatty Acid-Binding Protein Levels in Patients with Overt Hypothyroidism

    Directory of Open Access Journals (Sweden)

    Esra Tutal

    2017-03-01

    Full Text Available Purpose: Overt hypothyroidism affects mostly women with an increasing prevalence with age. Hypothyroidism is associated with accelerated atherosclerotic cardiovascular diseases possibly caused by the higher incidence of hyperlipidemia, insulin resistance, and hypertension. Heart-type fatty acid-binding protein (H-FABP is specific for cardiomyocytes and a sensitive marker of myocardial injury. The purpose of this study was examining the effect of hypothyroidism on H-FABP levels and carotid artery intima-media thickness (CIMT. Material and Method: We measured serum H-FABP levels in 33 patients with overt hypothyroidism and age, gender, and body mass index-matched 39 control subjects. The patients were newly diagnosed with Hashimoto’s thyroiditis. All participants underwent high-resolution B-mode ultrasonography for the measurement of CIMT. Results: There was no significant difference in serum levels of H-FABP between the patient group and controls (1515.87±2143.0 pg/mL vs. 953.0±416.0 pg/mL, respectively; p=0.15. CIMT level was significantly higher in the patient group than in the control group (0.53±0.08 mm vs. 0.48±0.05 mm; p=0.02. However, the homeostasis model assessment of insulin resistance and fasting insulin levels did not differ between the two groups. Discussion: Based on the results of this study, we assume that H-FABP is not a useful marker in detecting preclinical atherosclerosis in patients with overt hypothyroidism associated with Hashimoto’s thyroiditis, however, CIMT might be a useful marker in detecting early atherosclerosis.

  10. 3T3 fibroblasts transfected with a cDNA for mitochondrial aspartate aminotransferase express plasma membrane fatty acid-binding protein and saturable fatty acid uptake.

    OpenAIRE

    1995-01-01

    To explore the relationship between mitochondrial aspartate aminotransferase (mAspAT; EC 2.6.1.1) and plasma membrane fatty acid-binding protein (FABPpm) and their role in cellular fatty acid uptake, 3T3 fibroblasts were cotransfected with plasmid pMAAT2, containing a full-length mAspAT cDNA downstream of a Zn(2+)-inducible metallothionein promoter, and pFR400, which conveys methotrexate resistance. Transfectants were selected in methotrexate, cloned, and exposed to increasing methotrexate co...

  11. Short peptide nucleic acids bind strongly to homopurine tract of double helical RNA at pH 5.5.

    Science.gov (United States)

    Li, Ming; Zengeya, Thomas; Rozners, Eriks

    2010-06-30

    The important role that noncoding RNA plays in cell biology makes it an attractive target for molecular recognition. However, the discovery of small molecules that bind double helical RNA selectively and may serve as biochemical probes and potential drug leads has been relatively slow. Herein, we show that peptide nucleic acids, as short as six nucleobases, bind very strongly (K(a) > 10(7)) and sequence selectively to a homopurine tract of double helical RNA at pH 5.5. The isothermal titration calorimetry and circular dichroism experiments suggest that the binding mode may be a sequence selective triple helix formation. Our results have implications for development of biochemical probes to study function of noncoding RNAs and design of compounds with potential antibacterial and antiviral activity.

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

    Science.gov (United States)

    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.

  13. Characterization of Folic Acid and Poly(amidoamine) Dendrimer Interactions with Folate Binding Protein: A Force-Pulling Study.

    Science.gov (United States)

    Leroueil, Pascale R; DiMaggio, Stassi; Leistra, Abigail N; Blanchette, Craig D; Orme, Christine; Sinniah, Kumar; Orr, Bradford G; Banaszak Holl, Mark M

    2015-09-01

    Atomic force microscopy force-pulling experiments have been used to measure the binding forces between folic acid (FA) conjugated poly(amidoamine) (PAMAM) dendrimers and folate binding protein (FBP). The generation 5 (G5) PAMAM conjugates contained an average of 2.7, 4.7, and 7.2 FA per dendrimer. The most probable rupture force was measured to be 83, 201, and 189 pN for G5-FA2.7, G5-FA4.7, and G5-FA7.2, respectively. Folic acid blocking experiments for G5-FA7.2 reduced the frequency of successful binding events and increased the magnitude of the average rupture force to 274 pN. The force data are interpreted as arising from a network of van der Waals and electrostatic interactions that form between FBP and G5 PAMAM dendrimer, resulting in a binding strength far greater than that expected for an interaction between FA and FBP alone.

  14. Crystal structure and nucleic acid-binding activity of the CRISPR-associated protein Csx1 of Pyrococcus furiosus.

    Science.gov (United States)

    Kim, Young Kwan; Kim, Yeon-Gil; Oh, Byung-Ha

    2013-02-01

    In many prokaryotic organisms, chromosomal loci known as clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (CAS) genes comprise an acquired immune defense system against invading phages and plasmids. Although many different Cas protein families have been identified, the exact biochemical functions of most of their constituents remain to be determined. In this study, we report the crystal structure of PF1127, a Cas protein of Pyrococcus furiosus DSM 3638 that is composed of 480 amino acids and belongs to the Csx1 family. The C-terminal domain of PF1127 has a unique β-hairpin structure that protrudes out of an α-helix and contains several positively charged residues. We demonstrate that PF1127 binds double-stranded DNA and RNA and that this activity requires an intact β-hairpin and involve the homodimerization of the protein. In contrast, another Csx1 protein from Sulfolobus solfataricus P2 that is composed of 377 amino acids does not have the β-hairpin structure and exhibits no DNA-binding properties under the same experimental conditions. Notably, the C-terminal domain of these two Csx1 proteins is greatly diversified, in contrast to the conserved N-terminal domain, which appears to play a common role in the homodimerization of the protein. Thus, although P. furiosus Csx1 is identified as a nucleic acid-binding protein, other Csx1 proteins are predicted to exhibit different individual biochemical activities. Copyright © 2012 Wiley Periodicals, Inc.

  15. Light microscopic immunocytochemical localization of hepatic and intestinal types of fatty acid-binding proteins in rat small intestine.

    Science.gov (United States)

    Shields, H M; Bates, M L; Bass, N M; Best, C J; Alpers, D H; Ockner, R K

    1986-05-01

    Monospecific antisera to purified hepatic fatty acid-binding protein (hFABP) and gut fatty acid-binding protein (gFABP) have been used to localize these two proteins in the small intestine of fed rats at the light microscopic level. Pieces of duodenum, jejunum, and ileum were removed from 4-, 10-, 20-, 22-, and 60-day-old Sprague-Dawley rats. Both cryostat and paraffin sections were studied for the presence of hFABP or gFABP by the avidin-biotin immunoperoxidase method. Slides were graded blind for the intensity of staining. Despite the structural and immunological differences between these two proteins, we showed no major differences between their staining patterns or their staining intensity throughout the intestine during postnatal development. The staining for both fatty acid-binding proteins was cytoplasmic. No brush border staining was found. Staining was more intense in the proximal rather than distal intestine, in the villus rather than crypt cells, and in the apex rather than the base of intestinal cells. Shifts in staining patterns, and staining intensity occurring during development may be related to variations in dietary fat intake, rates of cell proliferation, intestinal anatomy, and mechanisms for fat absorption.

  16. Molybdenum (VI) binded to humic and nitrohumic acid models in aqueous solutions: phthalic, 3- and 4-nitrophthalic acids, catechol and 4-nitrocathecol, part 1

    Energy Technology Data Exchange (ETDEWEB)

    Merce, Ana Lucia R.; Greboge, Cristiane; Mendes, Giovani; Mangrich, Antonio S. [Parana Univ., Curitiba, PR (Brazil). Dept. de Quimica]. E-mail: anamerce@ufpr.br

    2005-02-01

    Many mathematical models have been tested in the literature in the search of how humic acids (HA) from many natural sources complex to metal ions. HA are composed of natural degradation sources of C, N, P and S, bearing hydroxyl and carboxyl aromatic units in their inner structure. The presence of metal ions binded to these basic sites promotes fertility to the soil as well as can hold metal ions to be slowly released as the mineralization of the soil occurs. Nitrohumic substances are a laboratory artifact with higher N content then humic acids with an electron withdrawing group - NO{sub 2}. However they still bear the main HA constituent chemical groups such as salicylate, catecholate and phthalate derivatives, all prone to bind to metal ions depending on the chemical conditions of the environment. This work intended to study the complexing behaviour of some HA models having very different Lewis basic binding sites in the presence of molybdenum (VI) ions, in aqueous systems, with varying pH values using some analytical tools. The formation constants of phthalic acid, 3- and 4-nitrophthalic acids, catechol and 4-nitrocatechol with Mo(VI) as well as the speciation of the complex species according to varying pH values were determined. Potentiometric and cyclic voltammetric titrations were employed to calculate the formation constants and to monitor the formation and decomposition of some complexed species. The results showed that although there is complexation between phthalic derived acids and molybdenum, the speciation favours it only until pH 6.0 at the best. On the other hand, salicylic and catechol derived models showed existence of complexation until basic pH values, allowing a compromising complexation pH range when humic and nitrohumic substances are involved. (author)

  17. Tannic acid binding of cell surfaces in normal, premalignant, and malignant squamous epithelium of the human uterine cervix.

    Science.gov (United States)

    Davina, J H; Lamers, G E; van Haelst, U J; Kenemans, P; Stadhouders, A M

    1984-01-01

    Alterations in tannic acid (TA) binding capacity of cell surface carbohydrates in normal, premalignant, and malignant squamous epithelium of the human uterine cervix have been studied using electron microscopic visualization in combination with microdensitometric evaluation. While in normal epithelium there is distinct binding in four to five cell layers of the deep intermediate zone, cells of carcinoma in situ and invasive cancer lesions lack TA binding. In moderate dysplasia an intermediate reacting pattern is found. Deep intermediate cells in areas bordering the carcinoma in situ lesions do not show any binding, although their ultrastructure cannot be distinguished from similar cells in normal tissue. The TA deposition within the deep intermediate zone is probably related to the presence here of glycoprotein-containing membrane-coating granules. The finding that TA binding discriminates between cells in normal squamous epithelium and morphologically normal cells in juxtaposition with lesional areas in premalignant and malignant epithelium opens the possibility for a more reliable cytologic diagnosis of cervical epithelial neoplasia.

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

    Science.gov (United States)

    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.

  19. Ab initio study of the binding of collagen amino acids to graphene and A-doped (A = H, Ca) graphene

    Energy Technology Data Exchange (ETDEWEB)

    Cazorla, Claudio, E-mail: c.silva@ucl.ac.u

    2010-09-30

    We present a theoretical study of the binding of collagen amino acids (AA, namely glycine, Gly; proline, Pro; and hydroxyproline, Hyp) to graphene (Gr), Ca-doped graphene and graphane (Gra) using density functional theory calculations and ab initio molecular dynamics (AIMD) simulations. It is found that binding of Gly, Pro and Hyp to Gr and Gra is thermodynamically favorable yet dependent on the amino acid orientation and always very weak (adsorption energies E{sub ads} range from -90 to -20 meV). AIMD simulations reveal that room-temperature thermal excitations are enough to induce detachment of Gly and Pro from Gr and of all three amino acids from Gra. Interestingly, we show that collagen AA binding to Gr is enhanced dramatically by doping the carbon surface with calcium atoms (corresponding E{sub ads} values decrease by practically two orders of magnitude with respect to the non-doped case). This effect is result of electronic charge transfers from the Ca impurity (donor) to Gr (acceptor) and the carboxyl group (COOH) of the amino acid (acceptor). The possibility of using Gr and Gra as nanoframes for sensing of collagen amino acids has also been investigated by performing electronic density of states analysis. It is found that, whether Gr is hardly sensitive, the electronic band gap of Gra can be modulated by attaching different number and species of AAs onto it. The results presented in this work provide fundamental insights on the quantum interactions of collagen protein components with carbon-based nanostructures and can be useful for developments in bio and nanotechnology fields.

  20. Production of a soluble single-chain variable fragment antibody against okadaic acid and exploration of its specific binding.

    Science.gov (United States)

    He, Kuo; Zhang, Xiuyuan; Wang, Lixia; Du, Xinjun; Wei, Dong

    2016-06-15

    Okadaic acid is a lipophilic marine algal toxin commonly responsible for diarrhetic shellfish poisoning (DSP). Outbreaks of DSP have been increasing and are of worldwide public health concern; therefore, there is a growing demand for more rapid, reliable, and economical analytical methods for the detection of this toxin. In this study, anti-okadaic acid single-chain variable fragment (scFv) genes were prepared by cloning heavy and light chain genes from hybridoma cells, followed by fusion of the chains via a linker peptide. An scFv-pLIP6/GN recombinant plasmid was constructed and transformed into Escherichia coli for expression, and the target scFv was identified with IC-CLEIA (chemiluminescent enzyme immunoassay). The IC15 was 0.012 ± 0.02 μg/L, and the IC50 was 0.25 ± 0.03 μg/L. The three-dimensional structure of the scFv was simulated with computer modeling, and okadaic acid was docked to the scFv model to obtain a putative structure of the binding complex. Two predicted critical amino acids, Ser32 and Thr187, were then mutated to verify this theoretical model. Both mutants exhibited significant loss of binding activity. These results help us to understand this specific scFv-antigen binding mechanism and provide guidance for affinity maturation of the antibody in vitro. The high-affinity scFv developed here also has potential for okadaic acid toxin detection.

  1. Fatty Acids Bind Tightly to the N-terminal Domain of Angiopoietin-like Protein 4 and Modulate Its Interaction with Lipoprotein Lipase*

    Science.gov (United States)

    Robal, Terje; Larsson, Mikael; Martin, Miina; Olivecrona, Gunilla; Lookene, Aivar

    2012-01-01

    Angiopoietin-like protein 4 (Angptl4), a potent regulator of plasma triglyceride metabolism, binds to lipoprotein lipase (LPL) through its N-terminal coiled-coil domain (ccd-Angptl4) inducing dissociation of the dimeric enzyme to inactive monomers. In this study, we demonstrate that fatty acids reduce the inactivation of LPL by Angptl4. This was the case both with ccd-Angptl4 and full-length Angptl4, and the effect was seen in human plasma or in the presence of albumin. The effect decreased in the sequence oleic acid > palmitic acid > myristic acid > linoleic acid > linolenic acid. Surface plasmon resonance, isothermal titration calorimetry, fluorescence, and chromatography measurements revealed that fatty acids bind with high affinity to ccd-Angptl4. The interactions were characterized by fast association and slow dissociation rates, indicating formation of stable complexes. The highest affinity for ccd-Angptl4 was detected for oleic acid with a subnanomolar equilibrium dissociation constant (Kd). The Kd values for palmitic and myristic acid were in the nanomolar range. Linoleic and linolenic acid bound with much lower affinity. On binding of fatty acids, ccd-Angptl4 underwent conformational changes resulting in a decreased helical content, weakened structural stability, dissociation of oligomers, and altered fluorescence properties of the Trp-38 residue that is located close to the putative LPL-binding region. Based on these results, we propose that fatty acids play an important role in modulating the effects of Angptl4. PMID:22773878

  2. iDNA-Prot|dis: identifying DNA-binding proteins by incorporating amino acid distance-pairs and reduced alphabet profile into the general pseudo amino acid composition.

    Directory of Open Access Journals (Sweden)

    Bin Liu

    Full Text Available Playing crucial roles in various cellular processes, such as recognition of specific nucleotide sequences, regulation of transcription, and regulation of gene expression, DNA-binding proteins are essential ingredients for both eukaryotic and prokaryotic proteomes. With the avalanche of protein sequences generated in the postgenomic age, it is a critical challenge to develop automated methods for accurate and rapidly identifying DNA-binding proteins based on their sequence information alone. Here, a novel predictor, called "iDNA-Prot|dis", was established by incorporating the amino acid distance-pair coupling information and the amino acid reduced alphabet profile into the general pseudo amino acid composition (PseAAC vector. The former can capture the characteristics of DNA-binding proteins so as to enhance its prediction quality, while the latter can reduce the dimension of PseAAC vector so as to speed up its prediction process. It was observed by the rigorous jackknife and independent dataset tests that the new predictor outperformed the existing predictors for the same purpose. As a user-friendly web-server, iDNA-Prot|dis is accessible to the public at http://bioinformatics.hitsz.edu.cn/iDNA-Prot_dis/. Moreover, for the convenience of the vast majority of experimental scientists, a step-by-step protocol guide is provided on how to use the web-server to get their desired results without the need to follow the complicated mathematic equations that are presented in this paper just for the integrity of its developing process. It is anticipated that the iDNA-Prot|dis predictor may become a useful high throughput tool for large-scale analysis of DNA-binding proteins, or at the very least, play a complementary role to the existing predictors in this regard.

  3. Urinary excretion of fatty acid-binding protein 4 is associated with albuminuria and renal dysfunction.

    Directory of Open Access Journals (Sweden)

    Yusuke Okazaki

    Full Text Available Fatty acid-binding protein 4 (FABP4/A-FABP/aP2 is expressed in not only adipocytes and macrophages but also peritubular capillaries in the normal kidney. We recently demonstrated that ectopic expression of FABP4, but not FABP1 known as liver FABP (L-FABP, in the glomerulus is associated with progression of proteinuria and renal dysfunction. However, urinary excretion of FABP4 has not been investigated.Subjects who participated in the Tanno-Sobetsu Study, a study with a population-based cohort design, in 2011 (n = 392, male/female: 166/226 were enrolled. Urinary FABP4 (U-FABP4 and urinary albumin-to-creatinine ratio (UACR were measured. Change in estimated glomerular filtration rate (eGFR was followed up one year later.In 93 (23.7% of the 392 subjects, U-FABP4 level was below the sensitivity of the assay. Subjects with undetectable U-FABP4 were younger and had lower UACR and higher eGFR levels than subjects with measurable U-FABP4. U-FABP4 level was positively correlated with age, systolic blood pressure and levels of serum FABP4 (S-FABP4, triglycerides, hemoglobin A1c (HbA1c, urinary FABP1 (U-FABP1 and UACR (r = 0.360, p<0.001. Age, S-FABP4, U-FABP1 and UACR were independent predictors of U-FABP4. On the other hand, systolic blood pressure, HbA1c and U-FABP4 were independently correlated with UACR. Reduction in eGFR after one year was significantly larger in a group with the highest tertile of baseline U-FABP4 than a group with the lowest tertile.Urinary FABP4 level is independently correlated with level of albuminuria and possibly predicts yearly decline of eGFR. U-FABP4 would be a novel biomarker of glomerular damage.

  4. Sterol regulation of human fatty acid synthase promoter I requires nuclear factor-Y- and Sp-1-binding sites.

    Science.gov (United States)

    Xiong, S; Chirala, S S; Wakil, S J

    2000-04-11

    To understand cholesterol-mediated regulation of human fatty acid synthase promoter I, we tested various 5'-deletion constructs of promoter I-luciferase reporter gene constructs in HepG2 cells. The reporter gene constructs that contained only the Sp-1-binding site (nucleotides -82 to -74) and the two tandem sterol regulatory elements (SREs; nucleotides -63 to -46) did not respond to cholesterol. Only the reporter gene constructs containing a nuclear factor-Y (NF-Y) sequence, the CCAAT sequence (nucleotides -90 to -86), an Sp-1 sequence, and the two tandem SREs responded to cholesterol. The NF-Y-binding site, therefore, is essential for cholesterol response. Mutating the SREs or the NF-Y site and inserting 4 bp between the Sp-1- and NF-Y-binding sites both resulted in a minimal cholesterol response of the reporter genes. Electrophoretic mobility-shift assays using anti-SRE-binding protein (SREBP) and anti-NF-Ya antibodies confirmed that these SREs and the NF-Y site bind the respective factors. We also identified a second Sp-1 site located between nucleotides -40 and -30 that can substitute for the mutated Sp-1 site located between nucleotides -82 and -74. The reporter gene expression of the wild-type promoter and the Sp-1 site (nucleotides -82 to -74) mutant promoter was similar when SREBP1a [the N-terminal domain of SREBP (amino acids 1-520)] was constitutively overexpressed, suggesting that Sp-1 recruits SREBP to the SREs. Under the same conditions, an NF-Y site mutation resulted in significant loss of reporter gene expression, suggesting that NF-Y is required to activate the cholesterol response.

  5. Binding of carboxylate and trimethylammonium salts to octa-acid and TEMOA deep-cavity cavitands

    Science.gov (United States)

    Sullivan, Matthew R.; Sokkalingam, Punidha; Nguyen, Thong; Donahue, James P.; Gibb, Bruce C.

    2017-01-01

    In participation of the fifth statistical assessment of modeling of proteins and ligands (SAMPL5), the strength of association of six guests ( 3- 8) to two hosts ( 1 and 2) were measured by 1H NMR and ITC. Each host possessed a unique and well-defined binding pocket, whilst the wide array of amphiphilic guests possessed binding moieties that included: a terminal alkyne, nitro-arene, alkyl halide and cyano-arene groups. Solubilizing head groups for the guests included both positively charged trimethylammonium and negatively charged carboxylate functionality. Measured association constants ( K a ) covered five orders of magnitude, ranging from 56 M-1 for guest 6 binding with host 2 up to 7.43 × 106 M-1 for guest 6 binding to host 1.

  6. Enhanced Binding Affinity for an i-Motif DNA Substrate Exhibited by a Protein Containing Nucleobase Amino Acids.

    Science.gov (United States)

    Bai, Xiaoguang; Talukder, Poulami; Daskalova, Sasha M; Roy, Basab; Chen, Shengxi; Li, Zhongxian; Dedkova, Larisa M; Hecht, Sidney M

    2017-03-17

    Several variants of a nucleic acid binding motif (RRM1) of putative transcription factor hnRNP LL containing nucleobase amino acids at specific positions have been prepared and used to study binding affinity for the BCL2 i-motif DNA. Molecular modeling suggested a number of amino acids in RRM1 likely to be involved in interaction with the i-motif DNA, and His24 and Arg26 were chosen for modification based on their potential ability to interact with G14 of the i-motif DNA. Four nucleobase amino acids were introduced into RRM1 at one or both of positions 24 and 26. The introduction of cytosine nucleobase 2 into position 24 of RRM1 increased the affinity of the modified protein for the i-motif DNA, consistent with the possible Watson-Crick interaction of 2 and G14. In comparison, the introduction of uracil nucleobase 3 had a minimal effect on DNA affinity. Two structurally simplified nucleobase analogues (1 and 4) lacking both the N-1 and the 2-oxo substituents were also introduced in lieu of His24. Again, the RRM1 analogue containing 1 exhibited enhanced affinity for the i-motif DNA, while the protein analogue containing 4 bound less tightly to the DNA substrate. Finally, the modified protein containing 1 in lieu of Arg26 also bound to the i-motif DNA more strongly than the wild-type protein, but a protein containing 1 both at positions 24 and 26 bound to the DNA less strongly than wild type. The results support the idea of using nucleobase amino acids as protein constituents for controlling and enhancing DNA-protein interaction. Finally, modification of the i-motif DNA at G14 diminished RRM1-DNA interaction, as well as the ability of nucleobase amino acid 1 to stabilize RRM1-DNA interaction.

  7. The Src homology 3 binding domain is required for lysophosphatidic acid 3 receptor-mediated cellular viability in melanoma cells.

    Science.gov (United States)

    Jia, Wei; Tran, Sterling K; Ruddick, Caitlin A; Murph, Mandi M

    2015-01-28

    The LPA3 receptor is a G protein-coupled receptor that binds extracellular lysophosphatidic acid and mediates intracellular signaling cascades. Although we previously reported that receptor inhibition using siRNA or chemical inhibition obliterates the viability of melanoma cells, the mechanism was unclear. Herein we hypothesized that amino acids comprising the Src homology 3 (SH3) ligand binding motif, R/K-X-X-V/P-X-X-P or (216)-KTNVLSP-(222), within the third intracellular loop of LPA3 were critical in mediating this outcome. Therefore, we performed site-directed mutagenesis of the lysine, valine and proline, replacing these amino acids with alanines, and evaluated the changes in viability, proliferation, ERK1/2 signaling and calcium in response to lysophosphatidic acid. Our results show that enforced LPA3 expression in SK-MEL-2 cells enhanced their resiliency by allowing these cells to oppose any loss of viability during growth in serum-free medium for up to 96 h, in contrast to parental SK-MEL-2 cells, which show a significant decline in viability. Similarly, site-directed alanine substitutions of valine and proline, V219A/P222A or 2aa-SK-MEL-2 cells, did not significantly alter viability, but adding a further alanine to replace the lysine, K216A/V219A/P222A or 3aa-SK-MEL-2 cells, obliterated this function. In addition, an inhibitor of the LPA3 receptor had no impact on the parental SK-MEL-2, 2aa-SK-MEL-2 or 3aa-SK-MEL-2 cells, but significantly reduced viability among wt-LPA3-SK-MEL-2 cells. Taken together, the data suggest that the SH3 ligand binding domain of LPA3 is required to mediate viability in melanoma cells.

  8. Pharmacology of morphine and morphine-3-glucuronide at opioid, excitatory amino acid, GABA and glycine binding sites

    Energy Technology Data Exchange (ETDEWEB)

    Bartlett, S.E.; Smith, M.T. (Department of Pharmacy, The University of Queensland (Australia)); Dood, P.R. (Clinical Research Centre, Royal Brisbane Hospital Foundation, Brisbane (Australia))

    1994-07-01

    Morphine in high doses and its major metabolite, morphine-3-glucuronide, cause CNS excitation following intrathecal and intracerebroventricular administration by an unknown mechanism. This study investigated whether morphine and morphine-3-glucuronide interact at major excitatory (glutamate), major inhibitory (GABA or glycine), or opioid binding sites. Homogenate binding assays were performed using specific radioligands. At opioid receptors, morphine-3-glucuronide and morphine caused an equipotent sodium shift, consistent with morphine-3-glucuronide behaving as an agonist. This suggests that morphine-3-glucuronide-mediated excitation is not caused by an interaction at opioid receptors. Morphine-3-glucuronide and morphine caused a weak inhibition of the binding of [sup 3]H-MK801 (non-competitive antagonist) and [sup 125]I-ifenprodil (polyamine site antagonist), but at unphysiologically high concentrations. This suggests that CNS excitation would not result from an interaction of morphine-3-glucuronide and high-dose morphine with these sites on the NMDA receptor. Morphine-3-glucuronide and morphine inhibited the binding of [sup 3]H-muscimol (GABA receptor agonist), [sup 3]H-diazepam and [sup 3]H-flunitraxepam (benzodiazepine agonists) binding very weakly, suggesting the excitatory effects of morphine-3-glucuronide and high-dose morphine are not elicited through GABA[sub A] receptors. Morphine-3-glucuronide and high-dose morphine did not prevent re-uptake of glutamate into presynaptic nerve terminals. In addition, morphine-3-glucuronide and morphine did not inhibit the binding of [sup 3]H-strychnine (glycine receptor antagonist) to synaptic membranes prepared from bovine spinal cord. It is concluded that excitation caused by high-dose morphine and morphine-3-glucuronide is not mediated by an interaction with postsynaptic amino acid receptors. (au) (30 refs.).

  9. Atomic model of human Rcd-1 reveals an armadillo-like-repeat protein with in vitro nucleic acid binding properties.

    Science.gov (United States)

    Garces, Robert G; Gillon, Wanda; Pai, Emil F

    2007-02-01

    Rcd-1, a protein highly conserved across eukaryotes, was initially identified as a factor essential for nitrogen starvation-invoked differentiation in fission yeast, and its Saccharomyces cerevisiae homolog, CAF40, has been identified as part of the CCR4-NOT transcription complex, where it interacts with the NOT1 protein. Mammalian homologs are involved in various cellular differentiation processes including retinoic acid-induced differentiation and hematopoetic cell development. Here, we present the 2.2 A X-ray structure of the highly conserved region of human Rcd-1 and investigate possible functional abilities of this and the full-length protein. The monomer is made up of six armadillo repeats forming a solvent-accessible, positively-charged cleft 21-22 A wide that, in contrast to other armadillo proteins, stays fully exposed in the dimer. Prompted by this finding, we established that Rcd-1 can bind to single- and double-stranded oligonucleotides in vitro with the affinity of G/C/T > A. Mutation of an arginine residue within the cleft strongly reduced or abolished oligonucleotide binding. Rcd-1's ability to bind to nucleic acids, in addition to the previously reported protein-protein interaction with NOT1, suggests a new feature in Rcd-1's role in regulation of overall cellular differentiation processes.

  10. Impact of size, secondary structure, and counterions on the binding of small ribonucleic acids to layered double hydroxide nanoparticles

    Science.gov (United States)

    Rodriguez, Blanca V.; Pescador, Jorge; Pollok, Nicole; Beall, Gary W.; Maeder, Corina; Lewis, L. Kevin

    2015-01-01

    Use of ribonucleic acid (RNA) interference to regulate protein expression has become an important research topic and gene therapy tool, and therefore, finding suitable vehicles for delivery of small RNAs into cells is of crucial importance. Layered double metal hydroxides such as hydrotalcite (HT) have shown great promise as nonviral vectors for transport of deoxyribose nucleic acid (DNA), proteins, and drugs into cells, but the adsorption of RNAs to these materials has been little explored. In this study, the binding of small RNAs with different lengths and levels of secondary structure to HT nanoparticles has been analyzed and compared to results obtained with small DNAs in concurrent experiments. Initial experiments established the spectrophotometric properties of HT in aqueous solutions and determined that HT particles could be readily sedimented with near 100% efficiencies. Use of RNA+HT cosedimentation experiments as well as electrophoretic mobility shift assays demonstrated strong adsorption of RNA 25mers to HT, with twofold greater binding of single-stranded RNAs relative to double-stranded molecules. Strong affinities were also observed with ssRNA and dsRNA 54mers and with more complex transfer RNA molecules. Competition binding and RNA displacement experiments indicated that RNA-HT associations were strong and were only modestly affected by the presence of high concentrations of inorganic anions. PMID:26620852

  11. Impact of size, secondary structure, and counterions on the binding of small ribonucleic acids to layered double hydroxide nanoparticles.

    Science.gov (United States)

    Rodriguez, Blanca V; Pescador, Jorge; Pollok, Nicole; Beall, Gary W; Maeder, Corina; Lewis, L Kevin

    2015-12-30

    Use of ribonucleic acid (RNA) interference to regulate protein expression has become an important research topic and gene therapy tool, and therefore, finding suitable vehicles for delivery of small RNAs into cells is of crucial importance. Layered double metal hydroxides such as hydrotalcite (HT) have shown great promise as nonviral vectors for transport of deoxyribose nucleic acid (DNA), proteins, and drugs into cells, but the adsorption of RNAs to these materials has been little explored. In this study, the binding of small RNAs with different lengths and levels of secondary structure to HT nanoparticles has been analyzed and compared to results obtained with small DNAs in concurrent experiments. Initial experiments established the spectrophotometric properties of HT in aqueous solutions and determined that HT particles could be readily sedimented with near 100% efficiencies. Use of RNA+HT cosedimentation experiments as well as electrophoretic mobility shift assays demonstrated strong adsorption of RNA 25mers to HT, with twofold greater binding of single-stranded RNAs relative to double-stranded molecules. Strong affinities were also observed with ssRNA and dsRNA 54mers and with more complex transfer RNA molecules. Competition binding and RNA displacement experiments indicated that RNA-HT associations were strong and were only modestly affected by the presence of high concentrations of inorganic anions.

  12. Iron Oxide Surface Chemistry: The Effect of Chemical Structure on Binding in Benzoic Acid and Catechol Derivatives.

    Science.gov (United States)

    Korpany, Katalin V; Majewski, Dorothy D; Chiu, Cindy T; Cross, Shoronia N; Blum, Amy Szuchmacher

    2017-02-18

    Excellent performance of functionalized iron oxide nanoparticles in nanomaterial and biomedical applications often relies on achieving attachment of ligands to the iron oxide surface both in sufficient number and with proper orientation. Towards this end, we determine relationships between ligand chemical structure and surface binding on magnetic iron oxide nanoparticles for a series of related benzoic acid and catechol derivatives. Ligand exchange was used to introduce the model ligands, and the resulting nanoparticles were characterized by FTIR-ATR, transmission electron microscopy (TEM), 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 iron oxide nanoparticles. The structure of the resulting ligand-surface complex was primarily influenced by the relative positioning of hydroxyl and carboxylic acid groups within the ligand as well as 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 towards realizing truly custom-built iron oxide nanoparticles.

  13. Comparative study of the fatty acid binding process of a new FABP from Cherax quadricarinatus by fluorescence intensity, lifetime and anisotropy.

    Directory of Open Access Journals (Sweden)

    Jiayao Li

    Full Text Available Fatty acid-binding proteins (FABPs are small cytosolic proteins, largely distributed in invertebrates and vertebrates, which accomplish uptake and intracellular transport of hydrophobic ligands such as fatty acids. Although long chain fatty acids play multiple crucial roles in cellular functions (structural, energy metabolism, regulation of gene expression, the precise functions of FABPs, especially those of invertebrate species, remain elusive. Here, we have identified and characterized a novel FABP family member, Cq-FABP, from the hepatopancreas of red claw crayfish Cherax quadricarinatus. We report the characterization of fatty acid-binding affinity of Cq-FABP by four different competitive fluorescence-based assays. In the two first approaches, the fluorescent probe 8-Anilino-1-naphthalenesulfonate (ANS, a binder of internal cavities of protein, was used either by directly monitoring its fluorescence emission or by monitoring the fluorescence resonance energy transfer occurring between the single tryptophan residue of Cq-FABP and ANS. The third and the fourth approaches were based on the measurement of the fluorescence emission intensity of the naturally fluorescent cis-parinaric acid probe or the steady-state fluorescence anisotropy measurements of a fluorescently labeled fatty acid (BODIPY-C16, respectively. The four methodologies displayed consistent equilibrium constants for a given fatty acid but were not equivalent in terms of analysis. Indeed, the two first methods were complicated by the existence of non specific binding modes of ANS while BODIPY-C16 and cis-parinaric acid specifically targeted the fatty acid binding site. We found a relationship between the affinity and the length of the carbon chain, with the highest affinity obtained for the shortest fatty acid, suggesting that steric effects primarily influence the interaction of fatty acids in the binding cavity of Cq-FABP. Moreover, our results show that the binding affinities

  14. High-throughput 1,536-well fluorescence polarization assays for α(1-acid glycoprotein and human serum albumin binding.

    Directory of Open Access Journals (Sweden)

    Adam Yasgar

    Full Text Available Two major plasma proteins in humans are primarily responsible for drug binding, the α(1-acid-glycoprotein (AGP and human serum albumin (HSA. The availability of at least a semiquantitative high-throughput assay for assessment of protein binding is expected to aid in bridging the current gap between high-throughput screening and early lead discovery, where cell-based and biochemical assays are deployed routinely to test up to several million compounds rapidly, as opposed to the late-stage candidate drug profiling methods which test at most dozens of compounds at a time. Here, we describe the miniaturization of a pair of assays based on the binding- and displacement-induced changes in fluorescence polarization (FP of fluorescent small molecule probes known to specifically target the drug-binding sites of these two proteins. A robust and reproducible assay performance was achieved in ≤4 µL assay volume in 1,536-well format. The assays were tested against a validation set of 10 known protein binders, and the results compared favorably with data obtained using protein-coated beads with high-performance liquid chromatography analysis. The miniaturized assays were taken to a high-throughput level in a screen of the LOPAC(1280 collection of 1,280 pharmacologically active compounds. The adaptation of the AGP and HSA FP assays to a 1,536-well format should allow their use in early-stage profiling of large-size compound sets.

  15. A novel injection strategy of flurbiprofen axetil by inhibiting protein binding with 6-methoxy-2-naphthylacetic acid.

    Science.gov (United States)

    Ogata, Kenji; Takamura, Norito; Tokunaga, Jin; Ikeda, Tetsuya; Setoguchi, Nao; Tanda, Kazuhiro; Yamasaki, Tetsuo; Nishio, Toyotaka; Kawai, Keiichi

    2016-04-01

    Flurbiprofen axetil (FPA) is an injection product and a prodrug of a non-steroidal anti-inflammatory drug (NSAID). After injection, it is rapidly hydrolyzed to the active form, flurbiprofen (FP). Since frequent injections of FPA can lead to abnormal physiology, an administration strategy is necessary to ensure there is enhancement of the analgesic efficiency of FP after a single dose and to reduce the total number of doses. FP strongly binds to site II of albumin, and thus the free (unbound) FP concentration is low. This study focused on 6-methoxy-2-naphthylacetic acid (6-MNA), the active metabolite of nabumetone (a prodrug of NSAID). We performed ultrafiltration experiments and pharmacokinetics analysis in rats to investigate whether the inhibitory effect of 6-MNA on FP binding to albumin increased the free FP concentration in vitro and in vivo. Results indicated that 6-MNA inhibited the binding of FP to albumin competitively. When 6-MNA was injected in rats, there was a significant increase in the free FP concentration and the area under concentration-time curve (AUC) calculated from the free FP concentration, while there was a significant decrease in the total (bound + free) FP concentration and the AUC calculated from the total FP concentration. These findings indicate that 6-MNA inhibits the protein binding of FP in vivo. This suggests that the frequency of FPA injections can be reduced when administered with nabumetone, as there is increase in the free FP concentration associated with pharmacological effect.

  16. Kinase Associated-1 Domains Drive MARK/PAR1 Kinases to Membrane Targets by Binding Acidic Phospholipids

    Energy Technology Data Exchange (ETDEWEB)

    Moravcevic, Katarina; Mendrola, Jeannine M.; Schmitz, Karl R.; Wang, Yu-Hsiu; Slochower, David; Janmey, Paul A.; Lemmon, Mark A. (UPENN-MED)

    2011-09-28

    Phospholipid-binding modules such as PH, C1, and C2 domains play crucial roles in location-dependent regulation of many protein kinases. Here, we identify the KA1 domain (kinase associated-1 domain), found at the C terminus of yeast septin-associated kinases (Kcc4p, Gin4p, and Hsl1p) and human MARK/PAR1 kinases, as a membrane association domain that binds acidic phospholipids. Membrane localization of isolated KA1 domains depends on phosphatidylserine. Using X-ray crystallography, we identified a structurally conserved binding site for anionic phospholipids in KA1 domains from Kcc4p and MARK1. Mutating this site impairs membrane association of both KA1 domains and intact proteins and reveals the importance of phosphatidylserine for bud neck localization of yeast Kcc4p. Our data suggest that KA1 domains contribute to coincidence detection, allowing kinases to bind other regulators (such as septins) only at the membrane surface. These findings have important implications for understanding MARK/PAR1 kinases, which are implicated in Alzheimer's disease, cancer, and autism.

  17. Exploring DNA binding and nucleolytic activity of few 4-aminoantipyrine based amino acid Schiff base complexes: A comparative approach

    Science.gov (United States)

    Raman, N.; Sakthivel, A.; Pravin, N.

    A series of novel Co(II), Cu(II), Ni(II) and Zn(II) complexes were synthesized from Schiff base(s), obtained by the condensation of 4-aminoantipyrine with furfural and amino acid (glycine(L1)/alanine(L2)/valine(L3)) and respective metal(II) chloride. Their structural features and other properties were explored from the analytical and spectral methods. The binding behaviors of the complexes to calf thymus DNA were investigated by absorption spectra, viscosity measurements and cyclic voltammetry. The intrinsic binding constants for the above synthesized complexes are found to be in the order of 102 to 105 indicating that most of the synthesized complexes are good intercalators. The binding constant values (Kb) clearly indicate that valine Schiff-base complexes have more intercalating ability than alanine and glycine Schiff-base complexes. The results indicate that the complexes bind to DNA through intercalation and act as efficient cleaving agents. The in vitro antibacterial and antifungal assay indicates that these complexes are good antimicrobial agents against various pathogens. The IC50 values of [Ni(L1)2] and [Zn(L1)2] complexes imply that these complexes have preferable ability to scavenge hydroxyl radical.

  18. Exploring DNA binding and nucleolytic activity of few 4-aminoantipyrine based amino acid Schiff base complexes: a comparative approach.

    Science.gov (United States)

    Raman, N; Sakthivel, A; Pravin, N

    2014-05-05

    A series of novel Co(II), Cu(II), Ni(II) and Zn(II) complexes were synthesized from Schiff base(s), obtained by the condensation of 4-aminoantipyrine with furfural and amino acid (glycine(L1)/alanine(L2)/valine(L3)) and respective metal(II) chloride. Their structural features and other properties were explored from the analytical and spectral methods. The binding behaviors of the complexes to calf thymus DNA were investigated by absorption spectra, viscosity measurements and cyclic voltammetry. The intrinsic binding constants for the above synthesized complexes are found to be in the order of 10(2) to 10(5) indicating that most of the synthesized complexes are good intercalators. The binding constant values (Kb) clearly indicate that valine Schiff-base complexes have more intercalating ability than alanine and glycine Schiff-base complexes. The results indicate that the complexes bind to DNA through intercalation and act as efficient cleaving agents. The in vitro antibacterial and antifungal assay indicates that these complexes are good antimicrobial agents against various pathogens. The IC50 values of [Ni(L1)2] and [Zn(L1)2] complexes imply that these complexes have preferable ability to scavenge hydroxyl radical.

  19. Development of a Novel Nonradiometric Assay for Nucleic Acid Binding to TDP-43 Suitable for High-Throughput Screening Using AlphaScreen® Technology

    OpenAIRE

    Cassel, Joel A.; Blass, Benjamin E.; Reitz, Allen B.; Pawlyk, Aaron C.

    2010-01-01

    TAR DNA binding protein 43 (TDP-43) is a nucleic acid binding protein that is associated with the pathology of cystic fibrosis and neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar dementia. We have developed a robust, quantitative, nonradiometric high-throughput assay measuring oligonucleotide binding to TDP-43 using AlphaScreen® technology. Biotinylated single-stranded TAR DNA (bt-TAR-32) and 6 TG repeats (bt-TG6) bound with high affinity to TDP-43, w...

  20. DNA binding protein identification by combining pseudo amino acid composition and profile-based protein representation

    Science.gov (United States)

    Liu, Bin; Wang, Shanyi; Wang, Xiaolong

    2015-10-01

    DNA-binding proteins play an important role in most cellular processes. Therefore, it is necessary to develop an efficient predictor for identifying DNA-binding proteins only based on the sequence information of proteins. The bottleneck for constructing a useful predictor is to find suitable features capturing the characteristics of DNA binding proteins. We applied PseAAC to DNA binding protein identification, and PseAAC was further improved by incorporating the evolutionary information by using profile-based protein representation. Finally, Combined with Support Vector Machines (SVMs), a predictor called iDNAPro-PseAAC was proposed. Experimental results on an updated benchmark dataset showed that iDNAPro-PseAAC outperformed some state-of-the-art approaches, and it can achieve stable performance on an independent dataset. By using an ensemble learning approach to incorporate more negative samples (non-DNA binding proteins) in the training process, the performance of iDNAPro-PseAAC was further improved. The web server of iDNAPro-PseAAC is available at http://bioinformatics.hitsz.edu.cn/iDNAPro-PseAAC/.

  1. Identification and functional characterization of the Arabidopsis Snf1-related protein kinase SnRK2.4 phosphatidic acid-binding domain

    NARCIS (Netherlands)

    Julkowska, M.M.; McLoughlin, F.; Galvan-Ampudia, C.S.; Rankenberg, J.M.; Kawa, D.; Klimecka, M.; Haring, M.A.; Munnik, T.; Kooijman, E.E.; Testerink, C.

    2015-01-01

    Phosphatidic acid (PA) is an important signalling lipid involved in various stress-induced signalling cascades. Two SnRK2 protein kinases (SnRK2.4 and SnRK2.10), previously identified as PA-binding proteins, are shown here to prefer binding to PA over other anionic phospholipids and to associate wit

  2. Prediction of the Occurrence of the ADP-binding βαβ-fold in Proteins, Using an Amino Acid Sequence Fingerprint

    NARCIS (Netherlands)

    Wierenga, Rik K.; Terpstra, Peter; Hol, Wim G.J.

    1986-01-01

    An amino acid sequence "fingerprint” has been derived that can be used to test if a particular sequence will fold into a βαβ-unit with ADP-binding properties. It was deduced from a careful analysis of the known three-dimensional structures of ADP-binding βαβ-folds. This fingerprint is in fact a set

  3. Effect of soil fulvic and humic acid on binding of Pb to goethite–water interface: Linear additivity and volume fractions of HS in the Stern layer

    NARCIS (Netherlands)

    Xiong, J.; Koopal, L.K.; Weng, L.; Wang, J.; Tan, W.

    2015-01-01

    The effects of soil fulvic (JGFA) and humic acid (JGHA) on Pb binding to goethite were investigated with batch experiments and modeling. The CD-MUSIC and NICA-Donnan model could describe the Pb binding to, respectively, the binary Pb–goethite and Pb–HS systems. The adsorption of humic substances (HS

  4. Mutations at positions 547-553 of rat glucocorticoid receptors reveal that hsp90 binding requires the presence, but not defined composition, of a seven-amino acid sequence at the amino terminus of the ligand binding domain.

    Science.gov (United States)

    Kaul, Sunil; Murphy, Patrick J M; Chen, Jun; Brown, Lloyd; Pratt, William B; Simons, S Stoney

    2002-09-27

    Glucocorticoid receptors (GRs) must heterocomplex with hsp90 to have an open steroid binding cleft that can be accessed by steroid. We reported that a seven-amino acid sequence (547-553 of rat GR) overlapping the amino-terminal end of the ligand binding domain is required for hsp90 binding to GR. We have now conducted saturation mutagenesis of this sequence, which appears to be part of the surface where the ligand binding cleft merges with the surface of the ligand binding domain. No single point mutation causes significant changes in any of a variety of biochemical and biological properties in addition to hsp90 binding. A triple mutation (P548A/T549A/V551A) increases by >100-fold the steroid concentration required for half-maximal induction without affecting the level of maximal induction or coactivator response. Interestingly, this triple mutant displays reduced binding of steroid and hsp90 in whole cells, but it possesses wild type affinity for steroid and normal hsp90 binding capacity under cell-free conditions. This phenotype of a dramatic shift in the dose response for transactivation would be expected from an increase in the rate of disassembly of the triple mutant GR.hsp90 heterocomplex in the cell. Mutation of the entire seven-amino acid region to CAAAAAC maintains the presence of a critical alpha-helical structure and heterocomplex formation with hsp90 but eliminates steroid binding and transcriptional activation, thus disconnecting hsp90 binding from opening of the ligand binding cleft and steroid binding.

  5. Preferential Lectin Binding of Cancer Cells upon Sialic Acid Treatment under Nutrient Deprivation

    OpenAIRE

    Badr, Haitham A.; ElSayed, Abdelaleim I.; Ahmed, Hafiz; Dwek, Miriam V.; Li, Chen-Zhong; Djansugurova, Leyla B.

    2013-01-01

    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 ...

  6. Verification of a novel NADH-binding motif: combinatorial mutagenesis of three amino acids in the cofactor-binding pocket of Corynebacterium 2,5-diketo-D-gluconic acid reductase.

    Science.gov (United States)

    Banta, Scott; Anderson, Stephen

    2002-12-01

    A screening method has been developed to support randomized mutagenesis of amino acids in the cofactor-binding pocket of the NADPH-dependent 2,5-diketo-D-gluconic acid (2,5-DKG) reductase. Such an approach could enable the isolation of an enzyme that can better catalyze the reduction of 2,5-DKG to 2-keto-L-gulonic acid (2-KLG) using NADH as a cofactor. 2-KLG is a valuable precursor to ascorbic acid, or vitamin C, and an enzyme with increased activity with NADH may be able to improve two potential vitamin C production processes. Previously we have identified three amino acid residues that can be mutated to improve activity with NADH as a cofactor. As a pilot study to show feasibility, a library was made with these three amino acids randomized, and 300 random colonies were screened for increased NADH activity. The activities of seven mutants with apparent improvements were verified using activity-stained native gels, and sequencing showed that the amino acids obtained were similar to some of those already discovered using rational design. The four most active mutants were purified and kinetically characterized. All of the new mutations resulted in apparent kcat values that were equal to or higher than that of the best mutant obtained through rational design. At saturating levels of cofactor, the best mutant obtained was almost twice as active with NADH as a cofactor as the wild-type enzyme is with NADPH. This screen is a valuable tool for improving 2,5-DKG reductase, and it could easily be modified for improving other aspects of this protein or similar enzymes.

  7. Nuclear magnetic resonance structure of the nucleic acid-binding domain of severe acute respiratory syndrome coronavirus nonstructural protein 3.

    Science.gov (United States)

    Serrano, Pedro; Johnson, Margaret A; Chatterjee, Amarnath; Neuman, Benjamin W; Joseph, Jeremiah S; Buchmeier, Michael J; Kuhn, Peter; Wüthrich, Kurt

    2009-12-01

    The nuclear magnetic resonance (NMR) structure of a globular domain of residues 1071 to 1178 within the previously annotated nucleic acid-binding region (NAB) of severe acute respiratory syndrome coronavirus nonstructural protein 3 (nsp3) has been determined, and N- and C-terminally adjoining polypeptide segments of 37 and 25 residues, respectively, have been shown to form flexibly extended linkers to the preceding globular domain and to the following, as yet uncharacterized domain. This extension of the structural coverage of nsp3 was obtained from NMR studies with an nsp3 construct comprising residues 1066 to 1181 [nsp3(1066-1181)] and the constructs nsp3(1066-1203) and nsp3(1035-1181). A search of the protein structure database indicates that the globular domain of the NAB represents a new fold, with a parallel four-strand beta-sheet holding two alpha-helices of three and four turns that are oriented antiparallel to the beta-strands. Two antiparallel two-strand beta-sheets and two 3(10)-helices are anchored against the surface of this barrel-like molecular core. Chemical shift changes upon the addition of single-stranded RNAs (ssRNAs) identified a group of residues that form a positively charged patch on the protein surface as the binding site responsible for the previously reported affinity for nucleic acids. This binding site is similar to the ssRNA-binding site of the sterile alpha motif domain of the Saccharomyces cerevisiae Vts1p protein, although the two proteins do not share a common globular fold.

  8. Phenylacetic acids and the structurally related non-steroidal anti-inflammatory drug diclofenac bind to specific gamma-hydroxybutyric acid sites in rat brain

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Høg, Signe; Skonberg, Christian

    2009-01-01

    with a twofold higher affinity than GHB. Measuring the affinities of structurally related NSAIDs for the [(3)H]NCS-382 site identified diclofenac, a clinically relevant NSAID (Voltaren, Diclon) of the phenylacetic acid (PAA) type, as a GHB ligand (K(i) value of 5.1 microM). Other non-NSAID PAAs also exhibited...... affinities similar to GHB. Our data raise the interesting possibility that the widely used over-the-counter drug compound, diclofenac, might affect GHB binding at relevant clinical dosages. Furthermore, the identification of PAAs as GHB ligands supplies new information about the structural preferences...

  9. A novel cold-regulated cold shock domain containing protein from scallop Chlamys farreri with nucleic acid-binding activity.

    Directory of Open Access Journals (Sweden)

    Chuanyan Yang

    Full Text Available BACKGROUND: The cold shock domain (CSD containing proteins (CSDPs are one group of the evolutionarily conserved nucleic acid-binding proteins widely distributed in bacteria, plants, animals, and involved in various cellular processes, including adaptation to low temperature, cellular growth, nutrient stress and stationary phase. METHODOLOGY: The cDNA of a novel CSDP was cloned from Zhikong scallop Chlamys farreri (designated as CfCSP by expressed sequence tag (EST analysis and rapid amplification of cDNA ends (RACE approach. The full length cDNA of CfCSP was of 1735 bp containing a 927 bp open reading frame which encoded an N-terminal CSD with conserved nucleic acids binding motif and a C-terminal domain with four Arg-Gly-Gly (RGG repeats. The CSD of CfCSP shared high homology with the CSDs from other CSDPs in vertebrate, invertebrate and bacteria. The mRNA transcripts of CfCSP were mainly detected in the tissue of adductor and also marginally detectable in gill, hepatopancreas, hemocytes, kidney, mantle and gonad of healthy scallop. The relative expression level of CfCSP was up-regulated significantly in adductor and hemocytes at 1 h and 24 h respectively after low temperature treatment (P<0.05. The recombinant CfCSP protein (rCfCSP could bind ssDNA and in vitro transcribed mRNA, but it could not bind dsDNA. BX04, a cold sensitive Escherichia coli CSP quadruple-deletion mutant, was used to examine the cold adaptation ability of CfCSP. After incubation at 17°C for 120 h, the strain of BX04 containing the vector pINIII showed growth defect and failed to form colonies, while strain containing pINIII-CSPA or pINIII-CfCSP grew vigorously, indicating that CfCSP shared a similar function with E. coli CSPs for the cold adaptation. CONCLUSIONS: These results suggest that CfCSP is a novel eukaryotic cold-regulated nucleic acid-binding protein and may function as an RNA chaperone in vivo during the cold adaptation process.

  10. The seven amino acids (547-553) of rat glucocorticoid receptor required for steroid and hsp90 binding contain a functionally independent LXXLL motif that is critical for steroid binding.

    Science.gov (United States)

    Giannoukos, G; Silverstein, A M; Pratt, W B; Simons, S S

    1999-12-17

    Hsp90 association with glucocorticoid receptors (GRs) is required for steroid binding. We recently reported that seven amino acids (547-553) overlapping the amino-terminal end of the rat GR ligand-binding domain are necessary for hsp90 binding, and consequently steroid binding. The role of a LXXLL motif at the COOH terminus of this sequence has now been analyzed by determining the properties of Leu to Ser mutations in full-length GR and glutathione S-transferase chimeras. Surprisingly, these mutations decreased steroid binding capacity without altering receptor levels, steroid binding affinity, or hsp90 binding. Single mutations in the context of the full-length receptor did not affect the transcriptional activity but the double mutant (L550S/L553S) was virtually inactive. This biological inactivity was found to be due to an increased rate of steroid dissociation from the activated mutant complex. These results, coupled with those from trypsin digestion studies, suggest a model in which the GR ligand-binding domain is viewed as having a "hinged pocket," with the hinge being in the region of the trypsin digestion site at Arg(651). The pocket would normally be kept shut via the intramolecular interactions of the LXXLL motif at amino acids 550-554 acting as a hydrophobic clasp.

  11. Rational Molecular Design of Potent PLK1 PBD Domain-binding Phosphopeptides Using Preferential Amino Acid Building Blocks.

    Science.gov (United States)

    Mao, Xin-Li; Wang, Kui-Feng; Zhu, Feng; Pan, Zhao-Hu; Wu, Guo-Min; Zhu, Hong-Yuan

    2016-08-01

    Polo-like kinase 1 (PLK1) is an important regulator in diverse aspects of the cell cycle and proliferation. The protein has a highly conserved polo-box domain (PBD) present in C-terminal noncatalytic region, which exhibits a relatively broad sequence specificity in recognizing and binding phosphorylated substrates to control substrate phosphorylation by the kinase. In order to elucidate the structural basis, thermodynamic property, and biological implication underlying PBD-substrate recognition and association, a systematic amino acid preference profile of phosphopeptide interaction with PLK1 PBD domain was established via virtual mutagenesis analysis and mutation energy calculation, from which the contribution of different amino acids at each residue position of two reference phosphopeptides to domain-peptide binding was characterized comprehensively and quantitatively. With the profile, we are able to determine the favorable, neutral, and unfavorable amino acid types for each position of PBD-binding phosphopeptides, and we also explored the molecular origin of the broad sequence specificity in PBD-substrate recognition. To practice computational findings, the profile was further employed to guide rational design of potent PBD binders; three 6-mer phosphopeptides (i.e., IQSpSPC, LQSpTPF, and LNSpTPT) were successfully developed, which can efficiently target PBD domain with high affinity (Kd = 5.7 ± 1.1, 0.75 ± 0.18, and 7.2 ± 2.6 μm, resp.) as measured by a fluorescence anisotropy assay. The complex structure of PLK1 PBD domain with a newly designed, potent phosphopeptide LQSpTPF as well as diverse noncovalent chemical forces, such as H-bonds and hydrophobic interactions at the complex interface, were examined in detail to reveal the molecular mechanism of high affinity and stability of the complex system.

  12. Hippocampal interneurons expressing glutamic acid decarboxylase and calcium-binding proteins decrease with aging in Fischer 344 rats.

    Science.gov (United States)

    Shetty, A K; Turner, D A

    1998-05-04

    Aging leads to alterations in the function and plasticity of hippocampal circuitry in addition to behavioral changes. To identify critical alterations in the substrate for inhibitory circuitry as a function of aging, we evaluated the numbers of hippocampal interneurons that were positive for glutamic acid decarboxylase and those that expressed calcium-binding proteins (parvalbumin, calbindin, and calretinin) in young adult (4-5 months old) and aged (23-25 months old) male Fischer 344 rats. Both the overall interneuron population and specific subpopulations of interneurons demonstrated a commensurate decline in numbers throughout the hippocampus with aging. Interneurons positive for glutamic acid decarboxylase were significantly depleted in the stratum radiatum of CA1, the strata oriens, radiatum and pyramidale of CA3, the dentate molecular layer, and the dentate hilus. Parvalbumin interneurons showed significant reductions in the strata oriens and pyramidale of CA1, the stratum pyramidale of CA3, and the dentate hilus. The reductions in calbindin interneurons were more pronounced than other calcium-binding protein-positive interneurons and were highly significant in the strata oriens and radiatum of both CA1 and CA3 subfields and in the dentate hilus. Calretinin interneurons were decreased significantly in the strata oriens and radiatum of CA3, in the dentate granule cell and molecular layers, and in the dentate hilus. However, the relative ratio of parvalbumin-, calbindin-, and calretinin-positive interneurons compared with glutamic acid decarboxylase-positive interneurons remained constant with aging, suggesting actual loss of interneurons expressing calcium-binding proteins with age. This loss contrasts with the reported preservation of pyramidal neurons with aging in the hippocampus. Functional decreases in inhibitory drive throughout the hippocampus may occur due to this loss, particularly alterations in the processing of feed-forward information through the

  13. Structure and function of Plasmodium falciparum malate dehydrogenase: role of critical amino acids in co-substrate binding pocket.

    Science.gov (United States)

    Pradhan, Anupam; Tripathi, Abhai K; Desai, Prashant V; Mukherjee, Prasenjit K; Avery, Mitchell A; Walker, Larry A; Tekwani, Babu L

    2009-01-01

    The malaria parasite thrives on anaerobic fermentation of glucose for energy. Earlier studies from our laboratory have demonstrated that a cytosolic malate dehydrogenase (PfMDH) with striking similarity to lactate dehydrogenase (PfLDH) might complement PfLDH function in Plasmodium falciparum. The N-terminal glycine motif, which forms a characteristic Rossman dinucleotide-binding fold in the co-substrate binding pocket, differentiates PfMDH (GlyXGlyXXGly) from other eukaryotic and prokaryotic malate dehydrogenases (GlyXXGlyXXGly). The amino acids lining the co-substrate binding pocket are completely conserved in MDHs from different species of human, primate and rodent malaria parasites. Based on this knowledge and conserved domains among prokaryotic and eukaryotic MDH, the role of critical amino acids lining the co-substrate binding pocket was analyzed in catalytic functions of PfMDH using site-directed mutagenesis. Insertion of Ala at the 9th or 10th position, which converts the N-terminal GlyXGlyXXGly motif (characteristic of malarial MDH and LDH) to GlyXXGlyXXGly (as in bacterial and eukaryotic MDH), uncoupled regulation of the enzyme through substrate inhibition. The dinucleotide fold GlyXGlyXXGly motif seems not to be responsible for the distinct affinity of PfMDH to 3-acetylpyridine-adenine dinucleotide (APAD, a synthetic analog of NAD), since Ala9 and Ala10 insertion mutants still utilized APADH. The Gln11Met mutation, which converts the signature glycine motif in PfMDH to that of PfLDH, did not change the enzyme function. However, the Gln11Gly mutant showed approximately a 5-fold increase in catalytic activity, and higher susceptibility to inhibition with gossypol. Asn119 and His174 participate in binding of both co-substrate and substrate. The Asn119Gly mutant exhibited approximately a 3-fold decrease in catalytic efficiency, while mutation of His174 to Asn or Ala resulted in an inactive enzyme. These studies provide critical insights into the co

  14. Liver fatty acid-binding protein (L-FABP) promotes cellular angiogenesis and migration in hepatocellular carcinoma

    OpenAIRE

    Ku, Chung-Yu; Liu, Yu-Huei; Lin, Hsuan-Yuan; Lu, Shao-Chun; Lin, Jung-Yaw

    2016-01-01

    Liver fatty acid-binding protein (L-FABP) is abundant in hepatocytes and known to be involved in lipid metabolism. Overexpression of L-FABP has been reported in various cancers; however, its role in hepatocellular carcinoma (HCC) remains unclear. In this study, we investigated L-FABP and its association with vascular endothelial growth factors (VEGFs) in 90 HCC patients. We found that L-FABP was highly expressed in their HCC tissues, and that this expression was positively correlated with tha...

  15. A new bile acid-derived lariat-ether: Design, synthesis and cation binding properties

    Indian Academy of Sciences (India)

    P Babu; Uday Maitra

    2003-10-01

    A new chola lariat ether (1, a 21-crown-6) was constructed from readily available precursors. The association constant of compound 1 with alkali metal picrates was measured using Cram’s extraction protocol. Evidence is presented for the involvement of the 3-methoxy group for the complexation. Energy minimised structures show that the A-ring gets slightly distorted upon metal ion binding.

  16. On-line flow displacement immunoassay for fatty acid-binding protein

    NARCIS (Netherlands)

    Kaptein, WA; Korf, J; Yang, M; Glatz, JFC; Renneberg, R

    1998-01-01

    In standard displacement flow immunoassays the analyte in the sample creates an active dissociation of labelled antigens (or antigen homologues) from an antigen binding site of an immobilized antibody, after which the labelled substance is measured downstream. Such systems have been described for mo

  17. Novel artificial metalloenzymes by in vivo incorporation of metal-binding unnatural amino acids

    NARCIS (Netherlands)

    Drienovska, Ivana; Rioz-Martinez, Ana; Draksharapu, Apparao; Roelfes, Gerard

    2015-01-01

    Artificial metalloenzymes have emerged as an attractive new approach to enantioselective catalysis. Herein, we introduce a novel strategy for preparation of artificial metalloenzymes utilizing amber stop codon suppression methodology for the in vivo incorporation of metal-binding unnatural amino aci

  18. Invariant Aspartic Acid in Muscle Nicotinic Receptor Contributes Selectively to the Kinetics of Agonist Binding

    Science.gov (United States)

    Lee, Won Yong; Sine, Steven M.

    2004-01-01

    We examined functional contributions of interdomain contacts within the nicotinic receptor ligand binding site using single channel kinetic analyses, site-directed mutagenesis, and a homology model of the major extracellular region. At the principal face of the binding site, the invariant αD89 forms a highly conserved interdomain contact near αT148, αW149, and αT150. Patch-clamp recordings show that the mutation αD89N markedly slows acetylcholine (ACh) binding to receptors in the resting closed state, but does not affect rates of channel opening and closing. Neither αT148L, αT150A, nor mutations at both positions substantially affects the kinetics of receptor activation, showing that hydroxyl side chains at these positions are not hydrogen bond donors for the strong acceptor αD89. However substituting a negative charge at αT148, but not at αT150, counteracts the effect of αD89N, demonstrating that a negative charge in the region of interdomain contact confers rapid association of ACh. Interpreted within the structural framework of ACh binding protein and a homology model of the receptor ligand binding site, these results implicate main chain amide groups in the domain harboring αW149 as principal hydrogen bond donors for αD89. The specific effect of αD89N on ACh association suggests that interdomain hydrogen bonding positions αW149 for optimal interaction with ACh. PMID:15504901

  19. High ω-3:ω-6 fatty acids ratio increases fatty acid binding protein 4 and extracellular secretory phospholipase A2IIa in human ectopic endometrial cells

    Science.gov (United States)

    Khanaki, Korosh; Sadeghi, Mohammad Reza; Akhondi, Mohammad Mehdi; Darabi, Masoud; Mehdizadeh, Amir; Shabani, Mahdi; Rahimipour, Ali; Nouri, Mohammad

    2014-01-01

    Background: Endometriosis, a common chronic inflammatory disorder, is defined by the atypical growth of endometrium- like tissue outside of the uterus. Secretory phospholipase A2 group IIa (sPLA2-IIa) and fatty acid binding protein4 (FABP4) play several important roles in the inflammatory diseases. Objective: Due to reported potential anti-inflammatory effects of ω-3 and ω-6 fatty acids, the purpose of the present study was to investigate the effects of ω-3 and ω-6 polyunsaturated fatty acids (PUFAs) on fatty acid binding protein 4 and extracellular secretory phospholipase A2IIa in cultured endometrial cells. Materials and Methods: Ectopic and eutopic endometrial tissues obtained from 15 women were snap frozen. After thawing and tissue digestion, primary mixed stromal and endometrial epithelial cell culture was performed for 8 days in culture mediums supplemented with normal and high ratios of ω-3 and ω-6 PUFA. sPLA2-IIa in the culture medium and FABP4 level was determined using enzyme immuno assay (EIA) technique. Results: Within ectopic endometrial cells group, the level of cellular FABP4 and extracellular sPLA2-IIa were remarkably increased under high ω-3 PUFA exposure compared with control condition (p=0.014 and p=0.04 respectively). Conclusion: ω-3 PUFAs may increase the level of cellular FABP4 and extracellular sPLA2-IIa in ectopic endometrial cells, since sPLAIIa and FABP4 may affect endometriosis via several mechanisms, more relevant studies are encouraged to know the potential effect of increased cellular FABP4 and extracellular sPLA2-IIa on endometriosis. PMID:25709631

  20. Crystal Structure of Okadaic Acid Binding Protein 2.1: A Sponge Protein Implicated in Cytotoxin Accumulation.

    Science.gov (United States)

    Ehara, Haruhiko; Makino, Marie; Kodama, Koichiro; Konoki, Keiichi; Ito, Takuhiro; Sekine, Shun-ichi; Fukuzawa, Seketsu; Yokoyama, Shigeyuki; Tachibana, Kazuo

    2015-07-06

    Okadaic acid (OA) is a marine polyether cytotoxin that was first isolated from the marine sponge Halichondria okadai. OA is a potent inhibitor of protein serine/threonine phosphatases (PP) 1 and 2A, and the structural basis of phosphatase inhibition has been well investigated. However, the role and mechanism of OA retention in the marine sponge have remained elusive. We have solved the crystal structure of okadaic acid binding protein 2.1 (OABP2.1) isolated from H. okadai; it has strong affinity for OA and limited sequence homology to other proteins. The structure revealed that OABP2.1 consists of two α-helical domains, with the OA molecule deeply buried inside the protein. In addition, the global fold of OABP2.1 was unexpectedly similar to that of aequorin, a jellyfish photoprotein. The presence of structural homologues suggested that, by using similar protein scaffolds, marine invertebrates have developed diverse survival systems adapted to their living environments.

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

    Directory of Open Access Journals (Sweden)

    Guy Caljon

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

  2. Evidence for differences in the binding of drugs to the two main genetic variants of human alpha 1-acid glycoprotein.

    Science.gov (United States)

    Herve, F; Gomas, E; Duche, J C; Tillement, J P

    1993-09-01

    1. Human alpha 1-acid glycoprotein (AAG), a plasma transport protein, has three main genetic variants. F1. S and A. Native commercial AAG (a mixture of almost equal proportions of these three variants) has been separated by chromatography into variants which correspond to the proteins of the two genes which code for AAG in humans: the A variant and a mixture of the F1 and S variants (60% F1 and 40% S). Their binding properties towards imipramine, warfarin and mifepristone were studied by equilibrium dialysis. 2. The F1S variant mixture strongly bound warfarin and mifepristone with an affinity of 1.89 and 2.06 x 10(6) l mol-1, respectively, but had a low affinity for imipramine. Conversely, the A variant strongly bound imipramine with an affinity of 0.98 x 10(6) l mol-1. The low degree of binding of warfarin and mifepristone to the A variant sample was explained by the presence of protein contaminants in this sample. These results indicate specific drug transport roles for each variant, with respect to its separate genetic origin. 3. Control binding experiments performed with (unfractionated) commercial AAG and with AAG isolated from individuals with either the F1/A or S/A phenotypes, agreed with these findings. The results for the binding of warfarin and mifepristone by the AAG samples were similar to those obtained with the F1S mixture: the mean high-affinity association constant of the AAG samples for each drug was of the same order as that of the F1S mixture: the decrease in the number of binding sites of the AAG samples, as compared with the F1S mixture, was explained by the smaller proportion of variants F1 and/or S in these samples. Conversely, results of the imipramine binding study with the AAG samples concurred with those for the binding of this basic drug by the A variant, with respect to the proportion of the A variant in these samples.

  3. A Peptide Mimetic of 5-Acetylneuraminic Acid-Galactose Binds with High Avidity to Siglecs and NKG2D.

    Directory of Open Access Journals (Sweden)

    Laura L Eggink

    Full Text Available We previously identified several peptide sequences that mimicked the terminal sugars of complex glycans. Using plant lectins as analogs of lectin-type cell-surface receptors, a tetravalent form of a peptide with the sequence NPSHPLSG, designated svH1C, bound with high avidity to lectins specific for glycans with terminal 5-acetylneuraminic acid (Neu5Ac-galactose (Gal/N-acetylgalactosamine (GalNAc sequences. In this report, we show by circular dichroism and NMR spectra that svH1C lacks an ordered structure and thus interacts with binding sites from a flexible conformation. The peptide binds with high avidity to several recombinant human siglec receptors that bind preferentially to Neu5Ac(α2,3Gal, Neu5Ac(α2,6GalNAc or Neu5Ac(α2,8Neu5Ac ligands. In addition, the peptide bound the receptor NKG2D, which contains a lectin-like domain that binds Neu5Ac(α2,3Gal. The peptide bound to these receptors with a KD in the range of 0.6 to 1 μM. Binding to these receptors was inhibited by the glycoprotein fetuin, which contains multiple glycans that terminate in Neu5Ac(α2,3Gal or Neu5Ac(α2,6Gal, and by sialyllactose. Binding of svH1C was not detected with CLEC9a, CLEC10a or DC-SIGN, which are lectin-type receptors specific for other sugars. Incubation of neuraminidase-treated human peripheral blood mononuclear cells with svH1C resulted in binding of the peptide to a subset of the CD14+ monocyte population. Tyrosine phosphorylation of siglecs decreased dramatically when peripheral blood mononuclear cells were treated with 100 nM svH1C. Subcutaneous, alternate-day injections of svH1C into mice induced several-fold increases in populations of several types of immune cells in the peritoneal cavity. These results support the conclusion that svH1C mimics Neu5Ac-containing sequences and interacts with cell-surface receptors with avidities sufficient to induce biological responses at low concentrations. The attenuation of inhibitory receptors suggests that svH1C

  4. A transition metal Lewis acid/base triad system for cooperative substrate binding.

    Science.gov (United States)

    Tutusaus, Oscar; Ni, Chengbao; Szymczak, Nathaniel K

    2013-03-06

    A frustrated Lewis pair accessory functionality is positioned in the secondary coordination sphere of a terpyridine ligand (Tpy(BN) = 6-morpholino-2,2':6',2″-terpyridine-6″-boronic acid pinacol ester) to promote directed Lewis acid/base interactions. Following metalation with VCl3, the utility of the metal Lewis acid/base triad (LABT) is highlighted with N2H4 as a cooperatively coordinated substrate, affording the first η(2)-[N2H3](-) vanadium complex.

  5. Molecular characterization and functions of fatty acid and retinoid binding protein gene (Ab-far-1 in Aphelenchoides besseyi.

    Directory of Open Access Journals (Sweden)

    Xi Cheng

    Full Text Available Rice white tip nematode, Aphelenchoides besseyi, is a kind of plant parasitic nematodes that cause serious losses in rice and many other crops. Fatty acid and retinoid binding protein (FAR is a specific protein in nematodes and is related to development, reproduction, infection to the host, and disruption of plant defense reactions, so the inhibition of FAR function is the potential approach to control A. besseyi. The full-length of Ab-far-1 cDNA is 805 bp, including 546 bp of ORF that encodes 181 amino acids. Software analysis revealed that the Ab-FAR-1 was rich in α-helix structure, contained a predicted consensus casein kinase II phosphorylation site and a hydrophobic secretory signal peptide, but did not have glycosylation sites. The Ab-FAR-1 had 52% homology to Gp-FAR-1 protein. The Ab-FAR-1 and Gp-FAR-1 were grouped in the same branch according to the phylogenetic tree. Fluorescence-based ligand binding analysis confirmed that the recombinant Ab-FAR-1 (rAb-FAR-1 bound with the fluorescent analogues 11-((5-dimethylaminonaphthalene-1-sulfonyl amino undecannoic acid (DAUDA, cis-parinaric acid and retinol, but the oleic acid would compete with the binding site. Quantitative PCR (qPCR was used to assess the expression level of Ab-far-1 at different development stages of A. besseyi, the highest expression was found in the females, followed by eggs, juveniles and males. Using in situ hybridization technique, Ab-far-1 mRNA was present in the hypodermis of juveniles and adults, the ovaries of females and the testes of males. When A. besseyi was treated with Ab-far-1 dsRNA for 48 h, the silencing efficiency of Ab-far-1 was the best and the number of nematodes on the carrot was the least. Thus FAR plays important roles in the development and reproduction of nematodes. This study is useful and helpful to figure out a new way to control the plant parasitic nematodes.

  6. Molecular characterization and functions of fatty acid and retinoid binding protein gene (Ab-far-1) in Aphelenchoides besseyi.

    Science.gov (United States)

    Cheng, Xi; Xiang, Yu; Xie, Hui; Xu, Chun-Ling; Xie, Teng-Fei; Zhang, Chao; Li, Yu

    2013-01-01

    Rice white tip nematode, Aphelenchoides besseyi, is a kind of plant parasitic nematodes that cause serious losses in rice and many other crops. Fatty acid and retinoid binding protein (FAR) is a specific protein in nematodes and is related to development, reproduction, infection to the host, and disruption of plant defense reactions, so the inhibition of FAR function is the potential approach to control A. besseyi. The full-length of Ab-far-1 cDNA is 805 bp, including 546 bp of ORF that encodes 181 amino acids. Software analysis revealed that the Ab-FAR-1 was rich in α-helix structure, contained a predicted consensus casein kinase II phosphorylation site and a hydrophobic secretory signal peptide, but did not have glycosylation sites. The Ab-FAR-1 had 52% homology to Gp-FAR-1 protein. The Ab-FAR-1 and Gp-FAR-1 were grouped in the same branch according to the phylogenetic tree. Fluorescence-based ligand binding analysis confirmed that the recombinant Ab-FAR-1 (rAb-FAR-1) bound with the fluorescent analogues 11-((5-dimethylaminonaphthalene-1-sulfonyl) amino) undecannoic acid (DAUDA), cis-parinaric acid and retinol, but the oleic acid would compete with the binding site. Quantitative PCR (qPCR) was used to assess the expression level of Ab-far-1 at different development stages of A. besseyi, the highest expression was found in the females, followed by eggs, juveniles and males. Using in situ hybridization technique, Ab-far-1 mRNA was present in the hypodermis of juveniles and adults, the ovaries of females and the testes of males. When A. besseyi was treated with Ab-far-1 dsRNA for 48 h, the silencing efficiency of Ab-far-1 was the best and the number of nematodes on the carrot was the least. Thus FAR plays important roles in the development and reproduction of nematodes. This study is useful and helpful to figure out a new way to control the plant parasitic nematodes.

  7. Plasma Levels of Fatty Acid-Binding Protein 4, Retinol-Binding Protein 4, High-Molecular-Weight Adiponectin, and Cardiovascular Mortality Among Men With Type 2 Diabetes: A 22-Year Prospective Study.

    Science.gov (United States)

    Liu, Gang; Ding, Ming; Chiuve, Stephanie E; Rimm, Eric B; Franks, Paul W; Meigs, James B; Hu, Frank B; Sun, Qi

    2016-11-01

    To examine select adipokines, including fatty acid-binding protein 4, retinol-binding protein 4, and high-molecular-weight (HMW) adiponectin in relation to cardiovascular disease (CVD) mortality among patients with type 2 diabetes mellitus. Plasma levels of fatty acid-binding protein 4, retinol-binding protein 4, and HMW adiponectin were measured in 950 men with type 2 diabetes mellitus in the Health Professionals Follow-up Study. After an average of 22 years of follow-up (1993-2015), 580 deaths occurred, of whom 220 died of CVD. After multivariate adjustment for covariates, higher levels of fatty acid-binding protein 4 were significantly associated with a higher CVD mortality: comparing extreme tertiles, the hazard ratio and 95% confidence interval of CVD mortality was 1.78 (1.22-2.59; P trend=0.001). A positive association was also observed for HMW adiponectin: the hazard ratio (95% confidence interval) was 2.07 (1.42-3.06; P trend=0.0002), comparing extreme tertiles, whereas higher retinol-binding protein 4 levels were nonsignificantly associated with a decreased CVD mortality with an hazard ratio (95% confidence interval) of 0.73 (0.50-1.07; P trend=0.09). A Mendelian randomization analysis suggested that the causal relationships of HMW adiponectin and retinol-binding protein 4 would be directionally opposite to those observed based on the biomarkers, although none of the Mendelian randomization associations achieved statistical significance. These data suggest that higher levels of fatty acid-binding protein 4 and HMW adiponectin are associated with elevated CVD mortality among men with type 2 diabetes mellitus. Biological mechanisms underlying these observations deserve elucidation, but the associations of HMW adiponectin may partially reflect altered adipose tissue functionality among patients with type 2 diabetes mellitus. © 2016 American Heart Association, Inc.

  8. Direct interaction between EgFABP1, a fatty acid binding protein from Echinococcus granulosus, and phospholipid membranes.

    Directory of Open Access Journals (Sweden)

    Jorge L Porfido

    Full Text Available BACKGROUND: Growth and maintenance of hydatid cysts produced by Echinococcus granulosus have a high requirement for host lipids for biosynthetic processes, membrane building and possibly cellular and developmental signalling. This requires a high degree of lipid trafficking facilitated by lipid transporter proteins. Members of the fatty acid binding protein (FABP family have been identified in Echinococcus granulosus, one of which, EgFABP1 is expressed at the tegumental level in the protoscoleces, but it has also been described in both hydatid cyst fluid and secretions of protoscoleces. In spite of a considerable amount of structural and biophysical information on the FABPs in general, their specific functions remain mysterious. METHODOLOGY/PRINCIPAL FINDINGS: We have investigated the way in which EgFABP1 may interact with membranes using a variety of fluorescence-based techniques and artificial small unilamellar vesicles. We first found that bacterial recombinant EgFABP1 is loaded with fatty acids from the synthesising bacteria, and that fatty acid binding increases its resistance to proteinases, possibly due to subtle conformational changes induced on EgFABP1. By manipulating the composition of lipid vesicles and the ionic environment, we found that EgFABP1 interacts with membranes in a direct contact, collisional, manner to exchange ligand, involving both ionic and hydrophobic interactions. Moreover, we observed that the protein can compete with cytochrome c for association with the surface of small unilamellar vesicles (SUVs. CONCLUSIONS/SIGNIFICANCE: This work constitutes a first approach to the understanding of protein-membrane interactions of EgFABP1. The results suggest that this protein may be actively involved in the exchange and transport of fatty acids between different membranes and cellular compartments within the parasite.

  9. Ferrocene-Boronic Acid-Fructose Binding Based on Dual-Plate Generator-Collector Voltammetry and Square-Wave Voltammetry.

    Science.gov (United States)

    Li, Meng; Xu, Su-Ying; Gross, Andrew J; Hammond, Jules L; Estrela, Pedro; Weber, James; Lacina, Karel; James, Tony D; Marken, Frank

    2015-06-10

    The interaction of ferrocene-boronic acid with fructose is investigated in aqueous 0.1 m phosphate buffer at pH 7, 8 and 9. Two voltammetric methods, based on 1) a dual-plate generator-collector micro-trench electrode (steady state) and 2) a square-wave voltammetry (transient) method, are applied and compared in terms of mechanistic resolution. A combination of experimental data is employed to obtain new insights into the binding rates and the cumulative binding constants for both the reduced ferrocene-boronic acid (pH dependent and weakly binding) and for the oxidised ferrocene-boronic acid (pH independent and strongly binding).

  10. Upregulation of peroxisome proliferator-activated receptors and liver fatty acid binding protein in hepatic cells of broiler chicken supplemented with conjugated linoleic acids

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    Suriya Kumari Ramiah

    2015-09-01

    Full Text Available Since conjugated linoleic acid (CLA has structural and physiological characteristics similar to peroxisome proliferators, it is hypothesized that CLA would upregulate peroxisome proliferator-activated receptor (PPAR and liver fatty acid binding protein (LFABP in the liver of broiler chicken. The aim of the present study was to determine fatty acid composition of liver in CLA-fed broiler chickens and the genes associated with hepatic lipid metabolism. A total of 180-day-old broiler chicks were randomly assigned to two diets containing 0 and 2.5% CLA and fed for 6 weeks. Fatty acid (FA composition of liver and PPAR α and γ and L-FABP were analyzed. It has been demonstrated that CLA was found in the liver of CLA-feed chicken compared to control group. Hepatic PPAR α and γ mRNA levels were upregulated 1.2 and 3-fold in CLA-fed chickens compared to chickens fed diet without CLA respectively. A similar response of upregulation was observed for L-FABP mRNA expression. Our data highlights the role of PPARs as a core regulator in the regulation of lipid metabolism in chicken liver.

  11. Long-Term Effect of Docosahexaenoic Acid Feeding on Lipid Composition and Brain Fatty Acid-Binding Protein Expression in Rats.

    Science.gov (United States)

    Elsherbiny, Marwa E; Goruk, Susan; Monckton, Elizabeth A; Richard, Caroline; Brun, Miranda; Emara, Marwan; Field, Catherine J; Godbout, Roseline

    2015-10-22

    Arachidonic (AA) and docosahexaenoic acid (DHA) brain accretion is essential for brain development. The impact of DHA-rich maternal diets on offspring brain fatty acid composition has previously been studied up to the weanling stage; however, there has been no follow-up at later stages. Here, we examine the impact of DHA-rich maternal and weaning diets on brain fatty acid composition at weaning and three weeks post-weaning. We report that DHA supplementation during lactation maintains high DHA levels in the brains of pups even when they are fed a DHA-deficient diet for three weeks after weaning. We show that boosting dietary DHA levels for three weeks after weaning compensates for a maternal DHA-deficient diet during lactation. Finally, our data indicate that brain fatty acid binding protein (FABP7), a marker of neural stem cells, is down-regulated in the brains of six-week pups with a high DHA:AA ratio. We propose that elevated levels of DHA in developing brain accelerate brain maturation relative to DHA-deficient brains.

  12. Long-Term Effect of Docosahexaenoic Acid Feeding on Lipid Composition and Brain Fatty Acid-Binding Protein Expression in Rats

    Directory of Open Access Journals (Sweden)

    Marwa E. Elsherbiny

    2015-10-01

    Full Text Available Arachidonic (AA and docosahexaenoic acid (DHA brain accretion is essential for brain development. The impact of DHA-rich maternal diets on offspring brain fatty acid composition has previously been studied up to the weanling stage; however, there has been no follow-up at later stages. Here, we examine the impact of DHA-rich maternal and weaning diets on brain fatty acid composition at weaning and three weeks post-weaning. We report that DHA supplementation during lactation maintains high DHA levels in the brains of pups even when they are fed a DHA-deficient diet for three weeks after weaning. We show that boosting dietary DHA levels for three weeks after weaning compensates for a maternal DHA-deficient diet during lactation. Finally, our data indicate that brain fatty acid binding protein (FABP7, a marker of neural stem cells, is down-regulated in the brains of six-week pups with a high DHA:AA ratio. We propose that elevated levels of DHA in developing brain accelerate brain maturation relative to DHA-deficient brains.

  13. Reversible and covalent binding of 5-(hydroxymethyl)-2-furaldehyde (HMF) with lysine and selected amino acids.

    Science.gov (United States)

    Nikolov, Plamen Y; Yaylayan, Varoujan A

    2011-06-08

    The chemical reactivity of 5-(hydroxymethyl)-2-furaldehyde (HMF) with lysine, glycine, and proline was studied using isotope labeling technique. To confirm the formation of HMF adducts in glucose amino acid model systems, a useful strategy was developed in which products simultaneously possessing six glucose (HMF moiety) and any number of amino acid carbon atoms in addition to nitrogen were targeted using specifically labeled precursors such as [(15)N(α)]lysine·2HCl, [(15)N(ε)]lysine·2HCl, [U-(13)C(6)]lysine·2HCl, [(13)C(6)]lysine·2HCl, and [U-(13)C(6)]glucose in the case of lysine model system. In addition, model systems containing HMF and amino acids were also studied to confirm specific adduct formation. Complete labeling studies along with structural analysis using appropriate synthetic precursors such as HMF Schiff base adducts of piperidine and glycine have indicated that HMF generated in the glucose/amino acid model systems initially forms a Schiff base adduct that can undergo decarboxylation through an oxazolidin-5-one intermediate and form two isomeric decarboxylated Schiff bases. Unlike the Schiff bases resulting from primary amines or amino acids such as glycine or lysine, those resulting from secondary amino acids such as proline or secondary amines such as piperidine can further undergo vinylogous Amadori rearrangement, forming N-substituted 5-(aminomethyl)furan-2-carbaldehyde derivatives.

  14. Nucleic acids encoding phloem small RNA-binding proteins and transgenic plants comprising them

    Science.gov (United States)

    Lucas, William J.; Yoo, Byung-Chun; Lough, Tony J.; Varkonyi-Gasic, Erika

    2007-03-13

    The present invention provides a polynucleotide sequence encoding a component of the protein machinery involved in small RNA trafficking, Cucurbita maxima phloem small RNA-binding protein (CmPSRB 1), and the corresponding polypeptide sequence. The invention also provides genetic constructs and transgenic plants comprising the polynucleotide sequence encoding a phloem small RNA-binding protein to alter (e.g., prevent, reduce or elevate) non-cell autonomous signaling events in the plants involving small RNA metabolism. These signaling events are involved in a broad spectrum of plant physiological and biochemical processes, including, for example, systemic resistance to pathogens, responses to environmental stresses, e.g., heat, drought, salinity, and systemic gene silencing (e.g., viral infections).

  15. Terminal sialic acids on CD44 N-glycans can block hyaluronan binding by forming competing intramolecular contacts with arginine sidechains

    Science.gov (United States)

    Faller, Christina E.; Guvench, Olgun

    2014-01-01

    Specific sugar residues and their linkages form the basis of molecular recognition for interactions of glycoproteins with other biomolecules. Seemingly small changes, like the addition of a single monosaccharide in the covalently attached glycan component of glycoproteins, can greatly affect these interactions. For instance, the sialic acid capping of glycans affects protein-ligand binding involved in cell-cell and cell-matrix interactions. CD44 is a single-pass transmembrane glycoprotein whose binding with its carbohydrate ligand hyaluronan (HA), an extracellular matrix component, mediates processes such as leukocyte homing, cell adhesion, and tumor metastasis. This binding is highly regulated by glycosylation of the N-terminal extracellular hyaluronan-binding domain (HABD); specifically, sialic acid capped N-glycans of HABD inhibit ligand binding. However, the molecular mechanism behind this sialic acid mediated regulation has remained unknown. Two of the five N-glycosyation sites of HABD have been previously identified as having the greatest inhibitory effect on HA binding, but only if the glycans contain terminal sialic acid residues. These two sites, Asn25 and Asn120, were chosen for in silico glycosylation in this study. Here, from extensive standard molecular dynamics simulations and biased simulations, we propose a molecular mechanism for this behavior based on spontaneously-formed charge-paired hydrogen bonding interactions between the negatively-charged sialic acid residues and positively-charged Arg sidechains known to be critically important for binding to HA, which itself is negatively charged. Such intramolecular hydrogen bonds would preclude associations critical to hyaluronan binding. This observation suggests how CD44 and related glycoprotein binding is regulated by sialylation as cellular environments fluctuate. PMID:25116630

  16. Identification and analysis of hepatitis C virus NS3 helicase inhibitors using nucleic acid binding assays

    OpenAIRE

    Mukherjee, Sourav; Alicia M Hanson; Shadrick, William R.; Ndjomou, Jean; Sweeney, Noreena L.; Hernandez, John J.; Bartczak, Diana; Li, Kelin; Frankowski, Kevin J.; Heck, Julie A.; Arnold, Leggy A.; Schoenen, Frank J.; Frick, David N.

    2012-01-01

    Typical assays used to discover and analyze small molecules that inhibit the hepatitis C virus (HCV) NS3 helicase yield few hits and are often confounded by compound interference. Oligonucleotide binding assays are examined here as an alternative. After comparing fluorescence polarization (FP), homogeneous time-resolved fluorescence (HTRF®; Cisbio) and AlphaScreen® (Perkin Elmer) assays, an FP-based assay was chosen to screen Sigma’s Library of Pharmacologically Active Compounds (LOPAC) for c...

  17. Excitatory amino acid binding sites in the hippocampal region of Alzheimer's disease and other dementias.

    OpenAIRE

    1990-01-01

    Quantitative receptor autoradiography was used to measure muscarinic cholinergic, benzodiazepine, kainate, phencyclidine (PCP), N-methyl-D-aspartate (NMDA) (measured in Tris acetate), quisqualate-sensitive, non-quisqualate-sensitive and total glutamate (measured in Tris chloride buffer) binding sites in adjacent sections of the hippocampal region of 10 Alzheimer's disease, nine control, and six demented, non-Alzheimer's disease postmortem human brains. The measurements were compared to the nu...

  18. Sugar-coated proteins: the importance of degree of polymerisation of oligo-galacturonic acid on protein binding and aggregation.

    Science.gov (United States)

    Xu, Amy Y; Melton, Laurence D; Ryan, Timothy M; Mata, Jitendra P; Jameson, Geoffrey B; Rekas, Agata; Williams, Martin A K; McGillivray, Duncan J

    2017-04-05

    We have simplified the structural heterogeneity of protein-polysaccharide binding by investigating protein binding to oligosaccharides. The interactions between bovine beta-lactoglobulin A (βLgA) and oligo-galacturonic acids (OGAs) with various numbers of sugar residues have been investigated with a range of biophysical techniques. We show that the βLgA-OGA interaction is critically dependent on the length of the oligosaccharide. Isothermal titration calorimetry results suggest that a minimum length of 7 or 8 sugar residues is required in order to exhibit appreciable exothermic interactions with βLgA - shorter oligosaccharides show no enthalpic interactions at any concentration ratio. When titrating βLgA into OGAs with more than 7-8 sugar residues the sample solution also became turbid with increasing amounts of βLgA, indicating the formation of macroscopic assemblies. Circular dichroism, thioflavin T fluorescence and small angle X-ray/neutron scattering experiments revealed two structural regimes during the titration. When OGAs were in excess, βLgA formed discrete assemblies upon OGA binding, and no subsequent aggregation was observed. However, when βLgA was present in excess, multi-scale structures were formed and this eventually led to the separation of the solution into two liquid-phases.

  19. Theoretical and experimental studies on binding mode of 3,5-pyrazoledicarboxylic acid in its new La(III) complex

    Science.gov (United States)

    Peica, Niculina; Kostova, Irena; Kiefer, Wolfgang

    2006-06-01

    A new La(III) complex with 3,5-pyrazoledicarboxylic acid (HPy) was synthesized and characterized with elemental analysis, IR, and Raman spectroscopies. Significant differences in the IR and Raman spectra of the complex were observed as compared to the spectra of the ligand. The metal-ligand binding mode was studied on the basis of theoretical and experimental data. B3PW91 and B3LYP methods with 6-311++G ∗∗ and LANL2DZ basis sets were successfully applied to study the molecular and vibrational structures as well as the conformational behavior of the neutral ligand and its new La(III) complex. The theoretical calculations of HPy suggested bidentate binding mode through the carboxylic oxygens. Detailed vibrational analysis of HPy and La(III)-Py systems based on both the calculated and experimental spectra confirmed the suggested metal-ligand binding mode. The density functional theory (DFT) calculated geometries, harmonic vibrational wavenumbers including IR and Raman scattering activities for the ligand and its La(III) complex were in good agreement with the experimental data, a complete vibrational assignment being proposed.

  20. BAD-lectins: boronic acid-decorated lectins with enhanced binding affinity for the selective enrichment of glycoproteins.

    Science.gov (United States)

    Lu, Ying-Wei; Chien, Chih-Wei; Lin, Po-Chiao; Huang, Li-De; Chen, Chang-Yang; Wu, Sz-Wei; Han, Chia-Li; Khoo, Kay-Hooi; Lin, Chun-Cheng; Chen, Yu-Ju

    2013-09-03

    The weak and variable binding affinities exhibited by lectin-carbohydrate interactions have often compromised the practical utility of lectin in capturing glycoproteins for glycoproteomic applications. We report here the development and applications of a new type of hybrid biomaterial, namely a boronic acid-decorated lectin (BAD-lectin), for efficient bifunctional glycoprotein labeling and enrichment. Our binding studies showed an enhanced affinity by BAD-lectin, likely to be mediated via the formation of boronate ester linkages between the lectin and glycan subsequent to the initial recognition process and thus preserving its glycan-specificity. Moreover, when attached to magnetic nanoparticles (BAD-lectin@MNPs), 2 to 60-fold improvement on detection sensitivity and enrichment efficiency for specific glycoproteins was observed over the independent use of either lectin or BA. Tested at the level of whole cell lysates for glycoproteomic applications, three different types of BAD-lectin@MNPs exhibited excellent specificities with only 6% overlapping among the 295 N-linked glycopeptides identified. As many as 236 N-linked glycopeptides (80%) were uniquely identified by one of the BAD-lectin@MNPs. These results indicated that the enhanced glycan-selective recognition and binding affinity of BAD-lectin@MNPs will facilitate a complementary identification of the under-explored glycoproteome.

  1. Effective DNA binding and cleaving tendencies of malonic acid coupled transition metal complexes

    Science.gov (United States)

    Pravin, Narayanaperumal; Utthra, Ponnukalai Ponya; Kumaravel, Ganesan; Raman, Natarajan

    2016-11-01

    Eight transition metal complexes were designed to achieve maximum biological efficacy. They were characterized by elemental analysis and various other spectroscopic techniques. The monomeric complexes were found to espouse octahedral geometry and non-electrolytic nature. The DNA interaction propensity of the complexes with calf thymus DNA (CT-DNA), studied at physiological pH by spectrophotometric, spectrofluorometric, cyclic voltammetry, and viscometric techniques revealed intercalation as the possible binding mode. Fascinatingly, the complexes were found to exhibit greater binding strength than that of the free ligands. A strong hypochromism and a slight red shift were exhibited by complex 5 among the other complexes. The intrinsic binding constant values of all the complexes compared to cisplatin reveal that they are excellent metallonucleases than that of cisplatin. The complexes were also shown to reveal displacement of the ethidium bromide, a strong intercalator using fluorescence titrations. Gel electrophoresis was used to divulge the competence of the complexes in cleaving the supercoiled pBR322 plasmid DNA. From the results, it is concluded that the complexes, especially 5, are excellent chemical nucleases in the presence of H2O2. Furthermore, the in vitro antimicrobial screening of the complexes exposes that these complexes are excellent antimicrobial agents. Overall the effect of coligands is evident from the results of all the investigations.

  2. Saturated Fatty Acid Induces Insulin Resistance Partially Through Nucleotide-binding Oligomerization Domain 1 Signaling Pathway in Adipocytes

    Institute of Scientific and Technical Information of China (English)

    Yi-jun Zhou; Yin-si Tang; Yu-ling Song; Ai Li; Hui Zhou; Yan Li

    2013-01-01

    Objective To investigate the potential role of nucleotide-binding oligomerization domain 1 (NOD1), a component of the innate immune system, in mediating lipid-induced insulin resistance in adipocytes. Methods Adipocytes from Toll-like receptor 4 deficiency mice were used for stimulation experiments. The effect of oleate/palmitate mixture on nuclear factor-κB (NF-κB) activation was analyzed by reporter plasmid assay. The release of proinflammatory chemokine/cytokines production was determined by using real-time PCR. Insulin-stimulated glucose uptake was measured by 2-deoxy-D-[3H] glucose uptake assay. Chemokine/cytokine expression and glucose uptake in adipocytes transfected with small interfering RNA (siRNA) targeting NOD1 upon fatty acids treatment were analyzed. Results Oleate/palmitate mixture activated the NF-κB pathway and induced interleukin-6, tumor necrosis factor-α, and monocyte chemoattractant protein-1 mRNA expressions in adipocytes from mice deficient in Toll-like receptor 4, and these effects were blocked by siRNA targeting NOD1. Furthermore, saturated fatty acids decreased the ability of insulin-stimulated glucose uptake. Importantly, siRNA targeting NOD1 partially reversed saturated fatty acid-induced suppression of insulin-induced glucose uptake. Conclusion NOD1 might play an important role in saturated fatty acid-induced insulin resistance in adipocytes, suggesting a mechanism by which reduced NOD1 activity confers beneficial effects on insulin action.

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

    Science.gov (United States)

    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.

  4. Synthesis and evaluation of boronic acids as inhibitors of Penicillin Binding Proteins of classes A, B and C.

    Science.gov (United States)

    Zervosen, Astrid; Bouillez, André; Herman, Alexandre; Amoroso, Ana; Joris, Bernard; Sauvage, Eric; Charlier, Paulette; Luxen, André

    2012-06-15

    In response to the widespread use of β-lactam antibiotics bacteria have evolved drug resistance mechanisms that include the production of resistant Penicillin Binding Proteins (PBPs). Boronic acids are potent β-lactamase inhibitors and have been shown to display some specificity for soluble transpeptidases and PBPs, but their potential as inhibitors of the latter enzymes is yet to be widely explored. Recently, a (2,6-dimethoxybenzamido)methylboronic acid was identified as being a potent inhibitor of Actinomadura sp. R39 transpeptidase (IC(50): 1.3 μM). In this work, we synthesized and studied the potential of a number of acylaminomethylboronic acids as inhibitors of PBPs from different classes. Several derivatives inhibited PBPs of classes A, B and C from penicillin sensitive strains. The (2-nitrobenzamido)methylboronic acid was identified as a good inhibitor of a class A PBP (PBP1b from Streptococcus pneumoniae, IC(50) = 26 μM), a class B PBP (PBP2xR6 from Streptococcus pneumoniae, IC(50) = 138 μM) and a class C PBP (R39 from Actinomadura sp., IC(50) = 0.6 μM). This work opens new avenues towards the development of molecules that inhibit PBPs, and eventually display bactericidal effects, on distinct bacterial species. Copyright © 2012 Elsevier Ltd. All rights reserved.

  5. Fatty Acid-binding Proteins Interact with Comparative Gene Identification-58 Linking Lipolysis with Lipid Ligand Shuttling.

    Science.gov (United States)

    Hofer, Peter; Boeszoermenyi, Andras; Jaeger, Doris; Feiler, Ursula; Arthanari, Haribabu; Mayer, Nicole; Zehender, Fabian; Rechberger, Gerald; Oberer, Monika; Zimmermann, Robert; Lass, Achim; Haemmerle, Guenter; Breinbauer, Rolf; Zechner, Rudolf; Preiss-Landl, Karina

    2015-07-24

    The coordinated breakdown of intracellular triglyceride (TG) stores requires the exquisitely regulated interaction of lipolytic enzymes with regulatory, accessory, and scaffolding proteins. Together they form a dynamic multiprotein network designated as the "lipolysome." Adipose triglyceride lipase (Atgl) catalyzes the initiating step of TG hydrolysis and requires comparative gene identification-58 (Cgi-58) as a potent activator of enzyme activity. Here, we identify adipocyte-type fatty acid-binding protein (A-Fabp) and other members of the fatty acid-binding protein (Fabp) family as interaction partners of Cgi-58. Co-immunoprecipitation, microscale thermophoresis, and solid phase assays proved direct protein/protein interaction between A-Fabp and Cgi-58. Using nuclear magnetic resonance titration experiments and site-directed mutagenesis, we located a potential contact region on A-Fabp. In functional terms, A-Fabp stimulates Atgl-catalyzed TG hydrolysis in a Cgi-58-dependent manner. Additionally, transcriptional transactivation assays with a luciferase reporter system revealed that Fabps enhance the ability of Atgl/Cgi-58-mediated lipolysis to induce the activity of peroxisome proliferator-activated receptors. Our studies identify Fabps as crucial structural and functional components of the lipolysome.

  6. Amino acid substitution in NPC1 that abolishes cholesterol binding reproduces phenotype of complete NPC1 deficiency in mice

    Science.gov (United States)

    Xie, Xuefen; Brown, Michael S.; Shelton, John M.; Richardson, James A.; Goldstein, Joseph L.; Liang, Guosheng

    2011-01-01

    Substitution mutations in adjacent amino acids of the N-terminal domain of NPC1, a lysosomal membrane protein, abolish its cholesterol binding activity and impair its ability to export cholesterol from lysosomes of cultured cells lacking npc1 [Kwon HJ, et al. (2009) Cell 137:1213–1224]. Here, we show that the same two mutations (proline-202 and phenylalanine-203, both changed to alanine) reproduce the phenotype of complete NPC1 deficiency when knocked into the mouse npc1 gene by homologous recombination. Homozygous npc1pf/pf mice exhibited neurodegeneration beginning at day 49 and died at a median age of 84 d, as previously reported for mice that lack npc1. Liver and other organs of the npc1pf/pf mice accumulated excess cholesterol in lysosomes. In liver, mRNAs encoding several lysosomal proteins were elevated, including NPC1 and NPC2 and several digestive enzymes (acid lipase, β-glucuronidase, and cathepsins B and D). Weekly treatment with hydroxypropyl-β-cyclodextrin (HPCD) beginning at 7 wk reduced hepatic cholesterol accumulation and diminished the lysosomal mRNAs. We conclude that the cholesterol binding site in the N-terminal domain of NPC1 is essential for cholesterol export from lysosomes in living animals as it is in cultured cells. The HPCD-mediated reduction of excess lysosomal enzymes may contribute to the ability of this drug to delay the progression of NPC disease in mice. PMID:21896731

  7. A 193-amino acid fragment of the SARS coronavirus S protein efficiently binds angiotensin-converting enzyme 2.

    Science.gov (United States)

    Wong, Swee Kee; Li, Wenhui; Moore, Michael J; Choe, Hyeryun; Farzan, Michael

    2004-01-30

    The coronavirus spike (S) protein mediates infection of receptor-expressing host cells and is a critical target for antiviral neutralizing antibodies. Angiotensin-converting enzyme 2 (ACE2) is a functional receptor for the coronavirus (severe acute respiratory syndrome (SARS)-CoV) that causes SARS. Here we demonstrate that a 193-amino acid fragment of the S protein (residues 318-510) bound ACE2 more efficiently than did the full S1 domain (residues 12-672). Smaller S protein fragments, expressing residues 327-510 or 318-490, did not detectably bind ACE2. A point mutation at aspartic acid 454 abolished association of the full S1 domain and of the 193-residue fragment with ACE2. The 193-residue fragment blocked S protein-mediated infection with an IC(50) of less than 10 nm, whereas the IC(50) of the S1 domain was approximately 50 nm. These data identify an independently folded receptor-binding domain of the SARS-CoV S protein.

  8. Caffeic acid phenethyl ester downregulates phospholipase D1 via direct binding and inhibition of NFκB transactivation

    Energy Technology Data Exchange (ETDEWEB)

    Park, Mi Hee; Kang, Dong Woo [Department of Molecular Biology, Pusan National University, Busan 609-735 (Korea, Republic of); Jung, Yunjin [College of Pharmacy, Pusan National University, Busan 609-735 (Korea, Republic of); Choi, Kang-Yell [Translational Research Center for Protein Function Control, Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul (Korea, Republic of); Min, Do Sik, E-mail: minds@pusan.ac.kr [Department of Molecular Biology, Pusan National University, Busan 609-735 (Korea, Republic of)

    2013-12-06

    Highlights: •We found CAFÉ, a natural product that suppresses expression and activity of PLD1. •CAPE decreased PLD1 expression by inhibiting NFκB transactivation. •CAPE rapidly inhibited PLD activity via its binding to a Cys837 of PLD1. •PLD1 downregulation by CAPE inhibited invasion and proliferation of glioma cells. -- Abstract: Upregulation of phospholipase D (PLD) is functionally linked with oncogenic signals and tumorigenesis. Caffeic acid phenethyl ester (CAPE) is an active compound of propolis extract that exhibits anti-proliferative, anti-inflammatory, anti-oxidant, and antineoplastic properties. In this study, we demonstrated that CAPE suppressed the expression of PLD1 at the transcriptional level via inhibition of binding of NFκB to PLD1 promoter. Moreover, CAPE, but not its analogs, bound to a Cys837 residue of PLD1 and inhibited enzymatic activity of PLD. CAPE also decreased activation of matrix metalloproteinases-2 induced by phosphatidic acid, a product of PLD activity. Ultimately, CAPE-induced downregulation of PLD1 suppressed invasion and proliferation of glioma cells. Taken together, the results of this study indicate that CAPE might contribute to anti-neoplastic effect by targeting PLD1.

  9. Single Amino Acid Changes Can Influence Titer, Heparin Binding, and Tissue Tropism in Different Adeno-Associated Virus Serotypes▿

    Science.gov (United States)

    Wu, Zhijian; Asokan, Aravind; Grieger, Joshua C.; Govindasamy, Lakshmanan; Agbandje-McKenna, Mavis; Samulski, R. Jude

    2006-01-01

    Despite the high degree of sequence homology between adeno-associated virus (AAV) serotype 1 and 6 capsids (99.2%), these viruses have different liver transduction profiles when tested as vectors. Examination of the six amino acid residues that differ between AAV1 and AAV6 revealed that a lysine-to-glutamate change (K531E) suppresses the heparin binding ability of AAV6. In addition, the same mutation in AAV6 reduces transgene expression to levels similar to those achieved with AAV1 in HepG2 cells in vitro and in mouse liver following portal vein administration. In corollary, the converse E531K mutation in AAV1 imparts heparin binding ability and increases transduction efficiency. Extraction of vector genomes from liver tissue suggests that the lysine 531 residue assists in preferential transduction of parenchymal cells by AAV6 vectors in comparison with AAV1. Lysine 531 is unique to AAV6 among other known AAV serotypes and is located in a basic cluster near the spikes that surround the icosahedral threefold axes of the AAV capsid. Similar to studies with autonomous parvoviruses, this study describes the first example of single amino acid changes that can explain differential phenotypes such as viral titer, receptor binding, and tissue tropism exhibited by closely related AAV serotypes. In particular, a single lysine residue appears to provide the critical minimum charged surface required for interacting with heparin through electrostatic interaction and simultaneously plays an unrelated yet critical role in the liver tropism of AAV6 vectors. PMID:16943302

  10. Chiral and structural discrimination in binding of polypeptides with condensed nucleic acid structures.

    Science.gov (United States)

    Reich, Z; Ittah, Y; Weinberger, S; Minsky, A

    1990-04-05

    In biological systems nucleic acids are invariably found in highly compact forms. These rather intricate forms raise questions of basic importance which are related to the various factors involved in the condensation processes, the chemical, physical, and structural features revealed by the packed species, and the effects of the extremely tight packaging upon interactions of the DNA molecules with proteins and drugs. A means for addressing these questions on a molecular level is provided by various procedures known to induce in vitro condensation of DNA molecules into highly compact species which, in turn, may serve as a model for the in vivo physical organization of nucleic acids. A study of the optical properties of the tightly packed DNA molecules indicates that the interactions of these species with polypeptides are characterized by distinct, hitherto unobserved, chiral and structural discrimination. Specifically, the polypeptides found to be selected against are composed of those amino acids that are not normally used in protein biosynthesis, such as D-lysine or ornithine. These findings provide new clues to long debated topics such as the specific universal chirality of amino acids in proteins or the correlation between conformational flexibility of polypeptides and their ability to form stable compact complexes with nucleic acids.

  11. Vanadyl ion (VO sup 2+ ) as a spectroscopic probe of metal binding to nitrohumic acid

    Energy Technology Data Exchange (ETDEWEB)

    Mangrich, A.S.; Vugman, N.V. (Universidad Federal do Parana, Parana (Brazil). Dept. de Quimica)

    1990-07-01

    A coal nitrohumic acid (CNHA) of high nitrogen content (compared with natural humic acid) was obtained by extraction from nitric acid oxidation products of a mineral coal. It was studied by e.s.r. and i.r. spectroscopies, using VO{sup 2+} as a probe of metal ion complexation sites. Spectroscopic data and a LCAO-MO ligand field calculation were used to evaluate the bond parameters of vanadyl-coal nitrohumic acid complex (VO-CNHA). In spite of the high nitrogen content of CNHA, plots of hyperfine coupling constants {lt}A{gt} versus isotropic {lt}g{gt} values indicate that nitrogen is not a donor atom in the complexation sites of these materials. The bond parameter values, {lt}A{gt} and {lt}g{gt}, and i.r. data suggest that VO{sup 2+} groups (in the CNHA molecules) are at sites with C{sub 4}{sub V}, symmetry having o-hydroxycarboxylic aromatic (salicylic) acids as equatorial ligands. 21 refs., 2 figs.

  12. Structural model for gamma-aminobutyric acid receptor noncompetitive antagonist binding: widely diverse structures fit the same site.

    Science.gov (United States)

    Chen, Ligong; Durkin, Kathleen A; Casida, John E

    2006-03-28

    Several major insecticides, including alpha-endosulfan, lindane, and fipronil, and the botanical picrotoxinin are noncompetitive antagonists (NCAs) for the GABA receptor. We showed earlier that human beta(3) homopentameric GABA(A) receptor recognizes all of the important GABAergic insecticides and reproduces the high insecticide sensitivity and structure-activity relationships of the native insect receptor. Despite large structural diversity, the NCAs are proposed to fit a single binding site in the chloride channel lumen lined by five transmembrane 2 segments. This hypothesis is examined with the beta(3) homopentamer by mutagenesis, pore structure studies, NCA binding, and molecular modeling. The 15 amino acids in the cytoplasmic half of the pore were mutated to cysteine, serine, or other residue for 22 mutants overall. Localization of A-1'C, A2'C, T6'C, and L9'C (index numbers for the transmembrane 2 region) in the channel lumen was established by disulfide cross-linking. Binding of two NCA radioligands [(3)H]1-(4-ethynylphenyl)-4-n-propyl-2,6,7-trioxabicyclo[2.2.2]octane and [(3)H] 3,3-bis-trifluoromethyl-bicyclo[2,2,1]heptane-2,2-dicarbonitrile was dramatically reduced with 8 of the 15 mutated positions, focusing attention on A2', T6', and L9' as proposed binding sites, consistent with earlier mutagenesis studies. The cytoplasmic half of the beta3 homopentamer pore was modeled as an alpha-helix. The six NCAs listed above plus t-butylbicyclophosphorothionate fit the 2' to 9' pore region forming hydrogen bonds with the T6' hydroxyl and hydrophobic interactions with A2', T6', and L9' alkyl substituents, thereby blocking the channel. Thus, widely diverse NCA structures fit the same GABA receptor beta subunit site with important implications for insecticide cross-resistance and selective toxicity between insects and mammals.

  13. Entrapment of alpha1-acid glycoprotein in high-performance affinity columns for drug-protein binding studies.

    Science.gov (United States)

    Bi, Cong; Jackson, Abby; Vargas-Badilla, John; Li, Rong; Rada, Giana; Anguizola, Jeanethe; Pfaunmiller, Erika; Hage, David S

    2016-05-15

    A slurry-based method was developed for the entrapment of alpha1-acid glycoprotein (AGP) for use in high-performance affinity chromatography to study drug interactions with this serum protein. Entrapment was achieved based on the physical containment of AGP in hydrazide-activated porous silica supports and by using mildly oxidized glycogen as a capping agent. The conditions needed for this process were examined and optimized. When this type of AGP column was used in binding studies, the association equilibrium constant (Ka) measured by frontal analysis at pH 7.4 and 37°C for carbamazepine with AGP was found to be 1.0 (±0.5)×10(5)M(-1), which agreed with a previously reported value of 1.0 (±0.1)×10(5)M(-1). Binding studies based on zonal elution were conducted for several other drugs with such columns, giving equilibrium constants that were consistent with literature values. An entrapped AGP column was also used in combination with a column containing entrapped HSA in a screening assay format to compare the binding of various drugs to AGP and HSA. These results also agreed with previous data that have been reported in literature for both of these proteins. The same entrapment method could be extended to other proteins and to the investigation of additional types of drug-protein interactions. Potential applications include the rapid quantitative analysis of biological interactions and the high-throughput screening of drug candidates for their binding to a given protein.

  14. Long-term fertilization modifies the structures of soil fulvic acids and their binding capability with Al.

    Science.gov (United States)

    Wu, Jun; Wu, Minjie; Li, Chunping; Yu, Guanghui

    2014-01-01

    The binding characteristics of organic ligands and minerals in fulvic acids (FAs) with Al are essential for understanding soil C sequestration, remain poorly understood. In this study, Fourier transform infrared (FTIR) spectroscopy combined with two-dimensional correlation spectroscopy (2DCOS) analysis was applied for the first time to explore the binding of Al with organic ligands and minerals in soil FAs. For these analyses, two contrasting treatments were selected from a long-term (i.e., 22-year) fertilization experiment: chemical (NPK) fertilization and swine manure (SM) fertilization. The results showed that the long-term application of organic and inorganic fertilizers to soils had little effect on the compositions of the fluorescent substances and organic ligands in the soil FAs. However, long-term SM fertilization increased the weathered Al and Si concentrations in the soil FAs compared with long-term chemical fertilization. Furthermore, organic ligands in the soil FAs were mainly bound with Al in the NPK treatment, whereas both organic ligands and minerals (Al-O-Si, Si-O) were bound with Al under the M fertilization conditions. Both transmission electron microscopy (TEM) images and X-ray diffraction spectra demonstrated that amorphous and short-range-ordered nanominerals were abundant in the soil FAs from the SM plot in contrast to the soil FAs from the NPK plot. This result illustrates the role nanominerals play in the preservation of soil FAs by during long-term organic fertilization. In summary, the combination of FTIR and 2D correlation spectroscopy is a promising approach for the characterization of the binding capability between soil FAs and Al, and a better understanding FA-Al binding capability will greatly contribute to global C cycling.

  15. No difference in high-magnification morphology and hyaluronic acid binding in the selection of euploid spermatozoa with intact DNA

    Institute of Scientific and Technical Information of China (English)

    Suchada Mongkolchaipak; Teraporn Vutyavanich

    2013-01-01

    In this study,we compared conventional sperm selection with high-magnification morphology based on the motile sperm organellar morphology examination (MSOME) criteria,and hyaluronic acid (HA) binding for sperm chromosome aneuploidy and DNA fragmentation rates.Semen from 50 severe male factor cases was processed through density gradient centrifugation,and subjected to sperm selection by using the conventional method (control),high magnification at x 6650 or HA binding.Aneuploidy was detected by fluorescence in situ hybridization with probes for chromosomes 13,18,21,X and Y,and DNA fragmentation by the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) method.Spermatozoa selected under high-magnification had a lower DNA fragmentation rate (2.6% vs.1.7%; P=0.032),with no significant difference in aneuploidy rate (0.8% vs 0.7%; P=0.583),than those selected by the HA binding method.Spermatozoa selected by both methods had much lower aneuploidy and DNA fragmentation rate than the controls (7% aneuploidy and 26.8% DNA fragmentation rates,respectively).In the high-magnification group,the aneuploidy rate was lower when the best spermatozoa were selected than when only the second-best spermatozoa were available for selection,but the DNA fragmentation rate was not different.In conclusion,sperm selection under high magnification was more effective than under HA binding in selecting spermatozoa with low DNA fragmentation rate,but the small difference (0.9%) might not be clinically meaningful.Both methods were better than the conventional method of sperm selection.

  16. The requirement of the glutamic acid residue at the third position from the carboxyl termini of the laminin gamma chains in integrin binding by laminins.

    Science.gov (United States)

    Ido, Hiroyuki; Nakamura, Aya; Kobayashi, Reiko; Ito, Shunsuke; Li, Shaoliang; Futaki, Sugiko; Sekiguchi, Kiyotoshi

    2007-04-13

    Laminins are the major cell-adhesive proteins in the basement membrane, consisting of three subunits termed alpha, beta, and gamma. The putative binding site for integrins has been mapped to the G domain of the alpha chain, although trimerization with beta and gamma chains is necessary for the G domain to exert its integrin binding activity. The mechanism underlying the requirement of beta and gamma chains in integrin binding by laminins remains poorly understood. Here, we show that the C-terminal region of the gamma chain is involved in modulation of the integrin binding activity of laminins. We found that deletion of the C-terminal three but not two amino acids within the gamma1 chain completely abrogated the integrin binding activity of laminin-511. Furthermore, substitution of Gln for Glu-1607, the amino acid residue at the third position from the C terminus of the gamma1 chain, also abolished the integrin binding activity, underscoring the role of Glu-1607 in integrin binding by the laminin. We also found that the conserved Glu residue of the gamma2 chain is necessary for integrin binding by laminin-332, suggesting that the same mechanism operates in the modulation of the integrin binding activity of laminins containing either gamma1 or gamma2 chains. However, the peptide segment modeled after the C-terminal region of gamma1 chain was incapable of either binding to integrin or inhibiting integrin binding by laminin-511, making it unlikely that the Glu residue is directly recognized by integrin. These results, together, indicate a novel mechanism operating in ligand recognition by laminin binding integrins.

  17. Vesiculoviral matrix (M) protein occupies nucleic acid binding site at nucleoporin pair (Rae1∙Nup98)

    Energy Technology Data Exchange (ETDEWEB)

    Quan, Beili; Seo, Hyuk-Soo; Blobel, Günter; Ren, Yi [Rockefeller

    2014-07-01

    mRNA export factor 1 (Rae1) and nucleoporin 98 (Nup98) are host cell targets for the matrix (M) protein of vesicular stomatitis virus (VSV). How Rae1 functions in mRNA export and how M protein targets both Rae1 and Nup98 are not understood at the molecular level. To obtain structural insights, we assembled a 1:1:1 complex of M•Rae1•Nup98 and established a crystal structure at 3.15-Å resolution. We found that the M protein contacts the Rae1•Nup98 heterodimer principally by two protrusions projecting from the globular domain of M like a finger and thumb. Both projections clamp to the side of the β-propeller of Rae1, with the finger also contacting Nup98. The most prominent feature of the finger is highly conserved Methionine 51 (Met51) with upstream and downstream acidic residues. The complementary surface on Rae1 displays a deep hydrophobic pocket, into which Met51 fastens like a bolt, and a groove of basic residues on either side, which bond to the acidic residues of the finger. Notably, the M protein competed for in vitro binding of various oligonucleotides to Rae1•Nup98. We localized this competing activity of M to its finger using a synthetic peptide. Collectively, our data suggest that Rae1 serves as a binding protein for the phosphate backbone of any nucleic acid and that the finger of M mimics this ligand. In the context of mRNA export, we propose that a given mRNA segment, after having been deproteinated by helicase, is transiently reproteinated by Nup98-tethered Rae1. We suggest that such repetitive cycles provide cytoplasmic stopover sites required for ratcheting mRNA across the nuclear pore.

  18. Polyunsaturated fatty acids synergize with lipid droplet binding thalidomide analogs to induce oxidative stress in cancer cells

    Directory of Open Access Journals (Sweden)

    Madácsi Ramóna

    2010-06-01

    Full Text Available Abstract Background Cytoplasmic lipid-droplets are common inclusions of eukaryotic cells. Lipid-droplet binding thalidomide analogs (2,6-dialkylphenyl-4/5-amino-substituted-5,6,7-trifluorophthalimides with potent anticancer activities were synthesized. Results Cytotoxicity was detected in different cell lines including melanoma, leukemia, hepatocellular carcinoma, glioblastoma at micromolar concentrations. The synthesized analogs are non-toxic to adult animals up to 1 g/kg but are teratogenic to zebrafish embryos at micromolar concentrations with defects in the developing muscle. Treatment of tumor cells resulted in calcium release from the endoplasmic reticulum (ER, induction of reactive oxygen species (ROS, ER stress and cell death. Antioxidants could partially, while an intracellular calcium chelator almost completely diminish ROS production. Exogenous docosahexaenoic acid or eicosapentaenoic acid induced calcium release and ROS generation, and synergized with the analogs in vitro, while oleic acid had no such an effect. Gene expression analysis confirmed the induction of ER stress-mediated apoptosis pathway components, such as GADD153, ATF3, Luman/CREB3 and the ER-associated degradation-related HERPUD1 genes. Tumor suppressors, P53, LATS2 and ING3 were also up-regulated in various cell lines after drug treatment. Amino-phthalimides down-regulated the expression of CCL2, which is implicated in tumor metastasis and angiogenesis. Conclusions Because of the anticancer, anti-angiogenic action and the wide range of applicability of the immunomodulatory drugs, including thalidomide analogs, lipid droplet-binding members of this family could represent a new class of agents by affecting ER-membrane integrity and perturbations of ER homeostasis.

  19. Modulation of phenytoin teratogenicity and embryonic covalent binding by acetylsalicylic acid, caffeic acid, and alpha-phenyl-N-t-butylnitrone: implications for bioactivation by prostaglandin synthetase

    Energy Technology Data Exchange (ETDEWEB)

    Wells, P.G.; Zubovits, J.T.; Wong, S.T.; Molinari, L.M.; Ali, S.

    1989-02-01

    Teratogenicity of the anticonvulsant drug phenytoin is thought to involve its bioactivation by cytochromes P-450 to a reactive arene oxide intermediate. We hypothesized that phenytoin also may be bioactivated to a teratogenic free radical intermediate by another enzymatic system, prostaglandin synthetase. To evaluate the teratogenic contribution of this latter pathway, an irreversible inhibitor of prostaglandin synthetase, acetylsalicylic acid (ASA), 10 mg/kg intraperitoneally (ip), was administered to pregnant CD-1 mice at 9:00 AM on Gestational Days 12 and 13, 2 hr before phenytoin, 65 mg/kg ip. Other groups were pretreated 2 hr prior to phenytoin administration with either the antioxidant caffeic acid or the free radical spin trapping agent alpha-phenyl-N-t-butylnitrone (PBN). Caffeic acid and PBN were given ip in doses that respectively were up to 1.0 to 0.05 molar equivalents to the dose of phenytoin. Dams were killed on Day 19 and the fetuses were assessed for teratologic anomalies. A similar study evaluated the effect of ASA on the in vivo covalent binding of radiolabeled phenytoin administered on Day 12, in which case dams were killed 24 hr later on Day 13. ASA pretreatment produced a 50% reduction in the incidence of fetal cleft palates induced by phenytoin (p less than 0.05), without significantly altering the incidence of resorptions or mean fetal body weight. Pretreatment with either caffeic acid or PBN resulted in dose-related decreases in the incidence of fetal cleft palates produced by phenytoin, with maximal respective reductions of 71 and 82% at the highest doses of caffeic acid and PBN (p less than 0.05).

  20. Ala54Thr fatty acid-binding protein 2 (FABP2 polymorphism in recurrent depression: associations with fatty acid concentrations and waist circumference.

    Directory of Open Access Journals (Sweden)

    Roel J T Mocking

    Full Text Available BACKGROUND: Fatty acid (FA-alterations may mediate the mutual association between Major Depressive Disorder (MDD and cardiovascular disease (CVD. However, etiology of observed FA-alterations in MDD and CVD remains largely unclear. An interesting candidate may be a mutation in the fatty acid-binding protein 2 (FABP2-gene, because it regulates dietary FA-uptake. Therefore, we aimed to test the hypotheses that in MDD-patients the FABP2 Ala54Thr-polymorphism would be (I more prevalent than in sex- and age-matched controls, (II associated with observed alterations in FA-metabolism, and (III associated with CVD-risk factor waist circumference. METHODS: We measured concentrations of 29 different erythrocyte FAs, FABP2-genotype, and waist circumference in recurrent MDD-patients and matched never-depressed controls. RESULTS: FABP2-genotype distribution did not significantly differ between the 137 MDD-patients and 73 matched controls. However, patients with the Ala54Thr-polymorphism had (I higher concentrations of especially eicosadienoic acid (C20:2ω6; P=.009 and other 20-carbon FAs, and associated (II lower waist circumference (P=.019. In addition, FABP2-genotype effects on waist circumference in patients seemed (I mediated by its effect on C20:2ω6, and (II different from controls. CONCLUSIONS: Although Ala54Thr-polymorphism distribution was not associated with recurrent MDD, our results indicate that FABP2 may play a role in the explanation of observed FA-alterations in MDD. For Ala54Thr-polymorphism patients, potentially adaptive conversion of increased bioavailable dietary precursors into eicosadienoic acid instead of arachidonic acid might be related to a low waist circumference. Because this is the first investigation of these associations, replication is warranted, preferably by nutrigenetic studies applying lipidomics and detailed dietary assessment.

  1. Fatty Acid-Binding Protein 5 at the Blood-Brain Barrier Regulates Endogenous Brain Docosahexaenoic Acid Levels and Cognitive Function.

    Science.gov (United States)

    Pan, Yijun; Short, Jennifer L; Choy, Kwok H C; Zeng, Annie X; Marriott, Philip J; Owada, Yuji; Scanlon, Martin J; Porter, Christopher J H; Nicolazzo, Joseph A

    2016-11-16

    Fatty acid-binding protein 5 (FABP5) at the blood-brain barrier contributes to the brain uptake of docosahexaenoic acid (DHA), a blood-derived polyunsaturated fatty acid essential for maintenance of cognitive function. Given the importance of DHA in cognition, the aim of this study was to investigate whether deletion of FABP5 results in cognitive dysfunction and whether this is associated with reduced brain endothelial cell uptake of exogenous DHA and subsequent attenuation in the brain levels of endogenous DHA. Cognitive function was assessed in male and female FABP5(+/+) and FABP5(-/-) mice using a battery of memory paradigms. FABP5(-/-) mice exhibited impaired working memory and short-term memory, and these cognitive deficits were associated with a 14.7 ± 5.7% reduction in endogenous brain DHA levels. The role of FABP5 in the blood-brain barrier transport of DHA was assessed by measuring (14)C-DHA uptake into brain endothelial cells and capillaries isolated from FABP5(+/+) and FABP5(-/-) mice. In line with a crucial role of FABP5 in the brain uptake of DHA, (14)C-DHA uptake into brain endothelial cells and brain capillaries of FABP5(-/-) mice was reduced by 48.4 ± 14.5% and 14.0 ± 4.2%, respectively, relative to those of FABP5(+/+) mice. These results strongly support the hypothesis that FABP5 is essential for maintaining brain endothelial cell uptake of DHA, and that cognitive deficits observed in FABP5(-/-) mice are associated with reduced CNS access of DHA. Genetic deletion of fatty acid-binding protein 5 (FABP5) in mice reduces uptake of exogenous docosahexaenoic acid (DHA) into brain endothelial cells and brain capillaries and reduces brain parenchymal levels of endogenous DHA. Therefore, FABP5 in the brain endothelial cell is a crucial contributor to the brain levels of DHA. Critically, lowered brain DHA levels in FABP5(-/-) mice occurred in tandem with cognitive deficits in a battery of memory paradigms. This study provides evidence of a critical role

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

    Science.gov (United States)

    Phenolic compounds at high concentrations are known to form insoluble complexes with proteins. We hypothesized that this complex formation could interfere with Western blot and ELISA assays for peanut allergens. To verify this, three simple phenolic compounds (ferulic, caffeic, and chlorogenic acids...

  3. Chemodynamics of metal complexation by natural soft colloids: Cu(II) binding by humic acid

    NARCIS (Netherlands)

    Town, R.M.; Duval, J.F.L.; Buffle, J.; Leeuwen, van H.P.

    2012-01-01

    The chemodynamics of Cu(II) complexation by humic acid is interpreted in terms of recently developed theory for permeable charged nanoparticles. Two opposing electric effects are operational with respect to the overall rate of association, namely, (i) the conductive enhancement of the diffusion of

  4. Characterization of DNA-binding sequences for CcaR in the cephamycin-clavulanic acid supercluster of Streptomyces clavuligerus.

    Science.gov (United States)

    Santamarta, I; López-García, M T; Kurt, A; Nárdiz, N; Alvarez-Álvarez, R; Pérez-Redondo, R; Martín, J F; Liras, P

    2011-08-01

    RT-PCR analysis of the genes in the clavulanic acid cluster revealed three transcriptional polycistronic units that comprised the ceaS2-bls2-pah2-cas2, cyp-fd-orf12-orf13 and oppA2-orf16 genes, whereas oat2, car, oppA1, claR, orf14, gcaS and pbpA were expressed as monocistronic transcripts. Quantitative RT-PCR of Streptomyces clavuligerus ATCC 27064 and the mutant S. clavuligerus ccaR::aph showed that, in the mutant, there was a 1000- to 10,000-fold lower transcript level for the ceaS2 to cas2 polycistronic transcript that encoded CeaS2, the first enzyme of the clavulanic acid pathway that commits arginine to clavulanic acid biosynthesis. Smaller decreases in expression were observed in the ccaR mutant for other genes in the cluster. Two-dimensional electrophoresis and MALDI-TOF analysis confirmed the absence in the mutant strain of proteins CeaS2, Bls2, Pah2 and Car that are required for clavulanic acid biosynthesis, and CefF and IPNS that are required for cephamycin biosynthesis. Gel shift electrophoresis using recombinant r-CcaR protein showed that it bound to the ceaS2 and claR promoter regions in the clavulanic acid cluster, and to the lat, cefF, cefD-cmcI and ccaR promoter regions in the cephamycin C gene cluster. Footprinting experiments indicated that triple heptameric conserved sequences were protected by r-CcaR, and allowed identification of heptameric sequences as CcaR binding sites.

  5. Acidic pH-Induced Conformations and LAMP1 Binding of the Lassa Virus Glycoprotein Spike.

    Directory of Open Access Journals (Sweden)

    Sai Li

    2016-02-01

    Full Text Available Lassa virus is an enveloped, bi-segmented RNA virus and the most prevalent and fatal of all Old World arenaviruses. Virus entry into the host cell is mediated by a tripartite surface spike complex, which is composed of two viral glycoprotein subunits, GP1 and GP2, and the stable signal peptide. Of these, GP1 binds to cellular receptors and GP2 catalyzes fusion between the viral envelope and the host cell membrane during endocytosis. The molecular structure of the spike and conformational rearrangements induced by low pH, prior to fusion, remain poorly understood. Here, we analyzed the three-dimensional ultrastructure of Lassa virus using electron cryotomography. Sub-tomogram averaging yielded a structure of the glycoprotein spike at 14-Å resolution. The spikes are trimeric, cover the virion envelope, and connect to the underlying matrix. Structural changes to the spike, following acidification, support a viral entry mechanism dependent on binding to the lysosome-resident receptor LAMP1 and further dissociation of the membrane-distal GP1 subunits.

  6. Acidic pH-Induced Conformations and LAMP1 Binding of the Lassa Virus Glycoprotein Spike.

    Science.gov (United States)

    Li, Sai; Sun, Zhaoyang; Pryce, Rhys; Parsy, Marie-Laure; Fehling, Sarah K; Schlie, Katrin; Siebert, C Alistair; Garten, Wolfgang; Bowden, Thomas A; Strecker, Thomas; Huiskonen, Juha T

    2016-02-01

    Lassa virus is an enveloped, bi-segmented RNA virus and the most prevalent and fatal of all Old World arenaviruses. Virus entry into the host cell is mediated by a tripartite surface spike complex, which is composed of two viral glycoprotein subunits, GP1 and GP2, and the stable signal peptide. Of these, GP1 binds to cellular receptors and GP2 catalyzes fusion between the viral envelope and the host cell membrane during endocytosis. The molecular structure of the spike and conformational rearrangements induced by low pH, prior to fusion, remain poorly understood. Here, we analyzed the three-dimensional ultrastructure of Lassa virus using electron cryotomography. Sub-tomogram averaging yielded a structure of the glycoprotein spike at 14-Å resolution. The spikes are trimeric, cover the virion envelope, and connect to the underlying matrix. Structural changes to the spike, following acidification, support a viral entry mechanism dependent on binding to the lysosome-resident receptor LAMP1 and further dissociation of the membrane-distal GP1 subunits.

  7. Acidic pH-Induced Conformations and LAMP1 Binding of the Lassa Virus Glycoprotein Spike.

    Directory of Open Access Journals (Sweden)

    Sai Li

    2016-02-01

    Full Text Available Lassa virus is an enveloped, bi-segmented RNA virus and the most prevalent and fatal of all Old World arenaviruses. Virus entry into the host cell is mediated by a tripartite surface spike complex, which is composed of two viral glycoprotein subunits, GP1 and GP2, and the stable signal peptide. Of these, GP1 binds to cellular receptors and GP2 catalyzes fusion between the viral envelope and the host cell membrane during endocytosis. The molecular structure of the spike and conformational rearrangements induced by low pH, prior to fusion, remain poorly understood. Here, we analyzed the three-dimensional ultrastructure of Lassa virus using electron cryotomography. Sub-tomogram averaging yielded a structure of the glycoprotein spike at 14-Å resolution. The spikes are trimeric, cover the virion envelope, and connect to the underlying matrix. Structural changes to the spike, following acidification, support a viral entry mechanism dependent on binding to the lysosome-resident receptor LAMP1 and further dissociation of the membrane-distal GP1 subunits.

  8. Combining substrate dynamics, binding statistics, and energy barriers to rationalize regioselective hydroxylation of octane and lauric acid by CYP102A1 and mutants.

    Science.gov (United States)

    Feenstra, K Anton; Starikov, Eugene B; Urlacher, Vlada B; Commandeur, Jan N M; Vermeulen, Nico P E

    2007-03-01

    Hydroxylations of octane and lauric acid by Cytochrome P450-BM3 (CYP102A1) wild-type and three active site mutants--F87A, L188Q/A74G, and F87V/L188Q/A74G--were rationalized using a combination of substrate orientation from docking, substrate binding statistics from molecular dynamics simulations, and barrier energies for hydrogen atom abstraction from quantum mechanical calculations. Wild-type BM3 typically hydroxylates medium- to long-chain fatty acids on subterminal (omega-1, omega-2, omega-3) but not the terminal (omega) positions. The known carboxylic anchoring site Y51/R47 for lauric acid, and hydrophobic interactions and steric exclusion, mainly by F87, for octane as well as lauric acid, play a role in the binding modes of the substrates. Electrostatic interactions between the protein and the substrate strongly modulate the substrate's regiodependent activation barriers. A combination of the binding statistics and the activation barriers of hydrogen-atom abstraction in the substrates is proposed to determine the product formation. Trends observed in experimental product formation for octane and lauric acid by wild-type BM3 and the three active site mutants were qualitatively explained. It is concluded that the combination of substrate binding statistics and hydrogen-atom abstraction barrier energies is a valuable tool to rationalize substrate binding and product formation and constitutes an important step toward prediction of product ratios.

  9. Molecular characterization, tissue expression, and polymorphism analysis of liver-type fatty acid binding protein in Landes geese.

    Science.gov (United States)

    Song, Z; Shao, D; Sun, X X; Niu, J W; Gong, D Q

    2015-01-01

    Liver weight is an important economic trait in the fatty goose liver industry. Liver-type fatty acid binding protein (L-FABP) is involved in the formation and metabolism of fatty acids. Thus, we hypothesized that sequence polymorphisms in L-FABP were associated with fatty liver weight in goose. We first isolated, sequenced, and characterized the goose L-FABP gene, which had not been previously reported. The goose L-FABP gene was 2490 bp and included 4 exons coding for a 126-amino acid protein. Analysis of expression levels of the goose L-FABP gene in different tissues showed that the expression level in the liver tissue was higher than in other tissues, and was significantly higher in the liver tissue of overfed geese than in control geese. Moreover, a single nucleotide polymorphism located at 774 bp in the gene was identified in a Landes goose population. To test whether this single nucleotide polymorphism was associated with fatty liver production, liver weight and the ratio of liver to carcass weights were determined for the 3 genotypes with this single nucleotide polymorphism (TT, TG, GG) in overfed Landes geese. Our data indicate that individuals with the GG genotype had higher values for the variables measured than those with the other 2 genotypes, suggesting that L-FABP can be a selection marker for the trait of fatty liver production in goose.

  10. Midkine, heparin-binding growth factor, blocks kainic acid-induced seizure and neuronal cell death in mouse hippocampus

    Directory of Open Access Journals (Sweden)

    Lim In J

    2010-03-01

    Full Text Available Abstract Background Midkine (MK, a member of the heparin-binding growth factor family, which includes MK and pleiotrophin, is known to possess neurotrophic and neuroprotective properties in the central nervous system. Previous studies have shown that MK is an effective neuroprotective agent in reducing retinal degeneration caused by excessive light and decreasing hippocampal neuronal death in ischemic gerbil brain. The present study was undertaken to investigate whether MK acts as an anticonvulsant in kainic acid (KA-induced seizure in mouse and blocks KA-mediated neuronal cell death in hippocampus. Results Increased expression of MK was found in hippocampus of mouse following seizures induced by intracerebroventricular injection of KA, and MK expression was found in glial fibrillary acidic protein (GFAP-positive astrocytes. Concurrent injection of MK and KA attenuated KA-induced seizure activity and cell death of hippocampal neurons including pyramidal cells and glutamic acid decarboxylase 67 (GAD67-positive GABAergic interneurons in the CA3 and hilar area. Conclusion The results of the present study indicate that MK functions as an anticonvulsant and neuroprotective agent in hippocampus during KA-induced seizures.

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

    Science.gov (United States)

    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. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. An acyl-CoA-binding protein (FcACBP) and a fatty acid binding protein (FcFABP) respond to microbial infection in Chinese white shrimp, Fenneropenaeus chinensis.

    Science.gov (United States)

    Ren, Qian; Du, Zhi-Qiang; Zhao, Xiao-Fan; Wang, Jin-Xing

    2009-12-01

    Acyl-CoA-binding protein (ACBP) and fatty acid-binding protein (FABP) are involved in lipid metabolism. ACBP plays a key role in multiple cellular tasks including modulation of fatty acid biosynthesis, enzyme regulation, vesicular trafficking, and gene regulation. In our study, a 536 bp cDNA of ACBP (FcACBP) was cloned and identified as a widely distributed gene in the Chinese white shrimp, Fenneropenaeus chinensis. Its expression in intestine was upregulated in response to white spot syndrome virus (WSSV) or Vibrio anguillarum infection. The expression patterns were confirmed by Western blot analysis. FABPs, members of the lipid-binding protein superfamily, play an important role in lipid metabolism and also participate in vertebrate innate immunity. A cDNA of FABP (FcFABP) cloned from the hepatopancreas of the shrimp was 715 bp in size and encoded a 14 kDa protein. FcFABP appeared to be a basic fatty acid binding protein with a predicted isoelectric point of 9.16. It showed sequence similarity to both vertebrate and invertebrate FABPs. Phylogenetic analysis showed that FcFABP, together with LvFABP, were clustered into one group. FcFABP was detected mainly in the hepatopancreas and expression level increased after a challenge with WSSV. FcFABP was down-regulated by V. anguillarum challenge. The protein also had bacterial binding activity. These two lipid metabolism related proteins may play important roles in shrimp innate immunity.

  13. Lysine-functionalized nanodiamonds: synthesis, physiochemical characterization, and nucleic acid binding studies

    Directory of Open Access Journals (Sweden)

    Kaur R

    2012-07-01

    Full Text Available Randeep Kaur,1 Jackson M Chitanda,2 Deborah Michel,1 Jason Maley,3 Ferenc Borondics,2,4 Peng Yang,5 Ronald E Verrall,2 Ildiko Badea11Drug Design and Discovery Research Group, College of Pharmacy and Nutrition, University of Saskatchewan, 2Department of Chemistry, University of Saskatchewan, 3Saskatchewan Structural Sciences Centre, University of Saskatchewan, 4Canadian Light Source, University of Saskatchewan, Saskatoon, SK, Canada; 5Department of Organic Chemistry, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang, People's Republic of ChinaPurpose: Detonation nanodiamonds (NDs are carbon-based nanomaterials that, because of their size (4–5 nm, stable inert core, alterable surface chemistry, fluorescence, and biocompatibility, are emerging as bioimaging agents and promising tools for the delivery of biochemical molecules into cellular systems. However, diamond particles possess a strong propensity to aggregate in liquid formulation media, restricting their applicability in biomedical sciences. Here, the authors describe the covalent functionalization of NDs with lysine in an attempt to develop nanoparticles able to act as suitable nonviral vectors for transferring genetic materials across cellular membranes.Methods: NDs were oxidized and functionalized by binding lysine moieties attached to a three-carbon-length linker (1,3-diaminopropane to their surfaces through amide bonds. Raman and Fourier transform infrared spectroscopy, zeta potential measurement, dynamic light scattering, atomic force microscopic imaging, and thermogravimetric analysis were used to characterize the lysine-functionalized NDs. Finally, the ability of the functionalized diamonds to bind plasmid DNA and small interfering RNA was investigated by gel electrophoresis assay and through size and zeta potential measurements.Results: NDs were successfully functionalized with the lysine linker, producing surface loading of 1.7 mmol g-1 of ND

  14. Visualization of Iron-Binding Micelles in Acidic Recombinant Biomineralization Protein, MamC

    OpenAIRE

    Sanjay Kashyap; Taylor Woehl; Carmen Valverde-Tercedor; Miguel Sánchez-Quesada; Concepción Jiménez López; Tanya Prozorov

    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, includin...

  15. Substitution of Ala564 in the first zinc cluster of the deoxyribonucleic acid (DNA)-binding domain of the androgen receptor by Asp, Asn, or Leu exerts differential effects on DNA binding

    NARCIS (Netherlands)

    H.T. Brüggenwirth (Hennie); A.L.M. Boehmer (Annemie); J.M. Lobaccaro; L. Chiche; C. Sultan; J. Trapman (Jan); A.O. Brinkmann (Albert)

    1998-01-01

    textabstractIn the androgen receptor of a patient with androgen insensitivity, the alanine residue at position 564 in the first zinc cluster of the DNA-binding domain was substituted by aspartic acid. In other members of the steroid receptor family, either valine or ala

  16. Binding site of ribosomal proteins on prokaryotic 5S ribonucleic acids: a study with ribonucleases

    DEFF Research Database (Denmark)

    Douthwaite, S; Christensen, A; Garrett, R A

    1982-01-01

    The binding sites of ribosomal proteins L18 and L25 on 5S RNA from Escherichia coli were probed with ribonucleases A, T1, and T2 and a double helix specific cobra venom endonuclease. The results for the protein-RNA complexes, which were compared with those for the free RNA [Douthwaite, S...... stearothermophilus 5S RNA. Several protein-induced changes in the RNA structures were identified; some are possibly allosteric in nature. The two prokaryotic 5S RNAs were also incubated with total 50S subunit proteins from E. coli and B. stearothermophilus ribosomes. Homologous and heterologous reconstitution....... stearothermophilus 5S RNA, which may have been due to a third ribosomal protein L5....

  17. RBscore&NBench: a high-level web server for nucleic acid binding residues prediction with a large-scale benchmarking database.

    Science.gov (United States)

    Miao, Zhichao; Westhof, Eric

    2016-07-08

    RBscore&NBench combines a web server, RBscore and a database, NBench. RBscore predicts RNA-/DNA-binding residues in proteins and visualizes the prediction scores and features on protein structures. The scoring scheme of RBscore directly links feature values to nucleic acid binding probabilities and illustrates the nucleic acid binding energy funnel on the protein surface. To avoid dataset, binding site definition and assessment metric biases, we compared RBscore with 18 web servers and 3 stand-alone programs on 41 datasets, which demonstrated the high and stable accuracy of RBscore. A comprehensive comparison led us to develop a benchmark database named NBench. The web server is available on: http://ahsoka.u-strasbg.fr/rbscorenbench/.

  18. MAZ-binding G4-decoy with locked nucleic acid and twisted intercalating nucleic acid modifications suppresses KRAS in pancreatic cancer cells and delays tumor growth in mice

    DEFF Research Database (Denmark)

    Cogoi, Susanna; Zorzet, Sonia; Rapozzi, Valentina;

    2013-01-01

    KRAS mutations are primary genetic lesions leading to pancreatic cancer. The promoter of human KRAS contains a nuclease-hypersensitive element (NHE) that can fold in G4-DNA structures binding to nuclear proteins, including MAZ (myc-associated zinc-finger). Here, we report that MAZ activates KRAS...... transcription. To knockdown oncogenic KRAS in pancreatic cancer cells, we designed oligonucleotides that mimic one of the G-quadruplexes formed by NHE (G4-decoys). To increase their nuclease resistance, two locked nucleic acid (LNA) modifications were introduced at the 3'-end, whereas to enhance the folding...... the Kaplan-Meier median survival time by 70%. Together, our data show that MAZ-specific G4-decoys mimicking a KRAS quadruplex are promising for pancreatic cancer therapy....

  19. Association of polymorphisms in adipocyte fatty acid binding protein gene with fat-related traits in chicken

    Institute of Scientific and Technical Information of China (English)

    Manhong YE; Jie WEN; Honghe CAO; Hongbin LI; Jilan CHEN; Guiping ZHAO; Maiqing ZHENG

    2008-01-01

    PCR-SSCP analysis was used to detect poly-morphic sites in chicken adipocyte fatty acid binding pro-tein (A-FABP) gene. Six Chinese local breeds, Beijing-You chicken, Dwarf chicken, Taihe silky chicken, Chong-renma chicken, Xiayan chicken, Luyuan chicken and an introduced foreign breed, Arbor Acre broiler, were used as test populations. Three PCR-SSCP loci were detected. Statistical results showed that frequencies of genotypes and alleles were significantly different in the test popula-tions. Sequence analysis revealed that C → T, G → A, and C → T transitions were responsible for the polymorph-isms. Some fat-related traits such as body weight, content of intramuscular fat (IMF) and percentage of abdominal fat (AFP) were measured in Dwarf chickens and male Beijing-You chickens. We found out that chicken quality was significantly related to different genotypes in these two populations.

  20. TGD4 involved in endoplasmic reticulum-to-chloroplast lipid trafficking is a phosphatidic acid binding protein

    Energy Technology Data Exchange (ETDEWEB)

    Wang Z.; Xu C.; Benning, C.

    2012-05-01

    The synthesis of galactoglycerolipids, which are prevalent in photosynthetic membranes, involves enzymes at the endoplasmic reticulum (ER) and the chloroplast envelope membranes. Genetic analysis of trigalactosyldiacylglycerol (TGD) proteins in Arabidopsis has demonstrated their role in polar lipid transfer from the ER to the chloroplast. The TGD1, 2, and 3 proteins resemble components of a bacterial-type ATP-binding cassette (ABC) transporter, with TGD1 representing the permease, TGD2 the substrate binding protein, and TGD3 the ATPase. However, the function of the TGD4 protein in this process is less clear and its location in plant cells remains to be firmly determined. The predicted C-terminal {beta}-barrel structure of TGD4 is weakly similar to proteins of the outer cell membrane of Gram-negative bacteria. Here, we show that, like TGD2, the TGD4 protein when fused to DsRED specifically binds phosphatidic acid (PtdOH). As previously shown for tgd1 mutants, tgd4 mutants have elevated PtdOH content, probably in extraplastidic membranes. Using highly purified and specific antibodies to probe different cell fractions, we demonstrated that the TGD4 protein was present in the outer envelope membrane of chloroplasts, where it appeared to be deeply buried within the membrane except for the N-terminus, which was found to be exposed to the cytosol. It is proposed that TGD4 is either directly involved in the transfer of polar lipids, possibly PtdOH, from the ER to the outer chloroplast envelope membrane or in the transfer of PtdOH through the outer envelope membrane.

  1. A Density Functional Tight Binding Study of Acetic Acid Adsorption on Crystalline and Amorphous Surfaces of Titania

    Directory of Open Access Journals (Sweden)

    Sergei Manzhos

    2015-02-01

    Full Text Available We present a comparative density functional tight binding study of an organic molecule attachment to TiO2 via a carboxylic group, with the example of acetic acid. For the first time, binding to low-energy surfaces of crystalline anatase (101, rutile (110 and (B-TiO2 (001, as well as to the surface of amorphous (a- TiO2 is compared with the same computational setup. On all surfaces, bidentate configurations are identified as providing the strongest adsorption energy, Eads = −1.93, −2.49 and −1.09 eV for anatase, rutile and (B-TiO2, respectively. For monodentate configurations, the strongest Eads = −1.06, −1.11 and −0.86 eV for anatase, rutile and (B-TiO2, respectively. Multiple monodentate and bidentate configurations are identified on a-TiO2 with a distribution of adsorption energies and with the lowest energy configuration having stronger bonding than that of the crystalline counterparts, with Eads up to −4.92 eV for bidentate and −1.83 eV for monodentate adsorption. Amorphous TiO2 can therefore be used to achieve strong anchoring of organic molecules, such as dyes, that bind via a -COOH group. While the presence of the surface leads to a contraction of the band gap vs. the bulk, molecular adsorption caused no appreciable effect on the band structure around the gap in any of the systems.

  2. Silkworm apolipophorin protein inhibits hemolysin gene expression of Staphylococcus aureus via binding to cell surface lipoteichoic acids.

    Science.gov (United States)

    Omae, Yosuke; Hanada, Yuichi; Sekimizu, Kazuhisa; Kaito, Chikara

    2013-08-30

    We previously reported that a silkworm hemolymph protein, apolipophorin (ApoLp), binds to the cell surface of Staphylococcus aureus and inhibits expression of the saePQRS operon encoding a two-component system, SaeRS, and hemolysin genes. In this study, we investigated the inhibitory mechanism of ApoLp on S. aureus hemolysin gene expression. ApoLp bound to lipoteichoic acids (LTA), an S. aureus cell surface component. The addition of purified LTA to liquid medium abolished the inhibitory effect of ApoLp against S. aureus hemolysin production. In an S. aureus knockdown mutant of ltaS encoding LTA synthetase, the inhibitory effects of ApoLp on saeQ expression and hemolysin production were attenuated. Furthermore, the addition of anti-LTA monoclonal antibody to liquid medium decreased the expression of S. aureus saeQ and hemolysin genes. In S. aureus strains expressing SaeS mutant proteins with a shortened extracellular domain, ApoLp did not decrease saeQ expression. These findings suggest that ApoLp binds to LTA on the S. aureus cell surface and inhibits S. aureus hemolysin gene expression via a two-component regulatory system, SaeRS.

  3. Structural basis for the assembly and nucleic acid binding of the TREX-2 transcription-export complex.

    Science.gov (United States)

    Ellisdon, Andrew M; Dimitrova, Lyudmila; Hurt, Ed; Stewart, Murray

    2012-02-19

    The conserved TREX-2 transcription-export complex integrates transcription and processing of many actively transcribed nascent mRNAs with the recruitment of export factors at nuclear pores and also contributes to transcriptional memory and genomic stability. We report the crystal structure of the Sac3-Thp1-Sem1 segment of Saccharomyces cerevisiae TREX-2 that interfaces with the gene expression machinery. Sac3-Thp1-Sem1 forms a previously uncharacterized PCI-domain complex characterized by the juxtaposition of Sac3 and Thp1 winged helix domains, forming a platform that mediates nucleic acid binding. Our structure-guided mutations support the idea that the Thp1-Sac3 interaction is an essential requirement for mRNA binding and for the coupling of transcription and processing to mRNP assembly and export. These results provide insight into how newly synthesized transcripts are efficiently transferred from TREX-2 to the principal mRNA export factor, and they reveal how Sem1 stabilizes PCI domain-containing proteins and promotes complex assembly.

  4. A highly Conserved Aspartic Acid Residue of the Chitosanase from Bacillus Sp. TS Is Involved in the Substrate Binding.

    Science.gov (United States)

    Zhou, Zhanping; Zhao, Shuangzhi; Liu, Yang; Chang, Zhengying; Ma, Yanhe; Li, Jian; Song, Jiangning

    2016-11-01

    The chitosanase from Bacillus sp. TS (CsnTS) is an enzyme belonging to the glycoside hydrolase family 8. The sequence of CsnTS shares 98 % identity with the chitosanase from Bacillus sp. K17. Crystallography analysis and site-direct mutagenesis of the chitosanase from Bacillus sp. K17 identified the important residues involved in the catalytic interaction and substrate binding. However, despite progress in understanding the catalytic mechanism of the chitosanase from the family GH8, the functional roles of some residues that are highly conserved throughout this family have not been fully elucidated. This study focused on one of these residues, i.e., the aspartic acid residue at position 318. We found that apart from asparagine, mutation of Asp318 resulted in significant loss of enzyme activity. In-depth investigations showed that mutation of this residue not only impaired enzymatic activity but also affected substrate binding. Taken together, our results showed that Asp318 plays an important role in CsnTS activity.

  5. Perbedaan Kadar Liver Fatty Acid Binding Protein (L-FABP Urine Penderita Diabetes Melitus Tipe 2 dengan Normoalbuminuria dan Mikroalbuminuria

    Directory of Open Access Journals (Sweden)

    Kristina Wiharjo

    2014-06-01

    Full Text Available Diabetes mellitus (DM is the leading cause of the end stage renal disease (ESRD. Around 20−40% patients with DM develop diabetic nephropathy and eventually progress into ESRD. Type 2 DM has a greater prevalence to develop diabetic nephropathy. Oxidative stress accumulation can increase permeability of the glomerulus which results in increased urine albumin excretion, which is divided into three groups: normoalbuminuria, microalbuminuria and macroalbuminuria. Glomerulus dysfunction occurs after tubulointerstisial renal dysfunction which decreases peritubular capillary flow that leads to tubulointerstisial hypoxia. Liver fatty acid binding protein function is to reduce hypoxia by binding oxidative stress and excretes it into urine. The aim of this study was to analyze the differences in the urine L-FABP level between normoalbuminuria and microalbuminuria type 2 DM patients. The study design was observational analytic using cross-sectional method. Subjects were 70 DM type 2 patients with normoalbuminuria (38 patients and microalbuminuria (32 patients. Statistical analysis was performed using the Mann Whitney test The results found that there were significant differences in levels of urine L-FABP between normoalbuminuria and microalbuminuria type 2 DM patients (ZM-W=3.513, p<0.001 with medians of 5 and 7 in normoalbuminuria and microalbuminuria, respectively. The urine L-FABP level increased because of the oxidative stress and hypoxia that happened before the glomerulus dysfunction. In conclusion, urine L-FABP level in patients DM type 2 with microalbuminuria is higher than that of the normoalbuminuria.

  6. Specific amino acid residues are involved in substrate discrimination and template binding of human REV1 protein.

    Science.gov (United States)

    Piao, Jinlian; Masuda, Yuji; Kamiya, Kenji

    2010-02-05

    REV1 is a member of the Y-family DNA polymerases, but is atypical in utilizing only dCTP with a preference for guanine (G) as the template. Crystallography of the REV1-DNA-dCTP ternary complex has revealed a unique mechanism by which template G is evicted from the DNA helix and incoming dCTP is recognized by an arginine residue in an alpha-loop, termed the N-digit. To better understand functions of its individual amino acid residues, we made a series of mutant human REV1 proteins. We found that R357 and L358 play vital roles in template binding. Furthermore, extensive mutation analysis revealed a novel function of R357 for substrate discrimination, in addition to previously proposed specific interaction with incoming dCTP. We found that the binding pocket for dCTP of REV1 has also significant but latent affinity for dGTP. The results suggest that the positive charge on R357 could prevent interaction with dGTP. We propose that both direct and indirect mechanisms mediated by R357 ensure specificity for dCTP.

  7. A single amino acid substitution in the group 1 Trypanosoma brucei gambiense haptoglobin-hemoglobin receptor abolishes TLF-1 binding.

    Directory of Open Access Journals (Sweden)

    E DeJesus

    Full Text Available Critical to human innate immunity against African trypanosomes is a minor subclass of human high-density lipoproteins, termed Trypanosome Lytic Factor-1 (TLF-1. This primate-specific molecule binds to a haptoglobin-hemoglobin receptor (HpHbR on the surface of susceptible trypanosomes, initiating a lytic pathway. Group 1 Trypanosoma brucei gambiense causes human African Trypanosomiasis (HAT, escaping TLF-1 killing due to reduced uptake. Previously, we found that group 1 T. b. gambiense HpHbR (TbgHpHbR mRNA levels were greatly reduced and the gene contained substitutions within the open reading frame. Here we show that a single, highly conserved amino acid in the TbgHpHbR ablates high affinity TLF-1 binding and subsequent endocytosis, thus evading TLF-1 killing. In addition, we show that over-expression of TbgHpHbR failed to rescue TLF-1 susceptibility. These findings suggest that the single substitution present in the TbgHpHbR directly contributes to the reduced uptake and resistance to TLF-1 seen in these important human pathogens.

  8. CLINICAL EXPERIENCE OF CANCER IMMUNOTHERAPY INTEGRATED WITH OLEIC ACID COMPLEXED WITH DE-GLYCOSYLATED VITAMIN D BINDING PROTEIN

    Directory of Open Access Journals (Sweden)

    Emma Ward

    2014-01-01

    Full Text Available Proteins highly represented in milk such as α-lactalbumin and lactoferrin bind Oleic Acid (OA to form complexes with selective anti-tumor activity. A protein present in milk, colostrum and blood, vitamin D binding protein is the precursor of a potent Macrophage Activating Factor (GcMAF and in analogy with other OA-protein complexes, we proposed that OA-GcMAF could demonstrate a greater immunotherapeutic activity than that of GcMAF alone. We describe a preliminary experience treating patients with advanced cancers, often labelled as “incurable” with an integrative immunotherapy centred on OA-GcMAF. Patients with advanced cancer were treated at the Immuno Biotech Treatment Centre with OA-GcMAF-based integrative immunotherapy in combination with a very low carbohydrate, high protein diet, fermented milk products containing naturally produced GcMAF, vitamin D3 and low-dose acetylsalicylic acid. When the primary tumor or a metastasis could be measured by ultrasonographic techniques, we observed, on average, a decrease of tumor volume of approximately 25% in a week. We also observed a consistent increase in splenic blood flow that was interpreted in the context of generalised immune system activation and allowed to assess the degree of responsiveness of the individual patient. The results reported here are consistent with the results previously described in the experimental animal harbouring a human hepatocellular carcinoma as well as with the results reported for neoadjuvant chemotherapy. OA-protein complexes are bound to play a leading role in cancer therapy thanks to selectivity of antitumoral effects, absence of any side effects, safety and oral availability. We hypothesise that OA-GcMAF, combines the known anticancer effects OA-protein complexes with the well established immune stimulating effects of GcMAF.

  9. Intestinal fatty acid binding protein Ala54Thr polymorphism is associated with peripheral atherosclerosis combined with type 2 diabetes mellitus.

    Science.gov (United States)

    Khattab, Salma A; Abo-Elmatty, Dina M; Ghattas, Maivel H; Mesbah, Noha M; Mehanna, Eman T

    2017-09-01

    Intestinal fatty acid-binding protein 2 (FABP2) is expressed in enterocytes and binds saturated and unsaturated long-chain fatty acids. The FABP2 Ala54Thr polymorphism has been reported to effect lipid metabolism. The aim of the present study was to assess the relationship between this polymorphism and peripheral atherosclerosis combined with type 2 diabetes mellitus (T2DM) in an Egyptian population. The study was performed on 100 T2DM patients with peripheral atherosclerosis and 100 control subjects. The Ala54Thr polymorphism was analyzed by polymerase chain reaction-restriction fragment length polymorphism, whereas serum FABP2 levels were determined using ELISA. Fasting blood glucose, fasting serum insulin concentrations, HbA1c, lipid profile, body mass index (BMI) and systolic and diastolic blood pressure (SBP and DBP, respectively) were determined. There was a higher frequency of the Thr54 allele among the patient group (P = 0.002). In Ala54/Thr54 heterozygotes and carriers of the rare Thr54/Thr54 genotype, there were significant increases in BMI and FABP2. Those with the Thr54/Thr54 genotype had significantly decreased high-density lipoprotein cholesterol (HDL-C) concentrations; in addition, those with the Thr54/Thr54 genotype had significantly higher SBP and DBP than subjects with the Ala54/Ala54 and Ala54/Thr54 genotypes. There was a positive correlation between FABP2 levels and BMI, SBP and DBP, and a negative correlation with HDL-C. The Thr54 allele of the FABP2 Ala54Thr polymorphism was associated with an increased incidence of peripheral atherosclerosis combined with T2DM in the population studied. © 2016 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and John Wiley & Sons Australia, Ltd.

  10. Fatty acid-binding proteins (FABPs) are intracellular carriers for Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD).

    Science.gov (United States)

    Elmes, Matthew W; Kaczocha, Martin; Berger, William T; Leung, KwanNok; Ralph, Brian P; Wang, Liqun; Sweeney, Joseph M; Miyauchi, Jeremy T; Tsirka, Stella E; Ojima, Iwao; Deutsch, Dale G

    2015-04-03

    Δ(9)-Tetrahydrocannabinol (THC) and cannabidiol (CBD) occur naturally in marijuana (Cannabis) and may be formulated, individually or in combination in pharmaceuticals such as Marinol or Sativex. Although it is known that these hydrophobic compounds can be transported in blood by albumin or lipoproteins, the intracellular carrier has not been identified. Recent reports suggest that CBD and THC elevate the levels of the endocannabinoid anandamide (AEA) when administered to humans, suggesting that phytocannabinoids target cellular proteins involved in endocannabinoid clearance. Fatty acid-binding proteins (FABPs) are intracellular proteins that mediate AEA transport to its catabolic enzyme fatty acid amide hydrolase (FAAH). By computational analysis and ligand displacement assays, we show that at least three human FABPs bind THC and CBD and demonstrate that THC and CBD inhibit the cellular uptake and catabolism of AEA by targeting FABPs. Furthermore, we show that in contrast to rodent FAAH, CBD does not inhibit the enzymatic actions of human FAAH, and thus FAAH inhibition cannot account for the observed increase in circulating AEA in humans following CBD consumption. Using computational molecular docking and site-directed mutagenesis we identify key residues within the active site of FAAH that confer the species-specific sensitivity to inhibition by CBD. Competition for FABPs may in part or wholly explain the increased circulating levels of endocannabinoids reported after consumption of cannabinoids. These data shed light on the mechanism of action of CBD in modulating the endocannabinoid tone in vivo and may explain, in part, its reported efficacy toward epilepsy and other neurological disorders.

  11. Fatty Acid-binding Proteins (FABPs) Are Intracellular Carriers for Δ9-Tetrahydrocannabinol (THC) and Cannabidiol (CBD)*

    Science.gov (United States)

    Elmes, Matthew W.; Kaczocha, Martin; Berger, William T.; Leung, KwanNok; Ralph, Brian P.; Wang, Liqun; Sweeney, Joseph M.; Miyauchi, Jeremy T.; Tsirka, Stella E.; Ojima, Iwao; Deutsch, Dale G.

    2015-01-01

    Δ9-Tetrahydrocannabinol (THC) and cannabidiol (CBD) occur naturally in marijuana (Cannabis) and may be formulated, individually or in combination in pharmaceuticals such as Marinol or Sativex. Although it is known that these hydrophobic compounds can be transported in blood by albumin or lipoproteins, the intracellular carrier has not been identified. Recent reports suggest that CBD and THC elevate the levels of the endocannabinoid anandamide (AEA) when administered to humans, suggesting that phytocannabinoids target cellular proteins involved in endocannabinoid clearance. Fatty acid-binding proteins (FABPs) are intracellular proteins that mediate AEA transport to its catabolic enzyme fatty acid amide hydrolase (FAAH). By computational analysis and ligand displacement assays, we show that at least three human FABPs bind THC and CBD and demonstrate that THC and CBD inhibit the cellular uptake and catabolism of AEA by targeting FABPs. Furthermore, we show that in contrast to rodent FAAH, CBD does not inhibit the enzymatic actions of human FAAH, and thus FAAH inhibition cannot account for the observed increase in circulating AEA in humans following CBD consumption. Using computational molecular docking and site-directed mutagenesis we identify key residues within the active site of FAAH that confer the species-specific sensitivity to inhibition by CBD. Competition for FABPs may in part or wholly explain the increased circulating levels of endocannabinoids reported after consumption of cannabinoids. These data shed light on the mechanism of action of CBD in modulating the endocannabinoid tone in vivo and may explain, in part, its reported efficacy toward epilepsy and other neurological disorders. PMID:25666611

  12. Chromatographic analysis of the effects of fatty acids and glycation on binding by probes for Sudlow sites I and II to human serum albumin.

    Science.gov (United States)

    Anguizola, Jeanethe; Debolt, Erin; Suresh, D; Hage, David S

    2016-05-15

    The primary endogenous ligands of human serum albumin (HSA) are non-esterified fatty acids, with 0.1-2mol of fatty acids normally being bound to HSA. In type II diabetes, fatty acid levels in serum are often elevated, and the presence of high glucose results in an increase in the non-enzymatic glycation of HSA. High-performance affinity chromatography (HPAC) was used to examine the combined effects of glycation and the presence of long chain fatty acids on the binding of HSA with R-warfarin and l-tryptophan (i.e., probes for Sudlow sites I and II, the major sites for drugs on this protein). Zonal elution competition studies were used to examine the interactions of myristic acid, palmitic acid and stearic acid with these probes on HSA. It was found that all these fatty acids had direct competition with R-warfarin at Sudlow site I of normal HSA and glycated HSA, with the glycated HSA typically having stronger binding for the fatty acids at this site. At Sudlow site II, direct competition was observed for all the fatty acids with l-tryptophan when using normal HSA, while glycated HSA gave no competition or positive allosteric interactions between these fatty acids and l-tryptophan. These data indicated that glycation can alter the interactions of drugs and fatty acids at specific binding sites on HSA. The results of this study should lead to a better understanding of how these interactions may change during diabetes and demonstrate how HPAC can be used to examine drug/solute-protein interactions in complex systems.

  13. In vitro bile acid-binding capacity of dietary fibre sources and their effects with bile acid on broiler chicken performance and lipid digestibility.

    Science.gov (United States)

    Hemati Matin, H R; Shariatmadari, F; Karimi Torshizi, M A; Chiba, L I

    2016-06-01

    A 4 × 2 factorial experiment was conducted to study the effect of feeding diets-containing dietary fibre (DF) sources and a source of bile acid (BA) on growth performance and lipid metabolism. In addition, in vitro BA-binding capacity of fibre sources was investigated. A total of 256 one-d-old male broiler chickens (Ross 308) were assigned to DF sources [maize-soybean meal (control, C), or 30 g/kg of wheat bran (WB), barley bran (BB) or soybean hulls (SH)] and BA (with or without 1.5 g Na-deoxycholate/kg). Each treatment was replicated 4 times with 8 broiler chickens per cage. The highest in vitro BA-binding capacity was observed with BB (8.76 mg/g BB). From 0 to 21 d, with the addition of BA, the average daily feed intake (ADFI) decreased in broiler chickens fed on the C, WB or BB diets, while there was no difference with the SH diet. With added BA, the average daily gain decreased in broiler chickens fed on the C or SH diets, but it did not change in those fed on the other diets. The addition of BA decreased feed conversion ratio (FCR) in broiler chickens fed on the BB or WB diets, but it increased in those fed on the C or SH diets. Interaction results indicated that the apparent ileal digestibility of lipid increased in broiler chickens fed the C and other DF diets with BA compared to those fed the diets without BA. The addition of BA decreased the pancreas lipase activity (PLA) in broiler chickens fed on the C diet compared to those fed the C diet without BA, while no changes observed in those fed the DF diets with or without BA. No interaction was observed in total liver bile acid (TLBA). The WB, BB and SH with little Na-deoxycholate-binding capacity (chickens. The magnitude of improvement in digestibility of lipid with the addition of BA depends on the source of fibre used and the addition of BA in DF diets had little effect on growth performance in young broiler chicken diets.

  14. TmiRUSite and TmiROSite scripts: searching for mRNA fragments with miRNA binding sites with encoded amino acid residues

    OpenAIRE

    Berillo, Olga; Régnier, Mireille; Ivashchenko, Anatoly

    2014-01-01

    microRNAs are small RNA molecules that inhibit the translation of target genes. microRNA binding sites are located in the untranslated regions as well as in the coding domains. We describe TmiRUSite and TmiROSite scripts developed using python as tools for the extraction of nucleotide sequences for miRNA binding sites with their encoded amino acid residue sequences. The scripts allow for retrieving a set of additional sequences at left and at right from the binding site. The scripts presents ...

  15. TmiRUSite and TmiROSite scripts: searching for mRNA fragments with miRNA binding sites with encoded amino acid residues

    OpenAIRE

    Berillo, Olga; Régnier, Mireille; Ivashchenko, Anatoly

    2014-01-01

    microRNAs are small RNA molecules that inhibit the translation of target genes. microRNA binding sites are located in the untranslated regions as well as in the coding domains. We describe TmiRUSite and TmiROSite scripts developed using python as tools for the extraction of nucleotide sequences for miRNA binding sites with their encoded amino acid residue sequences. The scripts allow for retrieving a set of additional sequences at left and at right from the binding site. The scripts presents ...

  16. Homology modeling of Homo sapiens lipoic acid synthase: Substrate docking and insights on its binding mode.

    Science.gov (United States)

    Krishnamoorthy, Ezhilarasi; Hassan, Sameer; Hanna, Luke Elizabeth; Padmalayam, Indira; Rajaram, Rama; Viswanathan, Vijay

    2017-05-07

    Lipoic acid synthase (LIAS) is an iron-sulfur cluster mitochondrial enzyme which catalyzes the final step in the de novo pathway for the biosynthesis of lipoic acid, a potent antioxidant. Recently there has been significant interest in its role in metabolic diseases and its deficiency in LIAS expression has been linked to conditions such as diabetes, atherosclerosis and neonatal-onset epilepsy, suggesting a strong inverse correlation between LIAS reduction and disease status. In this study we use a bioinformatics approach to predict its structure, which would be helpful to understanding its role. A homology model for LIAS protein was generated using X-ray crystallographic structure of Thermosynechococcus elongatus BP-1 (PDB ID: 4U0P). The predicted structure has 93% of the residues in the most favour region of Ramachandran plot. The active site of LIAS protein was mapped and docked with S-Adenosyl Methionine (SAM) using GOLD software. The LIAS-SAM complex was further refined using molecular dynamics simulation within the subsite 1 and subsite 3 of the active site. To the best of our knowledge, this is the first study to report a reliable homology model of LIAS protein. This study will facilitate a better understanding mode of action of the enzyme-substrate complex for future studies in designing drugs that can target LIAS protein. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. How does fatty acid influence anti-thyroid drugs binding and specificity toward protein human serum albumin? A blind docking simulation study

    Indian Academy of Sciences (India)

    Bijan K Paul; Nikhil Guchhait

    2014-11-01

    This study reports an AutoDock-based blind docking simulation investigation to characterize the binding interaction of a series of anti-thyroid drugs (2-mercapto-1-methylimidazole (MMI), 2-thiouracil (TU), 6-methyl-2-thiouracil (MTU), 6--propyl-2-thiouracil (PTU) with a model plasma protein Human SerumAlbumin (HSA) in the presence and absence of fatty acid (FA). The drug-protein binding efficiency is characterized in terms of binding free energy and the association constant (Ka, which is estimated as the reciprocal of the inhibition constant, Ki) of the drugs to the transport protein. The study also unveils the substantial impact of the presence of fatty acid (FA) on the binding interaction process. It is shown that in the presence of FA the drug-protein binding efficiency is markedly enhanced (except for MTU) and the binding location is changed. Hydrogen bonding interaction appears to play a governing role in the process of FA-induced modifications of binding efficiency and location.

  18. Linking Spermatid Ribonucleic Acid (RNA) Binding Protein and Retrogene Diversity to Reproductive Success*

    Science.gov (United States)

    Chapman, Karen M.; Powell, Heather M.; Chaudhary, Jaideep; Shelton, John M.; Richardson, James A.; Richardson, Timothy E.; Hamra, F. Kent

    2013-01-01

    Spermiogenesis is a postmeiotic process that drives development of round spermatids into fully elongated spermatozoa. Spermatid elongation is largely controlled post-transcriptionally after global silencing of mRNA synthesis from the haploid genome. Here, rats that differentially express EGFP from a lentiviral transgene during early and late steps of spermiogenesis were used to flow sort fractions of round and elongating spermatids. Mass-spectral analysis of 2D gel protein spots enriched >3-fold in each fraction revealed a heterogeneous RNA binding proteome (hnRNPA2/b1, hnRNPA3, hnRPDL, hnRNPK, hnRNPL, hnRNPM, PABPC1, PABPC4, PCBP1, PCBP3, PTBP2, PSIP1, RGSL1, RUVBL2, SARNP2, TDRD6, TDRD7) abundantly expressed in round spermatids prior to their elongation. Notably, each protein within this ontology cluster regulates alternative splicing, sub-cellular transport, degradation and/or translational repression of mRNAs. In contrast, elongating spermatid fractions were enriched with glycolytic enzymes, redox enzymes and protein synthesis factors. Retrogene-encoded proteins were over-represented among the most abundant elongating spermatid factors identified. Consistent with these biochemical activities, plus corresponding histological profiles, the identified RNA processing factors are predicted to collectively drive post-transcriptional expression of an alternative exome that fuels finishing steps of sperm maturation and fitness. PMID:23938467

  19. In vitro RNase and nucleic acid binding activities implicate coilin in U snRNA processing.

    Directory of Open Access Journals (Sweden)

    Hanna J Broome

    Full Text Available Coilin is known as the marker protein for Cajal bodies (CBs, subnuclear domains important for the biogenesis of small nuclear ribonucleoproteins (snRNPs which function in pre-mRNA splicing. CBs associate non-randomly with U1 and U2 gene loci, which produce the small nuclear RNA (snRNA component of the respective snRNP. Despite recognition as the CB marker protein, coilin is primarily nucleoplasmic, and the function of this fraction is not fully characterized. Here we show that coilin binds double stranded DNA and has RNase activity in vitro. U1 and U2 snRNAs undergo a processing event of the primary transcript prior to incorporation in the snRNP. We find that coilin displays RNase activity within the CU region of the U2 snRNA primary transcript in vitro, and that coilin knockdown results in accumulation of the 3' pre-processed U1 and U2 snRNA. These findings present new characteristics of coilin in vitro, and suggest additional functions of the protein in vivo.

  20. Molecular Properties of Guar Gum and Pectin Modify Cecal Bile Acids, Microbiota, and Plasma Lipopolysaccharide-Binding Protein in Rats.

    Science.gov (United States)

    Ghaffarzadegan, Tannaz; Marungruang, Nittaya; Fåk, Frida; Nyman, Margareta

    2016-01-01

    Bile acids (BAs) act as signaling molecules in various physiological processes, and are related to colonic microbiota composition as well as to different types of dietary fat and fiber. This study investigated whether guar gum and pectin-two fibers with distinct functional characteristics-affect BA profiles, microbiota composition, and gut metabolites in rats. Low- (LM) or high-methoxylated (HM) pectin, and low-, medium-, or high-molecular-weight (MW) guar gum were administered to rats that were fed either low- or high-fat diets. Cecal BAs, short-chain fatty acids (SCFA) and microbiota composition, and plasma lipopolysaccharide-binding protein (LBP) levels were analyzed, by using novel methodologies based on gas chromatography (BAs and SCFAs) and 16S rRNA gene sequencing on the Illumina MiSeq platform. Strong correlations were observed between cecal BA and SCFA levels, microbiota composition, and portal plasma LBP levels in rats on a high-fat diet. Notably, guar gum consumption with medium-MW increased the cecal amounts of cholic-, chenodeoxycholic-, and ursodeoxycholic acids as well as α-, β-, and ω-muricholic acids to a greater extent than other types of guar gum or the fiber-free control diet. In contrast, the amounts of cecal deoxycholic- and hyodeoxycholic acid were reduced with all types of guar gum independent of chain length. Differences in BA composition between pectin groups were less obvious, but cecal levels of α- and ω-muricholic acids were higher in rats fed LM as compared to HM pectin or the control diet. The inflammatory marker LBP was downregulated in rats fed medium-MW guar gum and HM pectin; these two fibers decreased the cecal abundance of Oscillospira and an unclassified genus in Ruminococcaceae, and increased that of an unclassified family in RF32. These results indicate that the molecular properties of guar gum and pectin are important for their ability to modulate cecal BA formation, gut microbiota composition, and high-fat diet induced

  1. Nucleic acid binding properties of a helix stabilising nucleoid protein from the thermoacidophilic archaeon Sulfolobus acidocaldarius that condenses DNA into compact structures.

    Science.gov (United States)

    Celestina, F; Suryanarayana, T

    1995-12-01

    Helix stabilising nucleoid protein (HSNP-C') from an acidothermophilic archaeon Sulfolobus acidocaldarius has been characterised with respect to interaction with nucleic acids by gel retardation assay, binding to nucleic acid columns, fluorescence titrations and electron microscopy. The protein exists in solution as very large multimeric aggregates as indicated by cross-linking studies. The protein binds strongly and co-operatively to double stranded DNA. Electron microscopy of the complexes of the protein with DNA shows compact structures suggesting that the protein condenses DNA.

  2. Conserved charged amino acids are key determinants for fatty acid binding proteins (FABPs)-membrane interactions. A multi-methodological computational approach.

    Science.gov (United States)

    Zamarreño, Fernando; Giorgetti, Alejandro; Amundarain, María Julia; Viso, Juan Francisco; Córsico, Betina; Costabel, Marcelo D

    2017-03-16

    Based on the analysis of the mechanism of ligand transfer to membranes employing in vitro methods, Fatty Acid Binding Protein (FABP) family has been divided in two subgroups: collisional and diffusional FABPs. Although the collisional mechanism has been well characterized employing in vitro methods, the structural features responsible for the difference between collisional and diffusional mechanisms remain uncertain. In this work, we have identified the amino acids putatively responsible for the interaction with membranes of both, collisional and diffusional, subgroups of FABPs. Moreover, we show how specific changes in FABPs' structure could change the mechanism of interaction with membranes. We have computed protein-membrane interaction energies for members of each subgroup of the family, and performed Molecular Dynamics simulations that have shown different configurations for the initial interaction between FABPs and membranes. In order to generalize our hypothesis, we extended the electrostatic and bioinformatics analysis over FABPs of different mammalian genus. Also, our methodological approach could be used for other systems involving protein-membrane interactions.

  3. Adaptation of human immunodeficiency virus type 1 to cells expressing a binding-deficient CD4 mutant (lysine 46 to aspartic acid).

    Science.gov (United States)

    Choe, H R; Sodroski, J

    1995-01-01

    Human immunodeficiency virus (HIV-1) was adapted to replicate efficiently in cells expressing an altered form of the CD4 viral receptor. The mutant CD4 (46 K/D) contained a single amino acid change (lysine 46 to aspartic acid) in the CDR2 loop of domain 1, which results in a 15-fold reduction in affinity for the viral gp120 glycoprotein. The ability of the adapted virus to replicate in CD4 46 K/D-expressing cells was independently enhanced by single amino acid changes in the V2 variable loop, the V3 variable loop, and the fourth conserved (C4) region of the gp120 glycoprotein. Combinations of these amino acids in the same envelope glycoprotein resulted in additive enhancement of virus replication in cells expressing the CD4 46 K/D molecule. In cells expressing the wild-type CD4 glycoproteins, the same V2 and V3 residue changes also increased the efficiency of replication of a virus exhibiting decreased receptor-binding ability due to an amino acid change (aspartic acid 368 to glutamic acid) in the gp120 glycoprotein. In neither instance did the adaptive changes restore the binding ability of the monomeric gp120 glycoprotein or the oligomeric envelope glycoprotein complex for the mutant or wild-type CD4 glycoproteins, respectively. Thus, particular conformations of the gp120 V2 and V3 variable loops and of the C4 region allow postreceptor binding events in the membrane fusion process to occur in the context of less than optimal receptor binding. These results suggest that the fusion-related functions of the V2, V3, and C4 regions of gp120 are modulated by CD4 binding. PMID:7707502

  4. Nanoscale Distribution of Sulfonic Acid Groups Determines Structure and Binding of Water in Nafion Membranes

    Science.gov (United States)

    Ling, Xiao; Bonn, Mischa

    2016-01-01

    Abstract The connection between the nanoscale structure of two chemically equivalent, yet morphologically distinct Nafion fuel‐cell membranes and their macroscopic chemical properties is demonstrated. Quantification of the chemical interactions between water and Nafion reveals that extruded membranes have smaller water channels with a reduced sulfonic acid head group density compared to dispersion‐cast membranes. As a result, a disproportionally large amount of non‐bulk water molecules exists in extruded membranes, which also exhibit larger proton conductivity and larger water mobility compared to cast membranes. The differences in the physicochemical properties of the membranes, that is, the chemical constitution of the water channels and the local water structure, and the accompanying differences in macroscopic water and proton transport suggest that the chemistry of nanoscale channels is an important, yet largely overlooked parameter that influences the functionality of fuel‐cell membranes. PMID:26895211

  5. Site-Selective Binding of Nanoparticles to Double-Stranded DNA via Peptide Nucleic Acid "Invasion"

    Energy Technology Data Exchange (ETDEWEB)

    Stadler, A.L.; van der Lelie, D.; Sun, D.; Maye, M. M.; Gang, O.

    2011-04-01

    We demonstrate a novel method for by-design placement of nano-objects along double-stranded (ds) DNA. A molecular intercalator, designed as a peptide nucleic acid (PNA)-DNA chimera, is able to invade dsDNA at the PNA-side due to the hybridization specificity between PNA and one of the duplex strands. At the same time, the single-stranded (ss) DNA tail of the chimera, allows for anchoring of nano-objects that have been functionalized with complementary ssDNA. The developed method is applied for interparticle attachment and for the fabrication of particle clusters using a dsDNA template. This method significantly broadens the molecular toolbox for constructing nanoscale systems by including the most conventional not yet utilized DNA motif, double helix DNA.

  6. A glucuronic acid binding leguminous lectin with mitogenic activity toward mouse splenocytes.

    Science.gov (United States)

    Chan, Yau Sang; Wong, Jack Ho; Ng, Tzi Bun

    2011-02-01

    A dimeric 64-kDa lectin was purified from seeds of French bean (Phaseolus vulgaris) cultivar number 1. The purification protocol entailed Q-Sepharose, Affi-gel blue gel, Mono S and Superdex 75. The lectin-enriched fraction was adsorbed on Q-Sepharose and Affi-gel blue gel and desorbed using 1M NaCl in the starting buffer. Hemagglutinating activity was adsorbed on Mono S and eluted with a linear 0.3-1 M NaCl gradient. Gel filtration on Superdex 75 yielded a single absorbance peak which appeared as a single 32-kDa in sodium dodecyl sulfate poylacylamide gel electrophoresis. Full hemagglutinating activity was observed when the lectin was exposed to a pH ranging from 3 to 11. About 50% activity remained at pH 12, and about 25% at pH 0 to pH 2. Activity was totally abolished at pH 13-14. The activity was completely preserved when the ambient temperature was 20 °C-60 °C. However, only 50% and 12.5% of the activity remained at 65 °C and 70 °C, respectively. Activity was barely discernible at 75 °C and completely abrogated at and above 80 °C. Hemagglutinating activity of the lectin was inhibited by glucuronic acid. Maximum mitogenic activity of the lectin toward murine splenocytes occurred at a lectin concentration of 0.488 µM. The mitogenic activity was nearly eliminated in the presence of 250 mM glucuronic acid. The lectin did not exhibit antiproliferative activity toward hepatoma (HepG2) cells, breast cancer (MCF7) cells, and nasopharynegeal carcinoma CNE stage 1 and stage 2 cells. It was also devoid of significant anti-HIV reverse transcriptase activity.

  7. A novel processing system of sterol regulatory element-binding protein-1c regulated by polyunsaturated fatty acid.

    Science.gov (United States)

    Nakakuki, Masanori; Kawano, Hiroyuki; Notsu, Tatsuto; Imada, Kazunori; Mizuguchi, Kiyoshi; Shimano, Hitoshi

    2014-05-01

    The proteolytic cascade is the key step in transactivation of sterol regulatory element-binding proteins (SREBPs), a transcriptional factor of lipid synthesis. Proteolysis of SREBP-2 is strictly regulated by sterols, but that of SREBP-1c was not strongly sterol-regulated, but inhibited by polyunsaturated fatty acids (PUFAs). In this study, the proteolytic processing of SREBP-1 and -2 was examined by transfection studies of cDNA-encoding mutants in which all the known cleavage sites were disrupted. In cultured cells, sterol-regulated SREBP-2 processing was completely eliminated by mutation of cleavage sites. In contrast, the corresponding SREBP-1c mutants as well as wild type exhibited large amounts of cleaved products in the nuclear extracts from culture cells and murine liver in vivo. The nuclear form of the mutant SREBP-1c was induced by delipidated condition and suppressed by eicosapentaenoic acid, an n-3 PUFA, but not by sterols. This novel processing mechanism was affected by neither SREBP cleavage-activating protein (SCAP) nor insulin-induced gene (Insig)-1, unlike SREBP-2, but abolished by a serine protease inhibitor. Through analysis of deletion mutant, a site-2 protease recognition sequence (DRSR) was identified to be involved in this novel processing. These findings suggest that SREBP-1c cleavage could be subjected to a novel PUFA-regulated cleavage system in addition to the sterol-regulatory SCAP/Insig system.

  8. Positive cooperativity in substrate binding of human prostatic acid phosphatase entrapped in AOT-isooctane-water reverse micelles.

    Science.gov (United States)

    Luchter-Wasylewska, Ewa; Iciek, Małgorzata

    2004-05-15

    The kinetics of 1-naphthyl phosphate and phenyl phosphate hydrolysis, catalyzed by human prostatic acid phosphatase (PAP) entrapped in AOT-isooctane-water reverse micelles, has been studied over surfactant hydration degree (w0) range 5 to 35. Continuous spectrophotometric acid phosphatase assays, previously prepared, were employed. PAP was catalytically active over the whole w0 studied range. In order to determine steady-state reaction constants the experimental data were fitted to Hill rate equation. Positive cooperativity in substrate binding was observed, as it was earlier found in aqueous solutions. The extent of cooperativity (expressed as the value of the Hill cooperation coefficient h) increased from 1 to 4, when the micellar water-pool size was growing, at fixed enzyme concentration. In the plots of catalytic activity (kcat) versus w0, the maxima have been found at w0=10 (pH 5.6) and 23 (pH 3.8). It is suggested that catalytically active monomeric and dimeric PAP forms are entrapped in reverse micelles of w0=10 and 23, respectively.

  9. Low heart-type fatty acid binding protein level during aging may protect down syndrome people against atherosclerosis

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    Vianello Elena

    2013-01-01

    Full Text Available Abstract Background Aging is considered an important independent risk factor for atherosclerosis. Down syndrome people (DS display an accelerated aging process compared to healthy subjects, anyway they are relatively resistant to developing atherosclerosis. The mechanisms involved in such protective effect are not well known. Since heart-type fatty acid binding protein (H-FABP is a protein involved in the transport of fatty acids and it has been recently correlated with the presence of atherosclerosis, we aimed to measure H-FABP level both in DS and in healthy subjects during aging to evaluate the association between this molecule, aging and atherosclerosis. Findings We quantified plasmatic H-FABP level in three groups of male DS and age-matched healthy subjects (children, age 2–14 years; adults, age 20–50 years; elderly, > 60 years using a biochip array analyzer. We observed that aging is associated with increased H-FABP level in healthy subjects but not in DS which display both the same protein level in the different ages of life and have also lower level compared to their age-matched healthy subjects. Conclusion Reduced H-FABP level during aging in DS may play a protective role against atherosclerosis. The potential involvement of H-FABP in the relationship between aging, atherosclerosis and development of coronary artery disease needs further investigations.

  10. The Formation of pH-Sensitive Wormlike Micelles in Ionic Liquids Driven by the Binding Ability of Anthranilic Acid

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    Qing You

    2015-11-01

    Full Text Available Wormlike micelles are typically formed by mixing cationic and anionic surfactants because of attractive interactions in oppositely charged head-groups. The structural transitions of wormlike micelles triggered by pH in ionic liquids composed of N-alkyl-N-methylpyrrolidinium bromide-based ILs (ionic liquids and anthranilic acid were investigated. These structures were found responsible for the variations in flow properties identified by rheology and dynamic light scattering, and account for the structures observed with cryogenic transmission electron microscopy (Cryo-TEM. High-viscosity, shear-thinning behavior, and Maxwell-type dynamic rheology shown by the system at certain pH values suggested that spherical micelles grow into entangled wormlike micelles. Light scattering profiles also supported the notion of pH-sensitive microstructural transitions in the solution. Cryo-TEM images confirmed the presence of spherical micelles in the low-viscosity sample and entangled wormlike micelles in the peak viscosity sample. Nuclear magnetic resonance spectroscopy analysis revealed that the pH sensitivity of ionic liquid systems originated from the pH-dependent binding ability of anthranilic acid to the cationic headgroup of ionic liquids.

  11. Binding of Na+ and K+ to the Headgroup of Palmitic Acid Monolayers Studied by Vibrational Sum Frequency Generation Spectroscopy

    Science.gov (United States)

    Huang, Zishuai; Allen, Heather C.

    2012-06-01

    Alkali cations are critical in biological systems due to their electrical interaction with cell membranes. While Na+ and K+ share similar chemical and physical properties, they can exhibit differences when interacting with biological membranes. These phenomena may be modeled using a Langmuir monolayer of surfactant on alkali chloride solutions. Vibrational sum frequency generation (VSFG) spectroscopy is an interface specific technique that is widely employed to study molecular organization at surfaces and interfaces. VSFG spectroscopy was used to probe the CO2- vibrational mode for the carboxylic acid headgroup of palmitic acid (PA) spread on the surface of NaCl and KCl solutions in the vibrational region between 1400 and 1500 cm-1. The ability of Na+ and K+ to bind with the carboxylic headgroup of PA is revealed by observing peak positions (˜1410 cm-1 and ˜1470 cm-1) and relative intensity for the CO2- peaks. These results are compared and discussed with perspective toward elucidating interfacial PA headgroup organization. The time evolution for the PA CO2- peaks is also monitored after monolayer spreading via VSFG and these results are presented as well.

  12. Plasma-free fatty acids, fatty acid-binding protein 4, and mortality in older adults (from the Cardiovascular Health Study).

    Science.gov (United States)

    Miedema, Michael D; Maziarz, Marlena; Biggs, Mary L; Zieman, Susan J; Kizer, Jorge R; Ix, Joachim H; Mozaffarian, Dariush; Tracy, Russell P; Psaty, Bruce M; Siscovick, David S; Mukamal, Kenneth J; Djousse, Luc

    2014-09-15

    Plasma-free fatty acids (FFAs) are largely derived from adipose tissue. Elevated levels of FFA and fatty acid-binding protein 4 (FABP4), a key cytoplasmic chaperone of fatty acids, have been associated with adverse cardiovascular outcomes, but limited data are available on the relation of these biomarkers with cardiovascular and total mortality. We studied 4,707 participants with a mean age of 75 years who had plasma FFA and FABP4 measured in 1992 to 1993 as part of the Cardiovascular Health Study, an observational cohort of community-dwelling older adults. Over a median follow-up of 11.8 years, 3,555 participants died. Cox proportional hazard regression was used to determine the association between FFA, FABP4, and mortality. In fully adjusted models, FFA were associated with dose-dependent significantly higher total mortality (hazard ratio [HR] per SD: 1.14, 95% confidence interval [CI] 1.09 to 1.18), but FABP4 levels were not (HR 1.04, 95% CI 0.98 to 1.09). In a cause-specific mortality analysis, higher concentrations of FFA were associated with significantly higher risk of death because of cardiovascular disease, dementia, infection, and respiratory causes but not cancer or trauma. We did not find evidence of an interaction between FFA and FABP4 (p = 0.45), but FABP4 appeared to be associated with total mortality differentially in men and women (HR 1.17, 95% CI 1.08 to 1.26 for men; HR 1.02, 95% CI 0.96 to 1.07 for women, interaction p value <0.001). In conclusion, in a cohort of community-dwelling older subjects, elevated plasma concentrations of FFA, but not FABP4, were associated with cardiovascular and noncardiovascular mortality.

  13. No need to be HAMLET or BAMLET to interact with histones: binding of monomeric alpha-lactalbumin to histones and basic poly-amino acids.

    Science.gov (United States)

    Permyakov, Serge E; Pershikova, Irina V; Khokhlova, Tatyana I; Uversky, Vladimir N; Permyakov, Eugene A

    2004-05-18

    The ability of a specific complex of human alpha-lactalbumin with oleic acid (HAMLET) to induce cell death with selectivity for tumor and undifferentiated cells was shown recently to be mediated by interaction of HAMLET with histone proteins irreversibly disrupting chromatin structure [Duringer, C., et al. (2003) J. Biol. Chem. 278, 42131-42135]. Here we show that monomeric alpha-lactalbumin (alpha-LA) in the absence of fatty acids is also able to bind efficiently to the primary target of HAMLET, histone HIII, regardless of Ca(2+) content. Thus, the modification of alpha-LA by oleic acid is not required for binding to histones. We suggest that interaction of negatively charged alpha-LA with the basic histone stabilizes apo-alpha-LA and destabilizes the Ca(2+)-bound protein due to compensation for excess negative charge of alpha-LA's Ca(2+)-binding loop by positively charged residues of the histone. Spectrofluorimetric curves of titration of alpha-LA by histone H3 were well approximated by a scheme of cooperative binding of four alpha-LA molecules per molecule of histone, with an equilibrium dissociation constant of 1.0 microM. Such a stoichiometry of binding implies that the binding process is not site-specific with respect to histone and likely is driven by just electrostatic interactions. Co-incubation of positively charged poly-amino acids (poly-Lys and poly-Arg) with alpha-LA resulted in effects which were similar to those caused by histone HIII, confirming the electrostatic nature of the alpha-LA-histone interaction. In all cases that were studied, the binding was accompanied by aggregation. The data indicate that alpha-lactalbumin can be used as a basis for the design of antitumor agents, acting through disorganization of chromatin structure due to interaction between alpha-LA and histone proteins.

  14. X-ray structure of Pur-alpha reveals a Whirly-like fold and an unusual nucleic-acid binding surface.

    Science.gov (United States)

    Graebsch, Almut; Roche, Stéphane; Niessing, Dierk

    2009-11-03

    The PUR protein family is a distinct and highly conserved class that is characterized by its sequence-specific RNA- and DNA-binding. Its best-studied family member, Pur-alpha, acts as a transcriptional regulator, as host factor for viral replication, and as cofactor for mRNP localization in dendrites. Pur-alpha-deficient mice show severe neurologic defects and die after birth. Nucleic-acid binding by Pur-alpha is mediated by its central core region, for which no structural information is available. We determined the x-ray structure of residues 40 to 185 from Drosophila melanogaster Pur-alpha, which constitutes a major part of the core region. We found that this region contains two almost identical structural motifs, termed "PUR repeats," which interact with each other to form a PUR domain. DNA- and RNA-binding studies confirmed that PUR domains are indeed functional nucleic-acid binding domains. Database analysis show that PUR domains share a fold with the Whirly class of nucleic-acid binding proteins. Structural analysis combined with mutational studies suggest that a PUR domain binds nucleic acids through two independent surface regions involving concave beta-sheets. Structure-based sequence alignment revealed that the core region harbors a third PUR repeat at its C terminus. Subsequent characterization by small-angle x-ray scattering (SAXS) and size-exclusion chromatography indicated that PUR repeat III mediates dimerization of Pur-alpha. Surface envelopes calculated from SAXS data show that the Pur-alpha dimer consisting of repeats I to III is arranged in a Z-like shape. This unexpected domain organization of the entire core domain of Pur-alpha has direct implications for ssDNA/ssRNA and dsDNA binding.

  15. Uncoupling of Obesity from Insulin Resistance Through a Targeted Mutation in aP2, the Adipocyte Fatty Acid Binding Protein

    Science.gov (United States)

    Hotamisligil, Gokhan S.; Johnson, Randall S.; Distel, Robert J.; Ellis, Ramsey; Papaioannou, Virginia E.; Spiegelman, Bruce M.

    1996-11-01

    Fatty acid binding proteins (FABPs) are small cytoplasmic proteins that are expressed in a highly tissue-specific manner and bind to fatty acids such as oleic and retinoic acid. Mice with a null mutation in aP2, the gene encoding the adipocyte FABP, were developmentally and metabolically normal. The aP2-deficient mice developed dietary obesity but, unlike control mice, they did not develop insulin resistance or diabetes. Also unlike their obese wild-type counterparts, obese aP2-/- animals failed to express in adipose tissue tumor necrosis factor-α (TNF-α), a molecule implicated in obesity-related insulin resistance. These results indicate that aP2 is central to the pathway that links obesity to insulin resistance, possibly by linking fatty acid metabolism to expression of TNF-α.

  16. Lipid Transport through the Fetoplacental Barrier by the Fatty Acid-Binding Proteins in Pregnant Women with Herpes Virus Infection in the third Trimester

    Directory of Open Access Journals (Sweden)

    Michael T. Lucenko, PhD, ScD

    2012-12-01

    Full Text Available In this study, the transport of the long chain polyunsaturated fatty acids (LCPUFAs from the lacunar blood through the syncytiotrophoblast of the placental villi to the fetal cord blood via a saturable protein-mediated mechanism by the heart-type fatty acid-binding proteins (H-FABPs has been examined. Exacerbation of the herpes simplex viruses (HSV-1 in the third trimester of gestation reduces the delivery of the fatty acid-binding proteins to the syncytiotrophoblast. During exacerbation of the HSV-1 infection, the selective transfer of the LCPUFAs across the syncytiotrophoblast basal plasma membrane into the fetal cord blood was observed. The supply of anti-inflammatory ω-3 PUFAs was reduced; however, the inflow of inflammatory arachidonic acid and other ω-6 PUFAs into the fetal blood was increased.

  17. Library screening by means of mass spectrometry (MS) binding assays-exemplarily demonstrated for a pseudostatic library addressing γ-aminobutyric acid (GABA) transporter 1 (GAT1).

    Science.gov (United States)

    Sindelar, Miriam; Wanner, Klaus T

    2012-09-01

    In the present study, the application of mass spectrometry (MS) binding assays as a tool for library screening is reported. For library generation, dynamic combinatorial chemistry (DCC) was used. These libraries can be screened by means of MS binding assays when appropriate measures are taken to render the libraries pseudostatic. That way, the efficiency of MS binding assays to determine ligand binding in compound screening with the ease of library generation by DCC is combined. The feasibility of this approach is shown for γ-aminobutyric acid (GABA) transporter 1 (GAT1) as a target, representing the most important subtype of the GABA transporters. For the screening, hydrazone libraries were employed that were generated in the presence of the target by reacting various sets of aldehydes with a hydrazine derivative that is delineated from piperidine-3-carboxylic acid (nipecotic acid), a common fragment of known GAT1 inhibitors. To ensure that the library generated is pseudostatic, a large excess of the nipecotic acid derivative is employed. As the library is generated in a buffer system suitable for binding and the target is already present, the mixtures can be directly analyzed by MS binding assays-the process of library generation and screening thus becoming simple to perform. The binding affinities of the hits identified by deconvolution were confirmed in conventional competitive MS binding assays performed with single compounds obtained by separate synthesis. In this way, two nipecotic acid derivatives exhibiting a biaryl moiety, 1-{2-[2'-(1,1'-biphenyl-2-ylmethylidene)hydrazine]ethyl}piperidine-3-carboxylic acid and 1-(2-{2'-[1-(2-thiophenylphenyl)methylidene]hydrazine}ethyl)piperidine-3-carboxylic acid, were found to be potent GAT1 ligands exhibiting pK(i) values of 6.186 ± 0.028 and 6.229 ± 0.039, respectively. This method enables screening of libraries, whether generated by conventional chemistry or DCC, and is applicable to all kinds of targets including

  18. Association analyses between brain-expressed fatty-acid binding protein (FABP) genes and schizophrenia and bipolar disorder.

    Science.gov (United States)

    Iwayama, Yoshimi; Hattori, Eiji; Maekawa, Motoko; Yamada, Kazuo; Toyota, Tomoko; Ohnishi, Tetsuo; Iwata, Yasuhide; Tsuchiya, Kenji J; Sugihara, Genichi; Kikuchi, Mitsuru; Hashimoto, Kenji; Iyo, Masaomi; Inada, Toshiya; Kunugi, Hiroshi; Ozaki, Norio; Iwata, Nakao; Nanko, Shinichiro; Iwamoto, Kazuya; Okazaki, Yuji; Kato, Tadafumi; Yoshikawa, Takeo

    2010-03-05

    Deficits in prepulse inhibition (PPI) are a biological marker for psychiatric illnesses such as schizophrenia and bipolar disorder. To unravel PPI-controlling mechanisms, we previously performed quantitative trait loci (QTL) analysis in mice, and identified Fabp7, that encodes a brain-type fatty acid binding protein (Fabp), as a causative gene. In that study, human FABP7 showed genetic association with schizophrenia. FABPs constitute a gene family, of which members FABP5 and FABP3 are also expressed in the brain. These FABP proteins are molecular chaperons for polyunsaturated fatty acids (PUFAs) such as arachidonic and docosahexaenoic acids. Additionally, the involvement of PUFAs has been documented in the pathophysiology of schizophrenia and mood disorders. Therefore in this study, we examined the genetic roles of FABP5 and 3 in schizophrenia (N = 1,900 in combination with controls) and FABP7, 5, and 3 in bipolar disorder (N = 1,762 in the case-control set). Three single nucleotide polymorphisms (SNPs) from FABP7 showed nominal association with bipolar disorder, and haplotypes of the same gene showed empirical associations with bipolar disorder even after correction of multiple testing. We could not perform association studies on FABP5, due to the lack of informative SNPs. FABP3 displayed no association with either disease. Each FABP is relatively small and it is assumed that there are multiple regulatory elements that control gene expression. Therefore, future identification of unknown regulatory elements will be necessary to make a more detailed analysis of their genetic contribution to mental illnesses.

  19. In situ study of binding of copper by fulvic acid: comparison of differential absorbance data and model predictions.

    Science.gov (United States)

    Yan, Mingquan; Dryer, Deborah; Korshin, Gregory V; Benedetti, Marc F

    2013-02-01

    This study examined the binding of copper(II) by Suwannee River fulvic acid (SRFA) using the method of differential absorbance that was used at environmentally-relevant concentrations of copper and SRFA. The pH- and metal-differential spectra were processed via numeric deconvolution to establish commonalities seen in the changes of absorbance caused by deprotonation of SRFA and its interactions with copper(II) ions. Six Gaussian bands were determined to be present in both the pH- and Cu-differential spectra. Their maxima were located, in the order of increasing wavelengths at 208 nm, 242 nm, 276 nm, 314 nm, 378 nm and 551 nm. The bands with these maxima were denoted as A0, A1, A2, A3, A4 and A5, respectively. Properties of these bands were compared with those existing in the spectra of model compounds such as sulfosalicylic acid (SSA), tannic acid (TA), and polystyrenesulfonic acid-co-maleic acid (PSMA). While none of the features observed in differential spectra of the model compound were identical to those present in the case of SRFA, Gaussian bands A1, A3 and possibly A2 were concluded to be largely attributable to a combination of responses of salicylic- and polyhydroxyphenolic groups. In contrast, bands A4 and A5 were detected in the differential spectra of SRFA only. Their nature remains to be elucidated. To examine correlations between the amount of copper(II) bound by SRFA and changes of its absorbance, differential absorbances measured at indicative wavelengths 250 nm and 400 nm were compared with the total amount of SRFA-bound copper estimated based on Visual MINTEQ calculations. This examination showed that the differential absorbances of SRFA in a wide range of pH values and copper concentrations were strongly correlated with the concentration of SRFA-bound copper. The approach presented in this study can be used to generate in situ information concerning the nature of functional groups in humic substances engaged in interactions with metals ions. This

  20. Sialic acid receptor detection in the human respiratory tract: evidence for widespread distribution of potential binding sites for human and avian influenza viruses

    Directory of Open Access Journals (Sweden)

    Guan Yi

    2007-10-01

    Full Text Available Abstract Background Influenza virus binds to cell receptors via sialic acid (SA linked glycoproteins. They recognize SA on host cells through their haemagglutinins (H. The distribution of SA on cell surfaces is one determinant of host tropism and understanding its expression on human cells and tissues is important for understanding influenza pathogenesis. The objective of this study therefore was to optimize the detection of α2,3-linked and α2,6-linked SA by lectin histochemistry by investigating the binding of Sambucus nigra agglutinin (SNA for SAα2,6Gal and Maackia amurensis agglutinin (MAA for SAα2,3Gal in the respiratory tract of normal adults and children. Methods We used fluorescent and biotinylated SNA and MAA from different suppliers on archived and prospectively collected biopsy and autopsy specimens from the nasopharynx, trachea, bronchus and lungs of fetuses, infants and adults. We compared different methods of unmasking for tissue sections to determine if these would affect lectin binding. Using serial sections we then compared the lectin binding of MAA from different suppliers. Results We found that unmasking using microwave treatment in citrate buffer produced increased lectin binding to the ciliated and glandular epithelium of the respiratory tract. In addition we found that there were differences in tissue distribution of the α2,3 linked SA when 2 different isoforms of MAA (MAA1 and MAA2 lectin were used. MAA1 had widespread binding throughout the upper and lower respiratory tract and showed more binding to the respiratory epithelium of children than in adults. By comparison, MAA2 binding was mainly restricted to the alveolar epithelial cells of the lung with weak binding to goblet cells. SNA binding was detected in bronchial and alveolar epithelial cells and binding of this lectin was stronger to the paediatric epithelium compared to adult epithelium. Furthermore, the MAA lectins from 2 suppliers (Roche and EY Labs tended

  1. Effects of retinoic acid and hydrogen peroxide on sterol regulatory element-binding protein-1a activation during adipogenic differentiation of 3T3-L1 cells

    OpenAIRE

    Eldaim, Mabrouk A. Abd; Okamatsu-Ogura, Yuko; Terao, Akira; Kimura, Kazuhiro

    2010-01-01

    Both retinoic acid (RA) and oxidative stress (H2O2) increased transcription and cleavage of membrane-bound sterol regulatory element-binding protein (SREBP)-1, leading to enhanced transcription of fatty acid synthase (FAS) in hepatoma cells. On the other hand, RA and H2O2 decreased and increased lipogenesis in adipocytes, respectively, although roles of SREBP-1 activation in these effects remain to be elucidated. To elucidate its involvement, we examined the activation of SREBP...

  2. Characterization of the N-Acetyl-5-neuraminic Acid-binding Site of the Extracytoplasmic Solute Receptor (SiaP) of Nontypeable Haemophilus influenzae Strain 2019

    Energy Technology Data Exchange (ETDEWEB)

    Johnston, Jason W.; Coussens, Nathan P.; Allen, Simon; Houtman, Jon C.D.; Turner, Keith H.; Zaleski, Anthony; Ramaswamy, S.; Gibson, Bradford W.; Apicella, Michael A. (Iowa); (Buck Inst.)

    2012-11-14

    Nontypeable Haemophilus influenzae is an opportunistic human pathogen causing otitis media in children and chronic bronchitis and pneumonia in patients with chronic obstructive pulmonary disease. The outer membrane of nontypeable H. influenzae is dominated by lipooligosaccharides (LOS), many of which incorporate sialic acid as a terminal nonreducing sugar. Sialic acid has been demonstrated to be an important factor in the survival of the bacteria within the host environment. H. influenzae is incapable of synthesizing sialic acid and is dependent on scavenging free sialic acid from the host environment. To achieve this, H. influenzae utilizes a tripartite ATP-independent periplasmic transporter. In this study, we characterize the binding site of the extracytoplasmic solute receptor (SiaP) from nontypeable H. influenzae strain 2019. A crystal structure of N-acetyl-5-neuraminic acid (Neu5Ac)-bound SiaP was determined to 1.4 {angstrom} resolution. Thermodynamic characterization of Neu5Ac binding shows this interaction is enthalpically driven with a substantial unfavorable contribution from entropy. This is expected because the binding of SiaP to Neu5Ac is mediated by numerous hydrogen bonds and has several buried water molecules. Point mutations targeting specific amino acids were introduced in the putative binding site. Complementation with the mutated siaP constructs resulted either in full, partial, or no complementation, depending on the role of specific residues. Mass spectrometry analysis of the O-deacylated LOS of the R127K point mutation confirmed the observation of reduced incorporation of Neu5Ac into the LOS. The decreased ability of H. influenzae to import sialic acid had negative effects on resistance to complement-mediated killing and viability of biofilms in vitro, confirming the importance of sialic acid transport to the bacterium.

  3. Characterization of the N-acetyl-5-neuraminic acid-binding site of the extracytoplasmic solute receptor (SiaP) of nontypeable Haemophilus influenzae strain 2019.

    Science.gov (United States)

    Johnston, Jason W; Coussens, Nathan P; Allen, Simon; Houtman, Jon C D; Turner, Keith H; Zaleski, Anthony; Ramaswamy, S; Gibson, Bradford W; Apicella, Michael A

    2008-01-11

    Nontypeable Haemophilus influenzae is an opportunistic human pathogen causing otitis media in children and chronic bronchitis and pneumonia in patients with chronic obstructive pulmonary disease. The outer membrane of nontypeable H. influenzae is dominated by lipooligosaccharides (LOS), many of which incorporate sialic acid as a terminal nonreducing sugar. Sialic acid has been demonstrated to be an important factor in the survival of the bacteria within the host environment. H. influenzae is incapable of synthesizing sialic acid and is dependent on scavenging free sialic acid from the host environment. To achieve this, H. influenzae utilizes a tripartite ATP-independent periplasmic transporter. In this study, we characterize the binding site of the extracytoplasmic solute receptor (SiaP) from nontypeable H. influenzae strain 2019. A crystal structure of N-acetyl-5-neuraminic acid (Neu5Ac)-bound SiaP was determined to 1.4A resolution. Thermodynamic characterization of Neu5Ac binding shows this interaction is enthalpically driven with a substantial unfavorable contribution from entropy. This is expected because the binding of SiaP to Neu5Ac is mediated by numerous hydrogen bonds and has several buried water molecules. Point mutations targeting specific amino acids were introduced in the putative binding site. Complementation with the mutated siaP constructs resulted either in full, partial, or no complementation, depending on the role of specific residues. Mass spectrometry analysis of the O-deacylated LOS of the R127K point mutation confirmed the observation of reduced incorporation of Neu5Ac into the LOS. The decreased ability of H. influenzae to import sialic acid had negative effects on resistance to complement-mediated killing and viability of biofilms in vitro, confirming the importance of sialic acid transport to the bacterium.

  4. Bile and unsaturated fatty acids inhibit the binding of cholera toxin and Escherichia coli heat-labile enterotoxin to GM1 receptor.

    Science.gov (United States)

    Chatterjee, Arpita; Chowdhury, Rukhsana

    2008-01-01

    Cholera toxin (CT) is an archetypal bacterial toxin that binds with a high affinity to the receptor ganglioside GM1 on the intestinal epithelial surface and that causes the severe watery diarrhea characteristic of the disease cholera. Blockage of the interaction of CT with the GM1 receptor is an attractive approach for therapeutic intervention. We report here that crude bile prevents the interaction of CT with GM1 and reduces CT-mediated fluid accumulation in the rabbit intestine. The unsaturated fatty acids detected in crude bile, arachidonic, linoleic, and oleic acids, were found to be the most effective. Crude bile and the unsaturated fatty acids interacted with CT but not GM1 to prevent CT-GM1 binding. Neither crude bile nor the unsaturated fatty acids had any effect on the subunit structure of CT. The binding of CT to unsaturated fatty acids resulted in a shift of the apparent pI of CT from 6.8 to 8.2 and a marked decrease in intrinsic fluorescence. The Kd was calculated from fluorescence quenching assays. It was demonstrated by the rabbit ileal loop model that practically no fluid accumulated in the intestinal loops when CT was administered together with inhibitory concentrations of linoleic acid. The bile present in the intestine was sufficient to inhibit the activity of up to 300 ng CT. Bile and unsaturated fatty acids also inhibited the binding of Escherichia coli heat-labile enterotoxin (LT) to GM1, and no fluid accumulation was observed in rabbit ileal loops when LT was administered together with linoleic acid.

  5. Human serum albumin binding to silica nanoparticles--effect of protein fatty acid ligand.

    Science.gov (United States)

    Ang, Joo Chuan; Henderson, Mark J; Campbell, Richard A; Lin, Jhih-Min; Yaron, Peter N; Nelson, Andrew; Faunce, Thomas; White, John W

    2014-06-07

    Neutron reflectivity shows that fatted (F-HSA) and defatted (DF-HSA) versions of human serum albumin behave differently in their interaction with silica nanoparticles premixed in buffer solutions although these proteins have close to the same surface excess when the silica is absent. In both cases a silica containing film is quickly established at the air-water interface. This film is stable for F-HSA at all relative protein-silica concentrations measured. This behaviour has been verified for two small silica nanoparticle radii (42 Å and 48 Å). Contrast variation and co-refinement have been used to find the film composition for the F-HSA-silica system. The film structure changes with protein concentration only for the DF-HSA-silica system. The different behaviour of the two proteins is interpreted as a combination of three factors: increased structural stability of F-HSA induced by the fatty acid ligand, differences in the electrostatic interactions, and the higher propensity of defatted albumin to self-aggregate. The interfacial structures of the proteins alone in buffer are also reported and discussed.

  6. Differential transcriptional modulation of duplicated fatty acid-binding protein genes by dietary fatty acids in zebrafish (Danio rerio: evidence for subfunctionalization or neofunctionalization of duplicated genes

    Directory of Open Access Journals (Sweden)

    Denovan-Wright Eileen M

    2009-09-01

    Full Text Available Abstract Background In the Duplication-Degeneration-Complementation (DDC model, subfunctionalization and neofunctionalization have been proposed as important processes driving the retention of duplicated genes in the genome. These processes are thought to occur by gain or loss of regulatory elements in the promoters of duplicated genes. We tested the DDC model by determining the transcriptional induction of fatty acid-binding proteins (Fabps genes by dietary fatty acids (FAs in zebrafish. We chose zebrafish for this study for two reasons: extensive bioinformatics resources are available for zebrafish at zfin.org and zebrafish contains many duplicated genes owing to a whole genome duplication event that occurred early in the ray-finned fish lineage approximately 230-400 million years ago. Adult zebrafish were fed diets containing either fish oil (12% lipid, rich in highly unsaturated fatty acid, sunflower oil (12% lipid, rich in linoleic acid, linseed oil (12% lipid, rich in linolenic acid, or low fat (4% lipid, low fat diet for 10 weeks. FA profiles and the steady-state levels of fabp mRNA and heterogeneous nuclear RNA in intestine, liver, muscle and brain of zebrafish were determined. Result FA profiles assayed by gas chromatography differed in the intestine, brain, muscle and liver depending on diet. The steady-state level of mRNA for three sets of duplicated genes, fabp1a/fabp1b.1/fabp1b.2, fabp7a/fabp7b, and fabp11a/fabp11b, was determined by reverse transcription, quantitative polymerase chain reaction (RT-qPCR. In brain, the steady-state level of fabp7b mRNAs was induced in fish fed the linoleic acid-rich diet; in intestine, the transcript level of fabp1b.1 and fabp7b were elevated in fish fed the linolenic acid-rich diet; in liver, the level of fabp7a mRNAs was elevated in fish fed the low fat diet; and in muscle, the level of fabp7a and fabp11a mRNAs were elevated in fish fed the linolenic acid-rich or the low fat diets. In all cases

  7. Investigation of the Binding Interaction of Fatty Acids with Human G Protein-Coupled Receptor 40 Using a Site-Specific Fluorescence Probe by Flow Cytometry.

    Science.gov (United States)

    Ren, Xiao-Min; Cao, Lin-Ying; Zhang, Jing; Qin, Wei-Ping; Yang, Yu; Wan, Bin; Guo, Liang-Hong

    2016-04-01

    Human G protein-coupled receptor 40 (hGPR40), with medium- and long-chain free fatty acids (FFAs) as its natural ligands, plays an important role in the enhancement of glucose-dependent insulin secretion. To date, information about the direct binding of FFAs to hGPR40 is very limited, and how carbon-chain length affects the activities of FFAs on hGPR40 is not yet understood. In this study, a fluorescein-fasiglifam analogue (F-TAK-875A) conjugate was designed and synthesized as a site-specific fluorescence probe to study the interaction of FFAs with hGPR40. hGPR40 was expressed in human embryonic kidney 293 cells and labeled with F-TAK-875A. By using flow cytometry, competitive binding of FFA and F-TAK-875A to hGPR40-expressed cells was measured. Binding affinities of 18 saturated FFAs, with carbon-chain lengths ranging from C6 to C23, were analyzed. The results showed that the binding potencies of FFAs to hGPR40 were dependent on carbon length. There was a positive correlation between length and binding potency for seven FFAs (C9-C15), with myristic acid (C15) showing the highest potency, 0.2% relative to TAK-875. For FFAs with a length of fewer than C9 or more than C15, they had very weak or no binding. Molecular docking results showed that the binding pocket of TAK-875 in hGPR40 could enclose FFAs with lengths of C15 or fewer. However, for FFAs with lengths longer than C15, part of the alkyl chain extended out of the binding pocket. This study provided insights into the structural dependence of FFAs binding to and activation of hGPR40.

  8. Acidity and lipolysis by group V secreted phospholipase A(2) strongly increase the binding of apoB-100-containing lipoproteins to human aortic proteoglycans.

    Science.gov (United States)

    Lähdesmäki, Katariina; Öörni, Katariina; Alanne-Kinnunen, Mervi; Jauhiainen, Matti; Hurt-Camejo, Eva; Kovanen, Petri T

    2012-02-01

    Local acidic areas characterize diffuse intimal thickening (DIT) and advanced atherosclerotic lesions. The role of acidity in the modification and extra- and intracellular accumulation of triglyceride-rich VLDL and IDL particles has not been studied before. Here, we examined the effects of acidic pH on the activity of recombinant human group V secreted phospholipase A(2) (sPLA(2)-V) toward small VLDL (sVLDL), IDL, and LDL, on the binding of these apoB-100-containing lipoproteins to human aortic proteoglycans, and on their uptake by human monocyte-derived macrophages. At acidic pH, the ability of sPLA(2)-V to lipolyze the apoB-100-containing lipoproteins was moderately, but significantly, increased while binding of the lipoproteins to proteoglycans increased >60-fold and sPLA(2)-V-modification further doubled the binding. Moreover, acidic pH more than doubled macrophage uptake of soluble complexes of sPLA(2)-V-LDL with aortic proteoglycans. Proteoglycan-affinity chromatography at pH 7.5 and 5.5 revealed that sVLDL, IDL, and LDL consisted of populations with different proteoglycan-binding affinities, and, surprisingly, the sVLDL fractions with the highest proteoglycan-affinity contained only low amounts of apolipoproteins E and C-III. Our results suggest that in atherosclerotic lesions with acidic extracellular pH, sPLA(2)-V is able to lipolyze sVLDL, IDL, and LDL, and increase their binding to proteoglycans. This is likely to provoke extracellular accumulation of lipids derived from these atherogenic lipoprotein particles and to increase the progression of the atherosclerotic lesions.

  9. Effect of nucleic acid binding dyes on DNA extraction, amplification, and STR typing.

    Science.gov (United States)

    Haines, Alicia M; Tobe, Shanan S; Kobus, Hilton J; Linacre, Adrian

    2015-10-01

    We report on the effects of six dyes used in the detection of DNA on the process of DNA extraction, amplification, and detection of STR loci. While dyes can be used to detect the presence of DNA, their use is restricted if they adversely affect subsequent DNA typing processes. Diamond™ Nucleic Acid Dye, GelGreen™, GelRed™, RedSafe™, SYBR(®) Green I, and EvaGreen™ were evaluated in this study. The percentage of dye removed during the extraction process was determined to be: 70.3% for SYBR(®) Green I; 99.6% for RedSafe™; 99.4% for EvaGreen™; 52.7% for Diamond™ Dye; 50.6% for GelRed™, and; could not be determined for GelGreen™. It was then assumed that the amount of dye in the fluorescent quantification assay had no effect on the DNA signal. The presence of all six dyes was then reviewed for their effect on DNA extraction. The t-test showed no significant difference between the dyes and the control. These extracts were then STR profiled and all dyes and control produced full DNA profiles. STR loci in the presence of GelGreen(TM) at 1X concentration showed increased amplification products in comparison to the control samples. Full STR profiles were detected in the presence of EvaGreen™ (1X), although with reduced amplification products. RedSafe™ (1X), Diamond™ Dye (1X), and SYBR(®) Green I (1X) all exhibited varying degrees of locus drop-out with GelRed™ generating no loci at all. We provide recommendations for the best dye to visualize the presence of DNA profile as a biological stain and its subsequent amplification and detection. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Plant Flavone Apigenin Binds to Nucleic Acid Bases and Reduces Oxidative DNA Damage in Prostate Epithelial Cells

    Science.gov (United States)

    Bhaskaran, Natarajan; Gupta, Sanjay

    2014-01-01

    Oxidative stress has been linked to prostate carcinogenesis as human prostate tissue is vulnerable to oxidative DNA damage. Apigenin, a dietary plant flavone, possesses anti-proliferative and anticancer effects; however, its antioxidant properties have not been fully elucidated. We investigated sub-cellular distribution of apigenin, it’s binding to DNA and protective effects against H2O2-induced DNA damage using transformed human prostate epithelial RWPE-1 cells and prostate cancer LNCaP, PC-3 and DU145 cells. Exposure of cells to apigenin exhibited higher accumulation in RWPE-1 and LNCaP cells, compared to PC-3 and DU145 cells. The kinetics of apigenin uptake in LNCaP cells was estimated with a Km value of 5 µmole/L and Vmax of 190 pmoles/million cells/h. Sub-cellular fractionation demonstrated that nuclear matrix retains the highest concentration of apigenin (45.3%), followed by cytosol (23.9%), nuclear membranes (17.9%) and microsomes (12.9%), respectively. Spectroscopic analysis of apigenin with calf-thymus DNA exhibited intercalation as the dominant binding mode to DNA duplex. Apigenin exposure resulted in significant genoprotective effects in H2O2-stressed RWPE-1 cells by reduction in reactive oxygen species levels. In addition, apigenin exposure suppressed the formation of 8-hydroxy-2′ deoxyguanosine and protected exposed cells from apoptosis. Our studies demonstrate that apigenin is readily taken up by normal prostatic epithelial cells and prostate cancer cells, and is incorporated into their nuclei, where its intercalation with nucleic acid bases may account for its antioxidant and chemopreventive activities. PMID:24614817

  11. Mechanistic studies for the role of cellular nucleic-acid-binding protein (CNBP) in regulation of c-myc transcription.

    Science.gov (United States)

    Chen, Siqi; Su, Lijuan; Qiu, Jun; Xiao, Nannan; Lin, Jing; Tan, Jia-Heng; Ou, Tian-Miao; Gu, Lian-Quan; Huang, Zhi-Shu; Li, Ding

    2013-10-01

    Guanine-rich sequence of c-myc nuclease hypersensitive element (NHE) III1 is known to fold in G-quadruplex and subsequently serves as a transcriptional silencer. Cellular nucleic-acid-binding protein (CNBP), a highly conserved zinc-finger protein with multiple biological functions, could bind to c-myc NHE III1 region, specifically to the single strand G-rich sequence. In the present study, a variety of methods, including cloning, expression and purification of protein, EMSA, CD, FRET, Ch-IP, RNA interference, luciferase reporter assay, SPR, co-immunoprecipitation, and co-transfection, were applied to investigate the mechanism for the role of CNBP in regulating c-myc transcription. We found that human CNBP specifically bound to the G-rich sequence of c-myc NHE III1 region both in vitro and in cellulo, and subsequently promoted the formation of G-quadruplex. CNBP could induce a transient decrease fo