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Sample records for acid fa binding

  1. Ligand Binding to the FA3-FA4 Cleft Inhibits the Esterase-Like Activity of Human Serum Albumin

    PubMed Central

    Ascenzi, Paolo; Leboffe, Loris; di Masi, Alessandra; Trezza, Viviana; Fanali, Gabriella; Gioia, Magda; Coletta, Massimo; Fasano, Mauro

    2015-01-01

    The hydrolysis of 4-nitrophenyl esters of hexanoate (NphOHe) and decanoate (NphODe) by human serum albumin (HSA) at Tyr411, located at the FA3-FA4 site, has been investigated between pH 5.8 and 9.5, at 22.0°C. Values of Ks, k+2, and k+2/Ks obtained at [HSA] ≥ 5×[NphOXx] and [NphOXx] ≥ 5×[HSA] (Xx is NphOHe or NphODe) match very well each other; moreover, the deacylation step turns out to be the rate limiting step in catalysis (i.e., k+3 << k+2). The pH dependence of the kinetic parameters for the hydrolysis of NphOHe and NphODe can be described by the acidic pKa-shift of a single amino acid residue, which varies from 8.9 in the free HSA to 7.6 and 7.0 in the HSA:NphOHe and HSA:NphODe complex, respectively; the pK>a-shift appears to be correlated to the length of the fatty acid tail of the substrate. The inhibition of the HSA-Tyr411-catalyzed hydrolysis of NphOHe, NphODe, and 4-nitrophenyl myristate (NphOMy) by five inhibitors (i.e., diazepam, diflunisal, ibuprofen, 3-indoxyl-sulfate, and propofol) has been investigated at pH 7.5 and 22.0°C, resulting competitive. The affinity of diazepam, diflunisal, ibuprofen, 3-indoxyl-sulfate, and propofol for HSA reflects the selectivity of the FA3-FA4 cleft. Under conditions where Tyr411 is not acylated, the molar fraction of diazepam, diflunisal, ibuprofen, and 3-indoxyl-sulfate bound to HSA is higher than 0.9 whereas the molar fraction of propofol bound to HSA is ca. 0.5. PMID:25790235

  2. Ligand binding to the FA3-FA4 cleft inhibits the esterase-like activity of human serum albumin.

    PubMed

    Ascenzi, Paolo; Leboffe, Loris; di Masi, Alessandra; Trezza, Viviana; Fanali, Gabriella; Gioia, Magda; Coletta, Massimo; Fasano, Mauro

    2015-01-01

    The hydrolysis of 4-nitrophenyl esters of hexanoate (NphOHe) and decanoate (NphODe) by human serum albumin (HSA) at Tyr411, located at the FA3-FA4 site, has been investigated between pH 5.8 and 9.5, at 22.0°C. Values of Ks, k+2, and k+2/Ks obtained at [HSA] ≥ 5×[NphOXx] and [NphOXx] ≥ 5×[HSA] (Xx is NphOHe or NphODe) match very well each other; moreover, the deacylation step turns out to be the rate limiting step in catalysis (i.e., k+3 < k+2). The pH dependence of the kinetic parameters for the hydrolysis of NphOHe and NphODe can be described by the acidic pKa-shift of a single amino acid residue, which varies from 8.9 in the free HSA to 7.6 and 7.0 in the HSA:NphOHe and HSA:NphODe complex, respectively; the pK>a-shift appears to be correlated to the length of the fatty acid tail of the substrate. The inhibition of the HSA-Tyr411-catalyzed hydrolysis of NphOHe, NphODe, and 4-nitrophenyl myristate (NphOMy) by five inhibitors (i.e., diazepam, diflunisal, ibuprofen, 3-indoxyl-sulfate, and propofol) has been investigated at pH 7.5 and 22.0°C, resulting competitive. The affinity of diazepam, diflunisal, ibuprofen, 3-indoxyl-sulfate, and propofol for HSA reflects the selectivity of the FA3-FA4 cleft. Under conditions where Tyr411 is not acylated, the molar fraction of diazepam, diflunisal, ibuprofen, and 3-indoxyl-sulfate bound to HSA is higher than 0.9 whereas the molar fraction of propofol bound to HSA is ca. 0.5. PMID:25790235

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

    PubMed

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

    2015-02-20

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

  4. Water-Mediated Differential Binding of Strontium and Cesium Cations in Fulvic Acid.

    PubMed

    Sadhu, Biswajit; Sundararajan, Mahesh; Bandyopadhyay, Tusar

    2015-08-27

    The migration of potentially harmful radionuclides, such as cesium ((137)Cs) and strontium ((90)Sr), in soil is governed by the chemical and biological reactivity of soil components. Soil organic matter (SOM) that can be modeled through fulvic acid (FA) is known to alter the mobility of radionuclide cations, Cs(+) and Sr(2+). Shedding light on the possible interaction mechanisms at the atomic level of these two ions with FA is thus vital to explain their transport behavior and for the design of new ligands for the efficient extraction of radionuclides. Here we have performed molecular dynamics, metadynamics simulations, and density-functional-theory-based calculations to understand the binding mechanism of Sr(2+) and Cs(+) cations with FA. Our studies predict that interaction of Cs(+) to FA is very weak as compared with Sr(2+). While the water-FA interaction is largely responsible for the weak binding of Cs(+) to FA, leading to the outer sphere complexation of the ion with FA, the interaction between Sr(2+) and FA is stronger and thus can surpass the existing secondary nonbonding interaction between coordinated waters and FA, leading to inner sphere complexation of the ion with FA. We also find that entropy plays a dominant role for Cs(+) binding to FA, whereas Sr(2+) binding is an enthalpy-driven process. Our predicted results are found to be in excellent agreement with the available experimental data on complexation of Cs(+) and Sr(2+) with SOM.

  5. Binding of Folic Acid Induces Specific Self-Aggregation of Lactoferrin: Thermodynamic Characterization.

    PubMed

    Tavares, Guilherme M; Croguennec, Thomas; Lê, Sébastien; Lerideau, Olivia; Hamon, Pascaline; Carvalho, Antônio F; Bouhallab, Saïd

    2015-11-17

    In the study presented here, we investigated the interaction at pH 5.5 between folic acid (FA) and lactoferrin (LF), a positively charged protein. We found a binding constant Ka of 10(5) M(-1) and a high stoichiometry of 10 mol of FA/mol of LF. The size and charge of the complexes formed evolved during titration experiments. Increasing the ionic strength to 50 mM completely abolished the isothermal titration calorimetry (ITC) signal, suggesting the predominance of electrostatic interactions in the exothermic binding obtained. We developed a theoretical model that explains the complex triphasic ITC profile. Our results revealed a two-step mechanism: FA/LF interaction followed by self-association of the complexes thus formed. We suggest that 10 FA molecules bind to LF to form saturated reactive complexes (FA10/LF) that further self-associate into aggregates with a finite size of around 15 nm. There is thus a critical saturation degree of the protein, above which the self-association can take place. We present here the first results that provide comprehensive details of the thermodynamics of FA/LF complexation-association. Given the high stoichiometry, allowing a load of 55 mg of FA/g of LF, we suggest that FA/LF aggregates would be an effective vehicle for FA in fortified drinks. PMID:26488446

  6. Field-Evolved Mode 1 Resistance of the Fall Armyworm to Transgenic Cry1Fa-Expressing Corn Associated with Reduced Cry1Fa Toxin Binding and Midgut Alkaline Phosphatase Expression

    PubMed Central

    Jakka, Siva R. K.; Gong, Liang; Hasler, James; Banerjee, Rahul; Sheets, Joel J.; Narva, Kenneth; Blanco, Carlos A.

    2015-01-01

    Insecticidal protein genes from the bacterium Bacillus thuringiensis (Bt) are expressed by transgenic Bt crops (Bt crops) for effective and environmentally safe pest control. The development of resistance to these insecticidal proteins is considered the most serious threat to the sustainability of Bt crops. Resistance in fall armyworm (Spodoptera frugiperda) populations from Puerto Rico to transgenic corn producing the Cry1Fa insecticidal protein resulted, for the first time in the United States, in practical resistance, and Bt corn was withdrawn from the local market. In this study, we used a field-collected Cry1Fa corn-resistant strain (456) of S. frugiperda to identify the mechanism responsible for field-evolved resistance. Binding assays detected reduced Cry1Fa, Cry1Ab, and Cry1Ac but not Cry1Ca toxin binding to midgut brush border membrane vesicles (BBMV) from the larvae of strain 456 compared to that from the larvae of a susceptible (Ben) strain. This binding phenotype is descriptive of the mode 1 type of resistance to Bt toxins. A comparison of the transcript levels for putative Cry1 toxin receptor genes identified a significant downregulation (>90%) of a membrane-bound alkaline phosphatase (ALP), which translated to reduced ALP protein levels and a 75% reduction in ALP activity in BBMV from 456 compared to that of Ben larvae. We cloned and heterologously expressed this ALP from susceptible S. frugiperda larvae and demonstrated that it specifically binds with Cry1Fa toxin. This study provides a thorough mechanistic description of field-evolved resistance to a transgenic Bt crop and supports an association between resistance and reduced Cry1Fa toxin binding and levels of a putative Cry1Fa toxin receptor, ALP, in the midguts of S. frugiperda larvae. PMID:26637593

  7. Field-Evolved Mode 1 Resistance of the Fall Armyworm to Transgenic Cry1Fa-Expressing Corn Associated with Reduced Cry1Fa Toxin Binding and Midgut Alkaline Phosphatase Expression.

    PubMed

    Jakka, Siva R K; Gong, Liang; Hasler, James; Banerjee, Rahul; Sheets, Joel J; Narva, Kenneth; Blanco, Carlos A; Jurat-Fuentes, Juan L

    2015-12-04

    Insecticidal protein genes from the bacterium Bacillus thuringiensis (Bt) are expressed by transgenic Bt crops (Bt crops) for effective and environmentally safe pest control. The development of resistance to these insecticidal proteins is considered the most serious threat to the sustainability of Bt crops. Resistance in fall armyworm (Spodoptera frugiperda) populations from Puerto Rico to transgenic corn producing the Cry1Fa insecticidal protein resulted, for the first time in the United States, in practical resistance, and Bt corn was withdrawn from the local market. In this study, we used a field-collected Cry1Fa corn-resistant strain (456) of S. frugiperda to identify the mechanism responsible for field-evolved resistance. Binding assays detected reduced Cry1Fa, Cry1Ab, and Cry1Ac but not Cry1Ca toxin binding to midgut brush border membrane vesicles (BBMV) from the larvae of strain 456 compared to that from the larvae of a susceptible (Ben) strain. This binding phenotype is descriptive of the mode 1 type of resistance to Bt toxins. A comparison of the transcript levels for putative Cry1 toxin receptor genes identified a significant downregulation (>90%) of a membrane-bound alkaline phosphatase (ALP), which translated to reduced ALP protein levels and a 75% reduction in ALP activity in BBMV from 456 compared to that of Ben larvae. We cloned and heterologously expressed this ALP from susceptible S. frugiperda larvae and demonstrated that it specifically binds with Cry1Fa toxin. This study provides a thorough mechanistic description of field-evolved resistance to a transgenic Bt crop and supports an association between resistance and reduced Cry1Fa toxin binding and levels of a putative Cry1Fa toxin receptor, ALP, in the midguts of S. frugiperda larvae.

  8. Evidence that Chemical Chaperone 4-Phenylbutyric Acid Binds to Human Serum Albumin at Fatty Acid Binding Sites

    PubMed Central

    James, Joel; Shihabudeen, Mohamed Sham; Kulshrestha, Shweta; Goel, Varun; Thirumurugan, Kavitha

    2015-01-01

    Endoplasmic reticulum stress elicits unfolded protein response to counteract the accumulating unfolded protein load inside a cell. The chemical chaperone, 4-Phenylbutyric acid (4-PBA) is a FDA approved drug that alleviates endoplasmic reticulum stress by assisting protein folding. It is found efficacious to augment pathological conditions like type 2 diabetes, obesity and neurodegeneration. This study explores the binding nature of 4-PBA with human serum albumin (HSA) through spectroscopic and molecular dynamics approaches, and the results show that 4-PBA has high binding specificity to Sudlow Site II (Fatty acid binding site 3, subdomain IIIA). Ligand displacement studies, RMSD stabilization profiles and MM-PBSA binding free energy calculation confirm the same. The binding constant as calculated from fluorescence spectroscopic studies was found to be kPBA = 2.69 x 105 M-1. Like long chain fatty acids, 4-PBA induces conformational changes on HSA as shown by circular dichroism, and it elicits stable binding at Sudlow Site II (fatty acid binding site 3) by forming strong hydrogen bonding and a salt bridge between domain II and III of HSA. This minimizes the fluctuation of HSA backbone as shown by limited conformational space occupancy in the principal component analysis. The overall hydrophobicity of W214 pocket (located at subdomain IIA), increases upon occupancy of 4-PBA at any FA site. Descriptors of this pocket formed by residues from other subdomains largely play a role in compensating the dynamic movement of W214. PMID:26181488

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

    PubMed Central

    Gajda, Angela M.; Storch, Judith

    2014-01-01

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

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

    PubMed Central

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

    2016-01-01

    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 is 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. PMID:26256755

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

    PubMed

    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.

  12. Δ(9)-THC modulation of fatty acid 2-hydroxylase (FA2H) gene expression: possible involvement of induced levels of PPARα in MDA-MB-231 breast cancer cells.

    PubMed

    Takeda, Shuso; Ikeda, Eriko; Su, Shengzhong; Harada, Mari; Okazaki, Hiroyuki; Yoshioka, Yasushi; Nishimura, Hajime; Ishii, Hiroyuki; Kakizoe, Kazuhiro; Taniguchi, Aya; Tokuyasu, Miki; Himeno, Taichi; Watanabe, Kazuhito; Omiecinski, Curtis J; Aramaki, Hironori

    2014-12-01

    We recently reported that Δ(9)-tetrahydrocannabinol (Δ(9)-THC), a major cannabinoid component in Cannabis Sativa (marijuana), significantly stimulated the expression of fatty acid 2-hydroxylase (FA2H) in human breast cancer MDA-MB-231 cells. Peroxisome proliferator-activated receptor α (PPARα) was previously implicated in this induction. However, the mechanisms mediating this induction have not been elucidated in detail. We performed a DNA microarray analysis of Δ(9)-THC-treated samples and showed the selective up-regulation of the PPARα isoform coupled with the induction of FA2H over the other isoforms (β and γ). Δ(9)-THC itself had no binding/activation potential to/on PPARα, and palmitic acid (PA), a PPARα ligand, exhibited no stimulatory effects on FA2H in MDA-MB-231 cells; thus, we hypothesized that the levels of PPARα induced were involved in the Δ(9)-THC-mediated increase in FA2H. In support of this hypothesis, we herein demonstrated that; (i) Δ(9)-THC activated the basal transcriptional activity of PPARα in a concentration-dependent manner, (ii) the concomitant up-regulation of PPARα/FA2H was caused by Δ(9)-THC, (iii) PA could activate PPARα after the PPARα expression plasmid was introduced, and (iv) the Δ(9)-THC-induced up-regulation of FA2H was further stimulated by the co-treatment with L-663,536 (a known PPARα inducer). Taken together, these results support the concept that the induced levels of PPARα may be involved in the Δ(9)-THC up-regulation of FA2H in MDA-MB-231 cells.

  13. Design of an effective bifunctional catalyst organotriphosphonic acid-functionalized ferric alginate (ATMP-FA) and optimization by Box-Behnken model for biodiesel esterification synthesis of oleic acid over ATMP-FA.

    PubMed

    Liu, Wei; Yin, Ping; Liu, Xiguang; Qu, Rongjun

    2014-12-01

    Biodiesel production has become an intense research area because of rapidly depleting energy reserves and increasing petroleum prices together with environmental concerns. This paper focused on the optimization of the catalytic performance in the esterification reaction of oleic acid for biodiesel production over the bifunctional catalyst organotriphosphonic acid-functionalized ferric alginate ATMP-FA. The reaction parameters including catalyst amount, ethanol to oleic acid molar ratio and reaction temperature have been optimized by response surface methodology (RSM) using the Box-Behnken model. It was found that the reaction temperature was the most significant factor, and the best conversion ratio of oleic acid could reach 93.17% under the reaction conditions with 9.53% of catalyst amount and 8.62:1 of ethanol to oleic acid molar ratio at 91.0 °C. The research results show that two catalytic species could work cooperatively to promote the esterification reaction, and the bifunctional ATMP-FA is a potential catalyst for biodiesel production.

  14. Binding of ferulic acid to cytochrome c enhances stability of the protein at physiological pH and inhibits cytochrome c-induced apoptosis.

    PubMed

    Yang, Fang; Zhou, Bing-Rui; Zhang, Peng; Zhao, Yan-Feng; Chen, Jie; Liang, Yi

    2007-12-15

    Ferulic acid (FA) is one of the most effective components of a traditional Chinese medicine, angelica, and cytochrome c plays a vital role in apoptosis. Here we report the application of fluorescence spectroscopy, isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), and circular dichroism (CD) to investigate the mechanism for the interaction of bovine heart cytochrome c with FA and the effect of the binding on native state stability of the protein at physiological pH. Fluorescence spectroscopic studies together with ITC measurements indicate that FA binds to cytochrome c with moderate affinity and quenches the intrinsic fluorescence of the protein in a static way. ITC experiments show that the interaction of cytochrome c with FA is driven by a moderately favorable entropy increase in combination with a less favorable enthalpy decrease for the first binding site of the protein. The melting temperature of cytochrome c in the presence of FA measured by DSC and CD increases 4.0 and 5.0 degrees C, respectively, compared with that in the absence of FA. Taken together, these results indicate that FA binds to and stabilizes cytochrome c at physiological pH. Furthermore, binding of FA to cytochrome c inhibits cytochrome c-induce apoptosis of human hepatoma cell line SMMC-7721. Our data provide insight into the mechanism of drug-protein interactions, and will be helpful to the understanding of the mechanism for FA-inhibited and cytochrome c-induced apoptosis.

  15. The mannose-binding lectin gene FaMBL1 is involved in the resistance of unripe strawberry fruits to Colletotrichum acutatum.

    PubMed

    Guidarelli, Michela; Zoli, Lisa; Orlandini, Alessandro; Bertolini, Paolo; Baraldi, Elena

    2014-10-01

    The fungal pathogen Colletotrichum acutatum is the causal agent of strawberry (Fragaria × ananassa) anthracnose. Although the fungus can infect strawberry fruits at both unripe and ripe stages, the symptoms appear only on red ripe fruits. On white unripe fruits, the pathogen becomes quiescent as melanized appressoria after 24 h of interaction. Previous transcriptome analysis has indicated that a mannose-binding lectin (MBL) gene is the most up-regulated gene in 24-h-infected white strawberries, suggesting a role for this gene in the low susceptibility of unripe stages. A time course analysis of the expression of this MBL gene, named FaMBL1 (Fragaria × ananassa MBL 1a), was undertaken to monitor its expression profile in white and red fruits at early interaction times: FaMBL1 was expressed exclusively in white fruit after 24 h, when the pathogen was quiescent. Agrobacterium-mediated transient transformation was used to silence and overexpress the FaMBL1 gene in 24-h-infected white and red strawberries, respectively. FaMBL1-silenced unripe fruits showed an increase in susceptibility to C. acutatum. These 24-h-infected tissues contained subcuticular hyphae, indicating pathogen penetration and active growth. In contrast, overexpression of FaMBL1 in ripe fruits decreased susceptibility; here, 24-h-infected tissues showed a high percentage of ungerminated appressoria, suggesting that the growth of the pathogen had slowed. These data suggest that FaMBL1 plays a crucial role in the resistance of unripe strawberry fruits to C. acutatum. PMID:24690196

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

    NASA Astrophysics Data System (ADS)

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

    2012-04-01

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

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

    PubMed Central

    Storch, Judith; McDermott, Lindsay

    2009-01-01

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

  18. Suppression of the ELO-2 FA elongation activity results in alterations of the fatty acid composition and multiple physiological defects, including abnormal ultradian rhythms, in Caenorhabditis elegans.

    PubMed Central

    Kniazeva, Marina; Sieber, Matt; McCauley, Scott; Zhang, Kang; Watts, Jennifer L; Han, Min

    2003-01-01

    While the general steps of fatty acid (FA) biosynthesis are well understood, the individual enzymes involved in the elongation of long chain saturated and polyunsaturated FA (PUFA) are largely unknown. Recent research indicates that these enzymes might be of considerable physiological importance for human health. We use Caenorhabditis elegans to study FA elongation activities and associated abnormal phenotypes. In this article we report that the predicted C. elegans F11E6.5/ELO-2 is a functional enzyme with the FA elongation activity. It is responsible for the elongation of palmitic acid and is involved in PUFA biosynthesis. RNAi-mediated suppression of ELO-2 causes an accumulation of palmitate and an associated decrease in the PUFA fraction in triacylglycerides and phospholipid classes. This imbalance in the FA composition results in multiple phenotypic defects such as slow growth, small body size, reproductive defects, and changes in rhythmic behavior. ELO-2 cooperates with the previously reported ELO-1 in 20-carbon PUFA production, and at least one of the enzymes must function to provide normal growth and development in C. elegans. The presented data indicate that suppression of a single enzyme of the FA elongation machinery is enough to affect various organs and systems in worms. This effect resembles syndromic disorders in humans. PMID:12586704

  19. Folate-binding protein and the absorption of folic acid in the small intestine of the suckling rat

    SciTech Connect

    Mason, J.B.; Selhub, J.

    1988-09-01

    The folate in milk is largely bound to high-affinity folate-binding protein (FBP). With an in vivo intestinal loop technique, we examined the absorption of folic acid bound to FBP (FA-FBP) in the small intestine of the suckling rat. In contrast to unbound folic acid (FA), FA-FBP is absorbed more avidly in the ileum than in the jejunum (p less than 0.025) and its absorption is not inhibited by 1 mmol sulfasalazine/L. Folate-binding activities in the mucosa of the proximal (duodenum and jejunum combined) and distal (ileum) small intestine were also examined and found to be 0.32 and 1.31 pmol/mg protein, respectively (p less than 0.001). A 6-h fast produced a 42% decrease in folate-binding activity in the distal small intestine (p less than 0.01) but did not change activity in the proximal portion. Collectively, these observations suggest that FA-FBP is absorbed by a mechanism that is distinct from that responsible for the absorption of FA and that absorption does not require prior dissociation of the vitamin-binding protein complex.

  20. The carbohydrate-binding module of Fragaria × ananassa expansin 2 (CBM-FaExp2) binds to cell wall polysaccharides and decreases cell wall enzyme activities "in vitro".

    PubMed

    Nardi, Cristina; Escudero, Cristian; Villarreal, Natalia; Martínez, Gustavo; Civello, Pedro Marcos

    2013-01-01

    A putative carbohydrate binding module (CBM) from strawberry (Fragaria × ananassa Duch.) expansin 2 (CBM-FaExp2) was cloned and the encoding protein was over-expressed in Escherichia coli and purified in order to evaluate its capacity to bind different cell wall polysaccharides "in vitro". The protein CBM-FaExp2 bound to microcrystalline cellulose, xylan and pectin with different affinities (K(ad) = 33.6 ± 0.44 mL g(-1), K(ad) = 11.37 ± 0.87 mL g(-1), K(ad) = 10.4 ± 0.19 mL g(-1), respectively). According to "in vitro" enzyme assays, this CBM is able to decrease the activity of cell wall degrading enzymes such as polygalacturonase, endo-glucanase, pectinase and xylanase, probably because the binding of CBM-FaExp2 to the different substrates interferes with enzyme activity. The results suggest that expansins would bind not only cellulose but also a wide range of cell wall polymers.

  1. Potent in vitro synergism of fusidic acid (FA) and berberine chloride (BBR) against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA).

    PubMed

    Liang, Rong-mei; Yong, Xiao-lan; Duan, Yu-qin; Tan, Yong-hong; Zeng, Ping; Zhou, Zi-ying; Jiang, Yan; Wang, Shi-hua; Jiang, Yun-ping; Huang, Xiao-chun; Dong, Zhao-hui; Hu, Ting-ting; Shi, Hui-qing; Li, Nan

    2014-11-01

    It was found in the present study that combined use of fusidic acid (FA) and berberine chloride (BBR) offered an in vitro synergistic action against 7 of the 30 clinical methicillin-resistant Staphylococcus aureus (MRSA) strains, with a fractional inhibitory concentration (FIC) index ranging from 0.5 to 0.19. This synergistic effect was most pronounced on MRSA 4806, an FA-resistant isolate, with a minimum inhibitory concentration (MIC) value of 1,024 μg/ml. The time-kill curve experiment showed that FA plus BBR yielded a 4.2 log10 c.f.u./ml reduction in the number of MRSA 4806 bacteria after 24-h incubation as compared with BBR alone. Viable count analysis showed that FA plus BBR produced a 3.0 log10 c.f.u./ml decrease in biofilm formation and a 1.5 log10 c.f.u./ml decrease in mature biofilm in viable cell density as compared with BBR alone. In addition, phase contrast micrographs confirmed that biofilm formation was significantly inhibited and mature biofilm was obviously destructed when FA was used in combination with BBR. These results provide evidence that combined use of FA and BBR may prove to be a promising clinical therapeutic strategy against MRSA.

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

    PubMed

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

    2014-06-01

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

  3. Calcium binding to an aquatic fulvic acid

    NASA Astrophysics Data System (ADS)

    Paxéus, Nicklas; Wedborg, Margareta

    The degree of binding of calcium to aquatic fulvic acid from the Göta River was estimated from potentiometric titrations. A pH-glass electrode and a calcium-selective electrode were used to monitor the free concentrations of the competing, central ions. The ionic strength and the temperature were maintained constant at 0.1 M and 25°C. The total concentration of fulvic acid was maintained at approximately 1 g 1-1, while the total calcium concentration was varied within the range 0-10-3 M. Two types of titrations were carried out: (1) back titration with hydrochloric acid from basic solution, roughly within the pH range 10.5-2.5; (2) titration with calcium chloride at a constant total hydrogen ion concentration. The model applied for the calcium binding was an extension of our previous model for the acid-base behaviour.

  4. Iodine binding to humic acid.

    PubMed

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

    2016-08-01

    The rate of reactions between humic acid (HA) and iodide (I(-)) and iodate (IO3(-)) have been investigated in suspensions spiked with (129)I at concentrations of 22, 44 and 88 μg L(-1) and stored at 10 °C. Changes in the speciation of (129)I(-), (129)IO3(-) and mixed ((129)I(-) + (129)IO3(-)) spikes were monitored over 77 days using liquid chromatography inductively coupled plasma mass spectrometry (LC-ICP-MS). In suspensions spiked with (129)I(-) 25% of the added I(-) was transformed into organic iodine (Org-(129)I) within 77 days and there was no evidence of (129)IO3(-) formation. By contrast, rapid loss of (129)IO3(-) and increase in both (129)I(-) and Org-(129)I was observed in (129)IO3(-)-spiked suspensions. However, the rate of Org-(129)I production was greater in mixed systems compared to (129)IO3(-)-spiked suspensions with the same total (129)I concentration, possibly indicating IO3(-)I(-) redox coupling. Size exclusion chromatography (SEC) demonstrated that Org-(129)I was present in both high and low molecular weight fractions of the HA although a slight preference to bond with the lower molecular weight fractions was observed indicating that, after 77 days, the spiked isotope had not fully mixed with the native (127)I pool. Iodine transformations were modelled using first order rate equations and fitted rate coefficients determined. However, extrapolation of the model to 250 days indicated that a pseudo-steady state would be attained after ∼200 days but that the proportion of (129)I incorporated into HA was less than that of (127)I indicating the presence of a recalcitrant pool of (127)I that was unavailable for isotopic mixing. PMID:27231879

  5. Characterization of DNA Binding and Retinoic Acid Binding Properties of Retinoic Acid Receptor

    NASA Astrophysics Data System (ADS)

    Yang, Na; Schule, Roland; Mangelsdorf, David J.; Evans, Ronald M.

    1991-05-01

    High-level expression of the full-length human retinoic acid receptor (RAR) α and the DNA binding domain of the RAR in Escherichia coli was achieved by using a T7 RNA polymerase-directed expression system. After induction, full-length RAR protein was produced at an estimated level of 20% of the total bacterial proteins. Both intact RAR molecules and the DNA binding domain bind to the cognate DNA response element with high specificity in the absence of retinoic acid. However, this binding is enhanced to a great extent upon the addition of eukaryotic cell extracts. The factor responsible for this enhancement is heat-sensitive and forms a complex with RAR that binds to DNA and exhibits a distinct migration pattern in the gel-mobility-shift assay. The interaction site of the factor with RAR is localized in the 70-amino acid DNA binding region of RAR. The hormone binding ability of the RARα protein was assayed by a charcoal absorption assay and the RAR protein was found to bind to retinoic acid with a K_d of 2.1 x 10-10 M.

  6. Linoleic acid binding properties of ovalbumin nanoparticles.

    PubMed

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

    2015-04-01

    In the present work, ovalbumin (OVA) solutions (10 g/L, 50 mM NaCl, pH 7.5) were heat-treated at 75, 80 and 85°C (namely, OVA-75, OVA-80 and OVA-85, respectively), from 0 to 25 min. OVA nanoparticles (OVAn) around 100 nm were obtained. For 3 min of heat treatment, OVAn sizes increased with temperature, but for a heating time longer than 10 min, OVA-75 showed the highest size values. OVAn surface hydrophobicity increased 6-8 folds in comparison with native OVA and wavelength blue shifts of 25-30 nm in maximum fluorescence intensity were registered. These results suggest that buried hydrophobic residues were exposed to the aqueous medium. Binding experiments with linoleic acid (LA) as polyunsaturated fatty acid (PUFA) model were carried out. Firstly, binding ability of OVAn was determined from LA titration curves of intrinsic fluorescence measurements. OVA-85 at 5 min presented the highest binding ability and it was used for further binding properties studies (turbidity, particle size distribution--PSD--analysis and ζ-potential measurements). Turbidity measurement and PSD analysis showed that OVAn-LA nanocomplexes were formed, avoiding LA supramolecular self-assembly formation. The union of LA to OVAn surface confers them significant lower ζ-potential and larger size. Hence, fluorescence and ζ-potential results showed that LA would bind to OVAn by mean of hydrophobic interactions. Information derived from this work could be important to potentially use OVAn as PUFA vehiculization with applications in several industrial sectors (food, pharmaceutical, cosmetics, etc.).

  7. Ligatin binds phosphohexose residues on acidic hydrolases.

    PubMed

    Jakoi, E R; Kempe, K; Gaston, S M

    1981-01-01

    Ligatin, a receptor that recognizes phosphorylated sugars, was isolated from plasma membranes of mouse macrophages, rat ileum, and rat brain. Several acidic hydrolases including N-acetyl beta-D-glucosaminidase (beta-NAG) were solubilized with this receptor. The solubilized beta-NAG bound to ligatin in vitro as demonstrated by affinity chromatography using the immobilized receptor. beta-N-Acetyl D-glucosaminidase-ligatin complexes were dissociated by low concentrations of mannose 6-phosphate (Man6P) and/or glucose 1-phosphate (Glc 1P). The effectiveness of these two phosphomonosaccharides varied depending on the source of the enzyme: ileal beta-NAG-ligatin complexes showed a four-fold preferential dissociation with Man6P; macrophage complexes showed a 160-fold preferential dissociation with Glc 1P. Brain complexes dissociated with nearly equal preference for Man6P and Glc 1P. Heterologous complexes displayed the specificity characteristic of the source of the enzyme regardless of the source of the ligatin. Treatment of the solubilized hydrolases with endoglucosaminidase H released phosphorous-32 label from these enzymes and prevented binding of beta-NAG to ligatin. However, treatment of the solubilized hydrolases with alkaline phosphatase reduced the binding of beta-NAG to ligatin by no more than 30%. This apparent resistance of beta-NAG to dephosphorylation was consistent with the chromatographic behavior of QAE of 3H-labeled acidic oligosaccharides isolated from the solubilized hydrolases. The oligosaccharides that contain phosphorylated hexose were less acidic than phosphomonoesters and were insensitive to alkaline phosphatase until subjected to acid hydrolysis. These results suggested the presence of a phosphodiester on beta-NAG analogous to the NAC glucosamine 1 P6 mannose present on beta-glucuronidase isolated from mouse lymphoma cells (Tabas I, Kornfield, S: J Biol Chem 255: 6633, 1980). PMID:7299841

  8. Liver Fatty Acid Binding Protein and Obesity

    PubMed Central

    Atshaves, B.P.; Martin, G.G.; Hostetler, H.A.; McIntosh, A.L.; Kier, A.B.; Schroeder, F.

    2010-01-01

    While low levels of unesterified long chain fatty acids (LCFAs) are normal metabolic intermediates of dietary and endogenous fat, LCFAs are also potent regulators of key receptors/enzymes, and at high levels become toxic detergents within the cell. Elevated levels of LCFAs are associated with diabetes, obesity, and metabolic syndrome. Consequently, mammals evolved fatty acid binding proteins (FABPs) that bind/sequester these potentially toxic free fatty acids in the cytosol and present them for rapid removal in oxidative (mitochondria, peroxisomes) or storage (endoplasmic reticulum, lipid droplets) organelles. Mammals have a large (15 member) family of FABPs with multiple members occurring within a single cell type. The first described FABP, liver-FABP (L-FABP, or FABP1), is expressed in very high levels (2-5% of cytosolic protein) in liver as well as intestine and kidney. Since L-FABP facilitates uptake and metabolism of LCFAs in vitro and in cultured cells, it was expected that abnormal function or loss of L-FABP would reduce hepatic LCFA uptake/oxidation and thereby increase LCFAs available for oxidation in muscle and/or storage in adipose. This prediction was confirmed in vitro with isolated liver slices and cultured primary hepatocytes from L-FABP gene-ablated mice. Despite unaltered food consumption when fed a control diet ad libitum, the L-FABP null mice exhibited age- and sex-dependent weight gain and increased fat tissue mass. The obese phenotype was exacerbated in L-FABP null mice pair-fed a high fat diet. Taken together with other findings, these data suggest that L-FABP could have an important role in preventing age- or diet-induced obesity. PMID:20537520

  9. Ala54Thr Fatty Acid-Binding Protein 2 (FABP2) Polymorphism in Recurrent Depression: Associations with Fatty Acid Concentrations and Waist Circumference

    PubMed Central

    Assies, Johanna; Koeter, Maarten W. J.; Visser, Ieke; Ruhé, Henricus G.; Bockting, Claudi L. H.; Schene, Aart H.

    2013-01-01

    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. PMID:24340071

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

    SciTech Connect

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

    2014-07-01

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

  11. Trans fatty acids (tFA): sources and intake levels, biological effects and content in commercial Spanish food.

    PubMed

    Fernández-San Juan, P-M

    2009-01-01

    Recent studies of dietary habits in children and adolescents performed in Spain show that a high percentage of the daily energy intake corresponds to fat (42.0-43.0%). These findings show an excessive contribution of saturated fatty acids and also a considerable supply of trans fatty acids. These compounds are formed generally during partial hydrogenation of vegetable oils, a process that converts vegetable oils into semisolid fats. Also, in some cases naturally occurring trans fatty acids in smaller amounts in meat and dairy products from ruminants (cows, sheep), these trans fatty acids are produced by the action of bacteria in the ruminant stomach by reactions of biohydrogenation. On the other hand, metabolic studies have clearly shown that trans fatty acids increase LDL cholesterol and reduce HDL cholesterol. Our results show that major sources of trans fatty acids in commercial Spanish foods are fast-food (hamburger, French fries), snacks, bakery products (cakes, donuts, biscuits), margarines and dehydrated soups. PMID:19893860

  12. Trans fatty acids (tFA): sources and intake levels, biological effects and content in commercial Spanish food.

    PubMed

    Fernández-San Juan, P-M

    2009-01-01

    Recent studies of dietary habits in children and adolescents performed in Spain show that a high percentage of the daily energy intake corresponds to fat (42.0-43.0%). These findings show an excessive contribution of saturated fatty acids and also a considerable supply of trans fatty acids. These compounds are formed generally during partial hydrogenation of vegetable oils, a process that converts vegetable oils into semisolid fats. Also, in some cases naturally occurring trans fatty acids in smaller amounts in meat and dairy products from ruminants (cows, sheep), these trans fatty acids are produced by the action of bacteria in the ruminant stomach by reactions of biohydrogenation. On the other hand, metabolic studies have clearly shown that trans fatty acids increase LDL cholesterol and reduce HDL cholesterol. Our results show that major sources of trans fatty acids in commercial Spanish foods are fast-food (hamburger, French fries), snacks, bakery products (cakes, donuts, biscuits), margarines and dehydrated soups.

  13. Echinococcus granulosus fatty acid binding proteins subcellular localization.

    PubMed

    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.

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

    PubMed Central

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

    2014-01-01

    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. PMID:25004958

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

    DOEpatents

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

    2015-03-17

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

  16. Copper binding to soil fulvic and humic acids: NICA-Donnan modeling and conditional affinity spectra.

    PubMed

    Xu, Jinling; Tan, Wenfeng; Xiong, Juan; Wang, Mingxia; Fang, Linchuan; Koopal, Luuk K

    2016-07-01

    Binding of Cu(II) to soil fulvic acid (JGFA), soil humic acids (JGHA, JLHA), and lignite-based humic acid (PAHA) was investigated through NICA-Donnan modeling and conditional affinity spectrum (CAS). It is to extend the knowledge of copper binding by soil humic substances (HS) both in respect of enlarging the database of metal ion binding to HS and obtaining a good insight into Cu binding to the functional groups of FA and HA by using the NICA-Donnan model to unravel the intrinsic and conditional affinity spectra. Results showed that Cu binding to HS increased with increasing pH and decreasing ionic strength. The amount of Cu bound to the HAs was larger than the amount bound to JGFA. Milne's generic parameters did not provide satisfactory predictions for the present soil HS samples, while material-specific NICA-Donnan model parameters described and predicted Cu binding to the HS well. Both the 'low' and 'high' concentration fitting procedures indicated a substantial bidentate structure of the Cu complexes with HS. By means of CAS underlying NICA isotherm, which was scarcely used, the nature of the binding at different solution conditions for a given sample and the differences in binding mode were illustrated. It was indicated that carboxylic group played an indispensable role in Cu binding to HS in that the carboxylic CAS had stronger conditional affinity than the phenolic distribution due to its large degree of proton dissociation. The fact was especially true for JGFA and JLHA which contain much larger amount of carboxylic groups, and the occupation of phenolic sites by Cu was negligible. Comparable amounts of carboxylic and phenolic groups on PAHA and JGHA, increased the occupation of phenolic type sites by Cu. The binding strength of PAHA-Cu and JGHA-Cu was stronger than that of JGFA-Cu and JLHA-Cu. The presence of phenolic groups increased the chance of forming more stable complexes, such as the salicylate-Cu or catechol-Cu type structures. PMID:27061366

  17. Nucleic acids encoding a cellulose binding domain

    DOEpatents

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

    1996-03-05

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 15 figs.

  18. Nucleic acids encoding a cellulose binding domain

    DOEpatents

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc A.; Doi, Roy H.

    1996-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  19. Poly(l-lysine)-graft-folic acid-coupled poly(2-methyl-2-oxazoline) (PLL-g-PMOXA-c-FA): a bioactive copolymer for specific targeting to folate receptor-positive cancer cells.

    PubMed

    Chen, Yin; Cao, Wenbin; Zhou, Junli; Pidhatika, Bidhari; Xiong, Bin; Huang, Lu; Tian, Qian; Shu, Yiwei; Wen, Weijia; Hsing, I-Ming; Wu, Hongkai

    2015-02-01

    In this study, we present the preparation, characterization and application of a novel bioactive copolymer poly(l-lysine)-graft-folic acid-coupled poly(2-methyl-2-oxazoline) (PLL-g-PMOXA-c-FA), which has a specific interaction with folate receptor (FR)-positive cancer cells. Glass surface immobilized with PLL-g-PMOXA-c-FA was demonstrated to be adhesive to FR-positive cancer cells (HeLa, JEG-3) while nonadhesive to FR-negative ones (MCF-7, HepG2) in 3 h. The specific interaction between conjugated FA on the substrate and FRs on the cells could hardly be inhibited unless a high concentration (5 mM) of free FA was used due to the multivalent nature of it. The FA functionality ratio of the copolymer on the substrate had a significant influence on the adhesion of HeLa cells, and our experiments revealed that the affinity of the substrate to the cells declined dramatically with the decrease of functionality ratio. This was believed to be caused by the polydispersity of PMOXA tethers, as supported by GPC and ToF-SIMS data. As a proof of concept in the application of our material, we demonstrated successful recovery of HeLa cells from mixture with MCF-7 (1:100) on the copolymer-coated glass, and our results showed that both high sensitivity (95.6 ± 13.3%) and specificity (24.3 ± 8.6%) were achieved.

  20. Imatinib binding to human serum albumin modulates heme association and reactivity.

    PubMed

    Di Muzio, Elena; Polticelli, Fabio; Trezza, Viviana; Fanali, Gabriella; Fasano, Mauro; Ascenzi, Paolo

    2014-10-15

    Imatinib, an inhibitor of the Bcr-Abl tyrosine kinase, is approximately 95% bound to plasma proteins, α1-acid glycoprotein (AGP) being the primary carrier. However, human serum albumin (HSA) may represent the secondary carrier of imatinib in pathological states characterized by low AGP levels, such as pancreatic cancer, hepatic cirrhosis, hepatitis, hyperthyroidism, nephrotic syndrome, malnutrition, and cachexia. Here, thermodynamics of imatinib binding to full-length HSA and its recombinant Asp1-Glu382 truncated form (containing only the FA1, FA2, FA6, and FA7 binding sites; trHSA), in the absence and presence of ferric heme (heme-Fe(III)), and the thermodynamics of heme-Fe(III) binding to HSA and trHSA, in the absence and presence of imatinib, has been investigated. Moreover, the effect of imatinib on kinetics of peroxynitrite detoxification by ferric human serum heme-albumin (HSA-heme-Fe(III)) and ferric truncated human serum heme-albumin (trHSA-heme-Fe(III)) has been explored. All data were obtained at pH 7.0, and 20.0 °C and 37.0 °C. Imatinib binding to the FA7 site of HSA and trHSA inhibits allosterically heme-Fe(III) association to the FA1 site and vice versa, according to linked functions. Moreover, imatinib binding to the secondary FA2 site of HSA-heme-Fe(III) inhibits allosterically peroxynitrite detoxification. Docking simulations and local structural comparison with other imatinib-binding proteins support functional data indicating the preferential binding of imatinib to the FA1 and FA7 sites of HSA, and to the FA2 and FA7 sites of HSA-heme-Fe(III). Present results highlight the allosteric coupling of the FA1, FA2, and FA7 sites of HSA, and may be relevant in modulating ligand binding and reactivity properties of HSA in vivo. PMID:25057771

  1. Folic acid binds DNA and RNA at different locations.

    PubMed

    Bourassa, P; Tajmir-Riahi, H A

    2015-03-01

    We located multiple binding sites for folic acid on DNA and tRNA at physiological conditions, using FTIR, CD, fluorescence spectroscopic methods and molecular modeling. Structural analysis revealed that folic acid binds DNA and tRNA at multiple sites via hydrophilic, hydrophobic and H-bonding contacts with overall binding constants of Kfolic acid-DNA=1.1 (±0.3)×10(4) M(-1) and Kfolic acid-tRNA=6.4 (±0.5)×10(3) M(-1). Molecular modeling showed the participation of several nucleobases in folic acid complexes with DNA and tRNA, stabilized by H-bonding network. Two types of complexes were located for folic acid-tRNA adducts, one at the major groove and the other with TΨC loop, while acid binding occurs at major and minor grooves of DNA duplex. Folic acid complexation induced more alterations of DNA structure than tRNA.

  2. Association of polymorphisms in solute carrier family 27, isoform A6 (SLC27A6) and fatty acid-binding protein-3 and fatty acid-binding protein-4 (FABP3 and FABP4) with fatty acid composition of bovine milk.

    PubMed

    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

    2013-09-01

    The main goal of this study was to develop tools for genetic selection of animals producing milk with a lower concentration of saturated fatty acids (SFA) and a higher concentration of unsaturated fatty acids (UFA). The reasons for changing milk fatty acid (FA) composition were to improve milk technological properties, such as for production of more spreadable butter, and milk nutritional value with respect to the potentially adverse effects of SFA on human health. We hypothesized that genetic polymorphisms in solute carrier family 27, isoform A6 (SLC27A6) fatty acid transport protein gene and fatty acid binding protein (FABP)-3 and FABP-4 (FABP3 and FABP4) would affect the selectivity of FA uptake into, and FA redistribution inside, mammary epithelial cells, resulting in altered FA composition of bovine milk. The objectives of our study were to discover genetic polymorphisms in SLC27A6, FABP3, and FABP4, and to test those polymorphisms for associations with milk FA composition. The results showed that after pairwise comparisons between SLC27A6 haplotypes for significantly associated traits, haplotype H3 was significantly associated with 1.37 weight percentage (wt%) lower SFA concentration, 0.091 lower SFA:UFA ratio, and 0.17 wt% lower lauric acid (12:0) concentration, but 1.37 wt% higher UFA and 1.24 wt% higher monounsaturated fatty acid (MUFA) concentrations compared with haplotype H1 during the first 3 mo of lactation. Pairwise comparisons between FABP4 haplotypes for significantly associated traits showed that haplotype H3 was significantly associated with 1.04 wt% lower SFA concentration, 0.079 lower SFA:UFA ratio, 0.15 wt% lower lauric acid (12:0), and 0.27 wt% lower myristic acid (14:0) concentrations, but 1.04 wt% higher UFA and 0.91 wt% higher MUFA concentrations compared with haplotype H1 during the first 3 mo of lactation. Percentages of genetic variance explained by H3 versus H1 haplotype substitutions for SLC27A6 and FABP4 ranged from 2.50 to 4.86% and

  3. Conformational transitions in human translin enable nucleic acid binding

    PubMed Central

    Pérez-Cano, Laura; Eliahoo, Elad; Lasker, Keren; Wolfson, Haim J.; Glaser, Fabian; Manor, Haim; Bernadó, Pau; Fernández-Recio, Juan

    2013-01-01

    Translin is a highly conserved RNA- and DNA-binding protein that plays essential roles in eukaryotic cells. Human translin functions as an octamer, but in the octameric crystallographic structure, the residues responsible for nucleic acid binding are not accessible. Moreover, electron microscopy data reveal very different octameric configurations. Consequently, the functional assembly and the mechanism of nucleic acid binding by the protein remain unclear. Here, we present an integrative study combining small-angle X-ray scattering (SAXS), site-directed mutagenesis, biochemical analysis and computational techniques to address these questions. Our data indicate a significant conformational heterogeneity for translin in solution, formed by a lesser-populated compact octameric state resembling the previously solved X-ray structure, and a highly populated open octameric state that had not been previously identified. On the other hand, our SAXS data and computational analyses of translin in complex with the RNA oligonucleotide (GU)12 show that the internal cavity found in the octameric assemblies can accommodate different nucleic acid conformations. According to this model, the nucleic acid binding residues become accessible for binding, which facilitates the entrance of the nucleic acids into the cavity. Our data thus provide a structural basis for the functions that translin performs in RNA metabolism and transport. PMID:23980029

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

    NASA Astrophysics Data System (ADS)

    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.

  5. Review: the liver bile acid-binding proteins.

    PubMed

    Monaco, Hugo L

    2009-12-01

    The liver bile acid-binding proteins, L-BABPs, formerly called the liver "basic" fatty acid-binding proteins, are a subfamily of the fatty acid-binding proteins, FABPs. All the members of this protein group share the same fold: a 10 stranded beta barrel in which two short helices are inserted in between the first and the second strand of antiparallel beta sheet. The barrel encloses the ligand binding cavity of the protein while the two helices are believed to be involved in ligand accessibility to the binding site. The L-BABP subfamily has been found to be present in the liver of several vertebrates: fish, amphibians, reptiles, and birds but not in mammals. The members of the FABP family present in mammals that appear to be more closely related to the L-BABPs are the liver FABPs and the ileal BABPs, both very extensively studied. Several L-BABP X-ray structures are available and chicken L-BABP has also been studied using NMR spectroscopy. The stoichiometry of ligand binding for bile acids, first determined by X-ray crystallography for the chicken liver protein, is of two cholates per protein molecule with the only exception of zebrafish L-BABP which, due to the presence of a disulfide bridge, has a stoichiometry of 1:1. The stoichiometry of ligand binding for fatty acids, determined with several different techniques, is 1:1. An unanswered question of great relevance is the identity of the protein that in mammals performs the function that in other vertebrates is carried out by the L-BABPS.

  6. Probing the Binding Site of Bile Acids in TGR5.

    PubMed

    Macchiarulo, Antonio; Gioiello, Antimo; Thomas, Charles; Pols, Thijs W H; Nuti, Roberto; Ferrari, Cristina; Giacchè, Nicola; De Franco, Francesca; Pruzanski, Mark; Auwerx, Johan; Schoonjans, Kristina; Pellicciari, Roberto

    2013-12-12

    TGR5 is a G-protein-coupled receptor (GPCR) mediating cellular responses to bile acids (BAs). Although some efforts have been devoted to generate homology models of TGR5 and draw structure-activity relationships of BAs, none of these studies has hitherto described how BAs bind to TGR5. Here, we present an integrated computational, chemical, and biological approach that has been instrumental to determine the binding mode of BAs to TGR5. As a result, key residues have been identified that are involved in mediating the binding of BAs to the receptor. Collectively, these results provide new hints to design potent and selective TGR5 agonists. PMID:24900622

  7. Probing the Binding Site of Bile Acids in TGR5

    PubMed Central

    2013-01-01

    TGR5 is a G-protein-coupled receptor (GPCR) mediating cellular responses to bile acids (BAs). Although some efforts have been devoted to generate homology models of TGR5 and draw structure–activity relationships of BAs, none of these studies has hitherto described how BAs bind to TGR5. Here, we present an integrated computational, chemical, and biological approach that has been instrumental to determine the binding mode of BAs to TGR5. As a result, key residues have been identified that are involved in mediating the binding of BAs to the receptor. Collectively, these results provide new hints to design potent and selective TGR5 agonists. PMID:24900622

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

    PubMed

    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.

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

    PubMed Central

    González, Javier M.; Fisher, S. Zoë

    2015-01-01

    Inhibition of human adipocyte fatty-acid binding protein (FABP4) has been proposed as a treatment for type 2 diabetes, fatty liver disease and atherosclerosis. However, FABP4 displays a naturally low selectivity towards hydrophobic ligands, leading to the possibility of side effects arising from cross-inhibition of other FABP isoforms. In a search for structural determinants of ligand-binding selectivity, the binding of FABP4 towards a group of small molecules structurally related to the nonsteroidal anti-inflammatory drug ibuprofen was analyzed through X-ray crystallography. Several specific hydrophobic interactions are shown to enhance the binding affinities of these compounds, whereas an aromatic edge-to-face interaction is proposed to determine the conformation of bound ligands, highlighting the importance of aromatic interactions in hydrophobic environments. PMID:25664790

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

    PubMed

    González, Javier M; Fisher, S Zoë

    2015-02-01

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

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

    PubMed

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

    2009-10-01

    Bile acid-binding proteins (BABPs) are cytosolic lipid chaperones that play central roles in driving bile flow, as well as in the adaptation to various pathological conditions, contributing to the maintenance of bile acid homeostasis and functional distribution within the cell. Understanding the mode of binding of bile acids with their cytoplasmic transporters is a key issue in providing a model for the mechanism of their transfer from the cytoplasm to the nucleus, for delivery to nuclear receptors. A number of factors have been shown to modulate bile salt selectivity, stoichiometry, and affinity of binding to BABPs, e.g. chemistry of the ligand, protein plasticity and, possibly, the formation of disulfide bridges. Here, the effects of the presence of a naturally occurring disulfide bridge on liver BABP ligand-binding properties and backbone dynamics have been investigated by NMR. Interestingly, the disulfide bridge does not modify the protein-binding stoichiometry, but has a key role in modulating recognition at both sites, inducing site selectivity for glycocholic and glycochenodeoxycholic acid. Protein conformational changes following the introduction of a disulfide bridge are small and located around the inner binding site, whereas significant changes in backbone motions are observed for several residues distributed over the entire protein, both in the apo form and in the holo form. Site selectivity appears, therefore, to be dependent on protein mobility rather than being governed by steric factors. The detected properties further establish a parallelism with the behaviour of human ileal BABP, substantiating the proposal that BABPs have parallel functions in hepatocytes and enterocytes. PMID:19754879

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

    PubMed

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

    2009-10-01

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

  13. THE INTEGRITY OF THE α-HELICAL DOMAIN OF INTESTINAL FATTY ACID BINDING PROTEIN IS ESSENTIAL FOR THE COLLISION-MEDIATED TRANSFER OF FATTY ACIDS TO PHOSPHOLIPID MEMBRANES

    PubMed Central

    Franchini, G. R.; Storch, J.; Corsico, B.

    2015-01-01

    Summary Intestinal FABP (IFABP) and liver FABP (LFABP), homologous proteins expressed at high levels in intestinal absorptive cells, employ markedly different mechanisms of fatty acid transfer to acceptor model membranes. Transfer from IFABP occurs during protein-membrane-collisional interactions, while for LFABP transfer occurs by diffusion through the aqueous phase. In addition, transfer from IFABP is markedly faster than from LFABP. The overall goal of this study was to further explore the structural differences between IFABP and LFABP which underlie their large functional differences in ligand transport. In particular, we addressed the role of the αI-helix domain in the unique transport properties of intestinal FABP. A chimeric protein was engineered with the ‘body’ (ligand binding domain) of IFABP and the αI-helix of LFABP (α(I)LβIFABP), and the fatty acid transfer properties of the chimeric FABP were examined using a fluorescence resonance energy transfer assay. The results showed a significant decrease in the absolute rate of FA transfer from α(I)LβIFABP compared to IFABP. The results indicate that the αI-helix is crucial for IFABP collisional FA transfer, and further indicate the participation of the αII-helix in the formation of a protein-membrane “collisional complex”. Photo-crosslinking experiments with a photoactivable reagent demonstrated the direct interaction of IFABP with membranes and further supports the importance of the αI helix of IFABP in its physical interaction with membranes. PMID:18284926

  14. Caffeic acid phenethyl ester exhibiting distinctive binding interaction with human serum albumin implies the pharmacokinetic basis of propolis bioactive components.

    PubMed

    Li, Hongliang; Wu, Fan; Tan, Jing; Wang, Kai; Zhang, Cuiping; Zheng, Huoqing; Hu, Fuliang

    2016-04-15

    Caffeic acid phenethyl ester (CAPE), as one of the major bioactive components present in propolis, exhibits versatile bioactivities, especially for its potent cytotoxic effects on several cancer cell models. To understand the pharmacokinetic characteristics of CAPE, the binding interaction between CAPE and human serum albumin (HSA) was investigated in vitro using multiple spectroscopic methods and molecular docking. The results reveal that CAPE exhibits a distinctive binding interaction with HSA comparing with other propolis components. The association constant K(A) (L mol(-1)) of the binding reaches 10(6) order of magnitude, which is significantly stronger than the other components of propolis. Based on the theory of fluorescence resonance energy transfer, the binding distance was calculated as 5.7 nm, which is longer than that of the other components of propolis. The thermodynamic results indicate that the binding is mainly driven by hydrogen bonds and van der Waals force. The docking and drugs (warfarin and ibuprofen) competitive results show that CAPE is located in the subdomain IIA (Sudlow's site I, FA7) of HSA, and Gln196 and Lys199 contribute to the hydrogen bonds. Circular dichroism spectra suggest an alteration of the secondary structure of HSA due to its partial unfolding in the presence of CAPE.

  15. Caffeic acid phenethyl ester exhibiting distinctive binding interaction with human serum albumin implies the pharmacokinetic basis of propolis bioactive components.

    PubMed

    Li, Hongliang; Wu, Fan; Tan, Jing; Wang, Kai; Zhang, Cuiping; Zheng, Huoqing; Hu, Fuliang

    2016-04-15

    Caffeic acid phenethyl ester (CAPE), as one of the major bioactive components present in propolis, exhibits versatile bioactivities, especially for its potent cytotoxic effects on several cancer cell models. To understand the pharmacokinetic characteristics of CAPE, the binding interaction between CAPE and human serum albumin (HSA) was investigated in vitro using multiple spectroscopic methods and molecular docking. The results reveal that CAPE exhibits a distinctive binding interaction with HSA comparing with other propolis components. The association constant K(A) (L mol(-1)) of the binding reaches 10(6) order of magnitude, which is significantly stronger than the other components of propolis. Based on the theory of fluorescence resonance energy transfer, the binding distance was calculated as 5.7 nm, which is longer than that of the other components of propolis. The thermodynamic results indicate that the binding is mainly driven by hydrogen bonds and van der Waals force. The docking and drugs (warfarin and ibuprofen) competitive results show that CAPE is located in the subdomain IIA (Sudlow's site I, FA7) of HSA, and Gln196 and Lys199 contribute to the hydrogen bonds. Circular dichroism spectra suggest an alteration of the secondary structure of HSA due to its partial unfolding in the presence of CAPE. PMID:26829518

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

    PubMed

    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.

  17. Interaction of perfluoroalkyl acids with human liver fatty acid-binding protein.

    PubMed

    Sheng, Nan; Li, Juan; Liu, Hui; Zhang, Aiqian; Dai, Jiayin

    2016-01-01

    Perfluoroalkyl acids (PFAAs) are highly persistent and bioaccumulative, resulting in their broad distribution in humans and the environment. The liver is an important target for PFAAs, but the mechanisms behind PFAAs interaction with hepatocyte proteins remain poorly understood. We characterized the binding of PFAAs to human liver fatty acid-binding protein (hL-FABP) and identified critical structural features in their interaction. The binding interaction of PFAAs with hL-FABP was determined by fluorescence displacement and isothermal titration calorimetry (ITC) assay. Molecular simulation was conducted to define interactions at the binding sites. ITC measurement revealed that PFOA/PFNA displayed a moderate affinity for hL-FABP at a 1:1 molar ratio, a weak binding affinity for PFHxS and no binding for PFHxA. Moreover, the interaction was mainly mediated by electrostatic attraction and hydrogen bonding. Substitution of Asn111 with Asp caused loss of binding affinity to PFAA, indicating its crucial role for the initial PFAA binding to the outer binding site. Substitution of Arg122 with Gly caused only one molecule of PFAA to bind to hL-FABP. Molecular simulation showed that substitution of Arg122 increased the volume of the outer binding pocket, making it impossible to form intensive hydrophobic stacking and hydrogen bonds with PFOA, and highlighting its crucial role in the binding process. The binding affinity of PFAAs increased significantly with their carbon number. Arg122 and Asn111 played a pivotal role in these interactions. Our findings may help understand the distribution pattern, bioaccumulation, elimination, and toxicity of PFAAs in humans.

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

    PubMed

    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. PMID:25639618

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

    PubMed

    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.

  20. Binding of carboxylic acids by fluorescent pyridyl ureas.

    PubMed

    Jordan, Lisa M; Boyle, Paul D; Sargent, Andrew L; Allen, William E

    2010-12-17

    Fluorescent pyrid-2-yl ureas were prepared by treating halogenated 2-aminopyridines with hexyl isocyanate, followed by Sonogashira coupling with arylacetylenes. The sensors emit light of ∼360 nm with quantum yields of 0.05-0.1 in acetonitrile solution. Addition of strong organic acids (pK(a) < 13 in CH(3)CN) shifts the fluorescence band to lower energy, and clean isoemissive behavior is observed. Fluorescence response curves (i.e., F/F(0) vs [acid](total)) are hyperbolic in shape for CCl(3)COOH and CF(3)COOH, with association constants on the order of 10(3) M(-1) for both acids. (1)H NMR titrations and DFT analyses indicate that trihaloacetic acids bind in ionized form to the receptors. Pyridine protonation disrupts an intramolecular H-bond, thereby unfolding an array of ureido NH donors for recognition of the corresponding carboxylates. Methanesulfonic acid protonates the sensors, but no evidence for conjugate base binding at the urea moiety is found by NMR. An isosteric control compound that lacks an integrated pyridine does not undergo significant fluorescence changes upon acidification.

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

    PubMed Central

    Swamy, Narasimha; Ray, Rahul

    2008-01-01

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

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

    SciTech Connect

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

    2010-08-31

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

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

    PubMed

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

    1982-01-01

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

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

    PubMed

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

    1982-01-01

    Sucrose density gradient analysis of cytosol from normal and neoplastic rat prostatic tissues exhibited a peak of (3H) retinoic acid binding in the 2S region, corresponding to the cytoplasmic retinoic acid binding protein (cRABP). In the Fisher-Copenhagen F1 rat, cRABP was present in the lateral lobe, but could not be detected in the ventral nor in the dorsal prostatic lobes. Four sublines of the R-3327 rat prostatic tumor contained similar levels of this binding protein. The absence of cRABP in the normal tissue of origin of the R-3327 tumor, the rat dorsal prostate, and reappearance in the neoplastic tissues follows a pattern described in other human and animal tumors. The occurrence of cRABP in the well-differentiated as well as in the anaplastic R-3327 tumors in which markers which reflect a state of differentiation and hormonal regulation, such as androgen receptor, 5 alpha reductase, and secretory acid phosphatase are either markedly reduced or absent, points to cRABP as a marker of malignant transformation. PMID:6283503

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

    NASA Astrophysics Data System (ADS)

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

    1995-07-01

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

  6. High-resolution neutron and X-ray diffraction room-temperature studies of an H-FABP-oleic acid complex: study of the internal water cluster and ligand binding by a transferred multipolar electron-density distribution.

    PubMed

    Howard, E I; Guillot, B; Blakeley, M P; Haertlein, M; Moulin, M; Mitschler, A; Cousido-Siah, A; Fadel, F; Valsecchi, W M; Tomizaki, Takashi; Petrova, T; Claudot, J; Podjarny, A

    2016-03-01

    Crystal diffraction data of heart fatty acid binding protein (H-FABP) in complex with oleic acid were measured at room temperature with high-resolution X-ray and neutron protein crystallography (0.98 and 1.90 Å resolution, respectively). These data provided very detailed information about the cluster of water molecules and the bound oleic acid in the H-FABP large internal cavity. The jointly refined X-ray/neutron structure of H-FABP was complemented by a transferred multipolar electron-density distribution using the parameters of the ELMAMII library. The resulting electron density allowed a precise determination of the electrostatic potential in the fatty acid (FA) binding pocket. Bader's quantum theory of atoms in molecules was then used to study interactions involving the internal water molecules, the FA and the protein. This approach showed H⋯H contacts of the FA with highly conserved hydrophobic residues known to play a role in the stabilization of long-chain FAs in the binding cavity. The determination of water hydrogen (deuterium) positions allowed the analysis of the orientation and electrostatic properties of the water molecules in the very ordered cluster. As a result, a significant alignment of the permanent dipoles of the water molecules with the protein electrostatic field was observed. This can be related to the dielectric properties of hydration layers around proteins, where the shielding of electrostatic interactions depends directly on the rotational degrees of freedom of the water molecules in the interface. PMID:27006775

  7. High-resolution neutron and X-ray diffraction room-temperature studies of an H-FABP-oleic acid complex: study of the internal water cluster and ligand binding by a transferred multipolar electron-density distribution.

    PubMed

    Howard, E I; Guillot, B; Blakeley, M P; Haertlein, M; Moulin, M; Mitschler, A; Cousido-Siah, A; Fadel, F; Valsecchi, W M; Tomizaki, Takashi; Petrova, T; Claudot, J; Podjarny, A

    2016-03-01

    Crystal diffraction data of heart fatty acid binding protein (H-FABP) in complex with oleic acid were measured at room temperature with high-resolution X-ray and neutron protein crystallography (0.98 and 1.90 Å resolution, respectively). These data provided very detailed information about the cluster of water molecules and the bound oleic acid in the H-FABP large internal cavity. The jointly refined X-ray/neutron structure of H-FABP was complemented by a transferred multipolar electron-density distribution using the parameters of the ELMAMII library. The resulting electron density allowed a precise determination of the electrostatic potential in the fatty acid (FA) binding pocket. Bader's quantum theory of atoms in molecules was then used to study interactions involving the internal water molecules, the FA and the protein. This approach showed H⋯H contacts of the FA with highly conserved hydrophobic residues known to play a role in the stabilization of long-chain FAs in the binding cavity. The determination of water hydrogen (deuterium) positions allowed the analysis of the orientation and electrostatic properties of the water molecules in the very ordered cluster. As a result, a significant alignment of the permanent dipoles of the water molecules with the protein electrostatic field was observed. This can be related to the dielectric properties of hydration layers around proteins, where the shielding of electrostatic interactions depends directly on the rotational degrees of freedom of the water molecules in the interface.

  8. Synthesis of kojic acid derivatives as secondary binding site probes of D-amino acid oxidase

    PubMed Central

    Raje, Mithun; Hin, Niyada; Duvall, Bridget; Ferraris, Dana V.; Berry, James F.; Thomas, Ajit G.; Alt, Jesse; Rojas, Camilo; Slusher, Barbara S.; Tsukamoto, Takashi

    2013-01-01

    A series of kojic acid (5-hydroxy-2-hydroxymethyl-4H-pyran-4-one) derivatives were synthesized and tested for their ability to inhibit D-amino acid oxidase (DAAO). Various substituents were incorporated into kojic acid at its 2-hydroxymethyl group. These analogs serve as useful molecular probes to explore the secondary binding site, which can be exploited in designing more potent DAAO inhibitors. PMID:23683589

  9. Hereditary folate malabsorption: A positively charged amino acid at position 113 of the proton-coupled folate transporter (PCFT/SLC46A1) is required for folic acid binding

    SciTech Connect

    Lasry, Inbal; Berman, Bluma; Glaser, Fabian; Jansen, Gerrit; Assaraf, Yehuda G.

    2009-08-28

    The proton-coupled folate transporter (PCFT/SLC46A1) mediates intestinal folate uptake at acidic pH. Some loss of folic acid (FA) transport mutations in PCFT from hereditary folate malabsorption (HFM) patients cluster in R113, thereby suggesting a functional role for this residue. Herein, unlike non-conservative substitutions, an R113H mutant displayed 80-fold increase in the FA transport Km while retaining parental Vmax, hence indicating a major fall in folate substrate affinity. Furthermore, consistent with the preservation of 9% of parental transport activity, R113H transfectants displayed a substantial decrease in the FA growth requirement relative to mock transfectants. Homology modeling based on the crystal structures of the Escherichia coli transporter homologues EmrD and glycerol-3-phosphate transporter revealed that the R113H rotamer properly protrudes into the cytoplasmic face of the minor cleft normally occupied by R113. These findings constitute the first demonstration that a basic amino acid at position 113 is required for folate substrate binding.

  10. Mass spectrometry of the lithium adducts of diacylglycerols containing hydroxy FA in castor oil and two normal FA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Castor oil can be used in industry. The molecular species of triacylglycerols containing hydroxy fatty acids (FA) in castor oil have been identified. We report here the identification of twelve diacylglycerols (DAG) containing hydroxy FA in castor oil using positive ion electrospray ionization mass ...

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

    PubMed

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

    1995-08-01

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

  12. Nucleic acid binding affinity of fd gene 5 protein in the cooperative binding mode.

    PubMed

    Bobst, A M; Ireland, J C; Bobst, E V

    1984-02-25

    A sensitive ESR method which allows a direct quantitative determination of nucleic acid binding affinities of proteins under physiologically relevant conditions has been applied to the gene 5 protein of bacteriophage fd. This was achieved with two spin-labeled nucleic acids, (ldT, dT)n and (lA,A)n, which served as macro-molecular spin probes in ESR competition experiments. With the two different macromolecular spin probes, it was possible to determine the relative apparent affinity constants, Kapp, over a large affinity domain. In 20 mM Tris X HCl (pH 8.1), 1 mM sodium EDTA, 0.1 mM dithiothreitol, 10% (w/v) glycerol, 0.05% Triton, and 125 mM NaCl, the following affinity relationship was observed: K(dT)napp = 10(3) KfdDNAapp = 2 X 10(4) K(A)napp = 6.6 X 10(4) KrRNAapp = 1.5 X 10(5) KR17RNAapp. Increasing the [NaCl] from 125 to 200 mM caused considerably less tight binding of gene 5 protein to (lA,A)n, and a typical cooperative binding isotherm was observed, whereas at the lower [NaCl] used for the competition experiments, the binding was essentially stoichiometric. A computer fit of the experimental titration data at 200 mM NaCl gave an intrinsic binding constant, Kint, of 1300 M-1 and a cooperativity factor, omega, of 60 (Kint omega = Kapp) for (lA,A)n.

  13. Spectroscopic investigations of lanthanide ion binding to nucleic acids.

    PubMed

    Morrow, Janet R; Andolina, Christopher M

    2012-01-01

    Luminescent lanthanide (Ln(III)) ions are valuable spectroscopic probes for metal ion binding sites in nucleic acids. In this chapter, we briefly review Ln(III) luminescence and the information available from these experiments. An emphasis is placed on direct excitation Eu(III) spectroscopy as a tool. Eu(III) excitation spectroscopy is used to show that solutions containing micromolar Eu(III), 100 mM NaCl, and 20 mM MES buffer contain predominantly a mononuclear Eu(III) aqua complex and an Eu(III) hydroxide complexes. The binding of these species to various RNA and DNA sequences are monitored by using Eu(III) excitation spectroscopy. Eu(III) luminescence lifetime data shows that the Eu(III) ion typically loses 1-3 water molecules to form innersphere complexes with RNA and DNA that contain tandem base pair mismatches or hairpin loops. In addition, early studies that used nucleobase-sensitized Eu(III) or Tb(III) luminescence within transfer RNA or in the hammerhead ribozyme are presented. Luminescence resonance energy transfer studies are shown to be useful for determining distances between bound Ln(III) ion and organic fluorophores or between two different Ln(III) ions. To supplement luminescence data, the binding sites of paramagnetic Ln(III) ions are determined by monitoring the chemical shifts of nucleotide protons. Binding sites are identified by following the protons that are influenced by the Ln(III) pseudo-contact shift.

  14. Fatty acid transfer between multilamellar liposomes and fatty acid-binding proteins.

    PubMed

    Brecher, P; Saouaf, R; Sugarman, J M; Eisenberg, D; LaRosa, K

    1984-11-10

    A simple experimental system was developed for studying the movement of long-chain fatty acids between multilamellar liposomes and soluble proteins capable of binding fatty acids. Oleic acid was incorporated into multilamellar liposomes containing cholesterol and egg yolk lecithin and incubated with albumin or hepatic fatty acid-binding protein. It was found that the fatty acid transferred from the liposomes to either protein rapidly and selectively under conditions where phospholipid and cholesterol transfer did not occur. More than 50% of the fatty acid contained within liposomes could become protein bound, suggesting that the fatty acid moved readily between and across phospholipid bilayers. Transfer was reduced at low pH, and this reduction appeared to result from decreased dissociation of the protonated fatty acid from the bilayer. Liposomes made with dimyristoyl or dipalmitoyl lecithin and containing 1 mol per cent palmitic acid were used to show the effect of temperature on fatty acid transfer. Transfer to either protein did not occur at temperatures where the liposomes were in a gel state but occurred rapidly at temperatures at or above the transition temperatures of the phospholipid used. PMID:6490659

  15. Inhibition of ornithine decarboxylase induction by retinobenzoic acids in relation to their binding affinities to cellular retinoid-binding proteins.

    PubMed

    Takagi, K; Suganuma, M; Kagechika, H; Shudo, K; Ninomiya, M; Muto, Y; Fujiki, H

    1988-01-01

    Retinobenzoic acids induce differentiation of human promyelocytic leukemia cells (HL-60). Like retinoic acid, 14 retinobenzoic acids inhibited the induction of ornithine decarboxylase (ODC) by teleocidin in mouse skin. The mechanism(s) of inhibition of ODC induction by 7 retinobenzoic acids, Am 80, Am 81, Am 580, Am 590, Am 68, Sa 80, and Ch 55 was compared with those by all-trans-retinoic acid and the arotinoid compound 19. Application of 114 nmol of Am 80, Am 81, Am 580, Am 590, Am 68, Sa 80, or Ch 55, 10 min before 11.4 nmol of teleocidin, resulted in 76.7%, 82.0%, 76.2%, 28.3%, 48.4%, 58.6%, and 85.1% inhibition of ODC induction, respectively. Since all-trans-retinoic acid and compound 19 were also inhibitory, we determined whether retinobenzoic acids bind to cellular retinoic acid-binding protein (CRABP) isolated from bovine adrenal glands. Am 80 and Am 580 inhibited the specific binding of 3H-retinoic acid to CRABP, but also showed less affinity than authentic unlabeled retinoic acid and compound 19. Am 81, Am 590, Am 68, Sa 80, and Ch 55 at up to 10 microM were not effective competitors of the binding of either 3H-retinoic acid or 3H-retinol. These results suggest that the inhibition of ODC induction can be mediated by pathways that do not involve CRABP or the cellular retinol-binding protein.

  16. Fatty acid binding protein in the intestine of the chicken.

    PubMed

    Katongole, J B; March, B E

    1979-03-01

    The mucosa of the mesenteric intestine of the chicken has been found to contain a fatty acid binding protein (FABP) with a molecular weight of less than 12,400. The protein is present in the newly hatched chick before ingestion of feed and in the adult bird. When a low-fat diet is fed, the concentration of the FABP is highest in the proximal portion of the intestine and decreases posteriorly. When a high-fat diet is fed, an increase occurs in the amount of FABP in the lower section of the intestine.

  17. Synthesis of Nanoporous Iminodiacetic Acid Sorbents for Binding Transition Metals

    PubMed Central

    Busche, Brad; Wiacek, Robert; Davidson, Joseph; Koonsiripaiboon, View; Yantasee, Wassana; Addleman, R. Shane; Fryxell, Glen E.

    2009-01-01

    Iminodiacetic acid (IDAA) forms strong complexes with a wide variety of metal ions. Using self-assembled monolayers in mesoporous supports (SAMMS) to present the IDAA ligand potentially allows for multiple metal-ligand interactions to enhance the metal binding affinity relative to that of randomly oriented polymer-based supports. This manuscript describes the synthesis of a novel nanostructured sorbent material built using self-assembly of a IDAA ligand inside a nanoporous silica, and demonstrates its use for capturing transition metal cations, and anionic metal complexes, such as PdCl4−2. PMID:22068901

  18. Identification and Investigation of Novel Binding Fragments in the Fatty Acid Binding Protein 6 (FABP6).

    PubMed

    Hendrick, Alan G; Müller, Ilka; Willems, Henriëtte; Leonard, Philip M; Irving, Steve; Davenport, Richard; Ito, Takashi; Reeves, Jenny; Wright, Susanne; Allen, Vivienne; Wilkinson, Stephen; Heffron, Helen; Bazin, Richard; Turney, Jennifer; Mitchell, Philip J

    2016-09-01

    Fatty acid binding protein 6 (FABP6) is a potential drug discovery target, which, if inhibited, may have a therapeutic benefit for the treatment of diabetes. Currently, there are no published inhibitors of FABP6, and with the target believed to be amenable to fragment-based drug discovery, a structurally enabled program was initiated. This program successfully identified fragment hits using the surface plasmon resonance (SPR) platform. Several hits were validated with SAR and were found to be displaced by the natural ligand taurocholate. We report the first crystal structure of human FABP6 in the unbound form, in complex with cholate, and with one of the key fragments. PMID:27500412

  19. Involvement of Acidic Amino Acid Residues in Zn(2+) Binding to Respiratory Complex I.

    PubMed

    Kriegel, Sébastien; Srour, Batoul; Steimle, Stefan; Friedrich, Thorsten; Hellwig, Petra

    2015-09-21

    Proton transfer across membranes and membrane proteins is a central process in biological systems. Zn(2+) ions are capable of binding to acidic residues, often found within such specific pathways, thereby leading to a blockage. Here we probed Zn(2+) inhibition of the proton-pumping NADH:ubiquinone oxidoreductase from Escherichia coli by means of electrochemically induced FTIR difference spectroscopy. Numerous conformational changes were identified including those that arise from the reorganization of the membrane arm upon electron transfer in the peripheral arm of the protein. Signals at very high wavenumbers (1781 and 1756 cm(-1)) point to the perturbation of acidic residues in a highly hydrophobic environment upon Zn(2+) binding. In variant D563N(L), which lacks part of the proton pumping activity (residue located on the horizontal amphipathic helix), the spectral signature of Zn(2+) binding is changed. Our data support a role for this residue in proton translocation.

  20. MAN or FA from n-butane

    SciTech Connect

    Di Cio, A.; Verde, L.

    1985-08-01

    Unsaturated polyester resins were first produced mostly from fumaric acid (FA) rather than from maleic anhydride (MAN). This is perfectly understandable if we consider that, using fumaric acid as raw material, polycondensates with a more homogeneous (less branched) structure are obtained, thus producing resins characterized by a more uniform and reproducible chemical and mechanical properties. Presently, for economical reasons, fumaric acid is used marginally as a MAN substitute in the production of polyester resins. These resins account for a major share (50%) of the overall MAN consumption in the U.S. and in Western Europe.

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

    PubMed Central

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

    1994-01-01

    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. Images Figure 2 Figure 3 PMID:8141774

  2. Characterization and amino acid sequence of a fatty acid-binding protein from human heart.

    PubMed Central

    Offner, G D; Brecher, P; Sawlivich, W B; Costello, C E; Troxler, R F

    1988-01-01

    The complete amino acid sequence of a fatty acid-binding protein from human heart was determined by automated Edman degradation of CNBr, BNPS-skatole [3'-bromo-3-methyl-2-(2-nitrobenzenesulphenyl)indolenine], hydroxylamine, Staphylococcus aureus V8 proteinase, tryptic and chymotryptic peptides, and by digestion of the protein with carboxypeptidase A. The sequence of the blocked N-terminal tryptic peptide from citraconylated protein was determined by collisionally induced decomposition mass spectrometry. The protein contains 132 amino acid residues, is enriched with respect to threonine and lysine, lacks cysteine, has an acetylated valine residue at the N-terminus, and has an Mr of 14768 and an isoelectric point of 5.25. This protein contains two short internal repeated sequences from residues 48-54 and from residues 114-119 located within regions of predicted beta-structure and decreasing hydrophobicity. These short repeats are contained within two longer repeated regions from residues 48-60 and residues 114-125, which display 62% sequence similarity. These regions could accommodate the charged and uncharged moieties of long-chain fatty acids and may represent fatty acid-binding domains consistent with the finding that human heart fatty acid-binding protein binds 2 mol of oleate or palmitate/mol of protein. Detailed evidence for the amino acid sequences of the peptides has been deposited as Supplementary Publication SUP 50143 (23 pages) at the British Library Lending Division, Boston Spa, Yorkshire LS23 7BQ, U.K., from whom copies may be obtained as indicated in Biochem. J. (1988) 249, 5. PMID:3421901

  3. Role of fatty acid binding protein on hepatic palmitate uptake.

    PubMed

    Burczynski, F J; Zhang, M N; Pavletic, P; Wang, G Q

    1997-12-01

    Expression of hepatic fatty acid binding protein (FABP) mRNA is regulated by growth hormone. In the absence of growth hormone, there is a 60% reduction in FABP mRNA levels (S.A. Berry, J.-B Yoon, U. List, and S. Seelig. J. Am. Coll. Nutr. 12:638-642. 1995). Previous work in our laboratory focused on the role of extracellular binding proteins in the hepatic uptake of long chain fatty acids. In the present study we were interested to determine the role of FABP in the transmembrane flux of long chain fatty acids. Using hepatocyte monolayers from control (n = 9) and hypophysectomized (n = 6) rats, we investigated the uptake of [3H]palmitate in the presence and absence of albumin. In the absence of albumin, total hepatocyte [3H]palmitate clearance rates from control (17.2 +/- 1.5 microL.mg-1 protein.s-1; mean +/- SEM; n = 9) and hypophysectomized (15.5 +/- 2.1 microL.mg-1 protein.s-1; n = 6) animals were similar (p > 0.05). In the presence of 2 microM albumin the total [3H]palmitate clearance rate from control hepatocytes (1.63 +/- 0.11 microL.mg-1 protein.s-1; n = 9) was significantly larger (40%) than from hepatocytes obtained from hypophysectomized (0.97 +/- 0.15 microL.mg-1 protein.s-1; n = 6; p < 0.01) animals. SDS-PAGE electrophoresis revealed that plasma membrane FABP levels from control and hypophysectomized animals were similar. However, there was a 49% decrease in the cytosolic FABP levels of hepatocytes isolated from hypophysectomized as compared with control animals. The decreased cytosolic FABB levels paralleled the decrease in palmitate uptake. We conclude that in the absence of extracellular binding proteins the rate-limiting step in the overall uptake of long chain fatty acids is diffusion to the cell surface. However, in the presence of albumin, the rate of palmitate uptake is determined primarily by cytosolic FABP levels.

  4. Characterization of a fatty acid-binding protein from rat heart.

    PubMed

    Offner, G D; Troxler, R F; Brecher, P

    1986-04-25

    A fatty acid-binding protein has been isolated from rat heart and purified by gel filtration chromatography on Sephadex G-75 and anion-exchange chromatography on DE52. The circular dichroic spectrum of this protein was not affected by protein concentration, suggesting that it does not aggregate into multimers. Computer analyses of the circular dichroic spectrum predicted that rat heart fatty acid-binding protein contains approximately 22% alpha-helix, 45% beta-form and 33% unordered structure. Immunological studies showed that the fatty acid-binding proteins from rat heart and rat liver are immunochemically unrelated. The amino acid composition and partial amino acid sequence of the heart protein indicated that it is structurally related to, but distinct from, other fatty acid-binding proteins from liver, intestine, and 3T3 adipocytes. Using a binding assay which measures the transfer of fatty acids between donor liposomes and protein (Brecher, P., Saouaf, R., Sugarman, J. M., Eisenberg, D., and LaRosa, K. (1984) J. Biol. Chem. 259, 13395-13401), it was shown that both rat heart and liver fatty acid-binding proteins bind 2 mol of oleic acid or palmitic acid/mol of protein. The structural and functional relationship of rat heart fatty acid-binding protein to fatty acid-binding proteins from other tissues is discussed. PMID:3957934

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

    NASA Astrophysics Data System (ADS)

    Ritchie, Jason D.; Perdue, E. Michael

    2003-01-01

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

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

    PubMed

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

    2009-12-18

    Bile acid molecules are transferred vectorially between basolateral and apical membranes of hepatocytes and enterocytes in the context of the enterohepatic circulation, a process regulating whole body lipid homeostasis. This work addresses the role of the cytosolic lipid binding proteins in the intracellular transfer of bile acids between different membrane compartments. We present nuclear magnetic resonance (NMR) data describing the ternary system composed of the bile acid binding protein, bile acids, and membrane mimetic systems, such as anionic liposomes. This work provides evidence that the investigated liver bile acid binding protein undergoes association with the anionic membrane and binding-induced partial unfolding. The addition of the physiological ligand to the protein-liposome mixture is capable of modulating this interaction, shifting the equilibrium towards the free folded holo protein. An ensemble of NMR titration experiments, based on nitrogen-15 protein and ligand observation, confirm that the membrane and the ligand establish competing binding equilibria, modulating the cytoplasmic permeability of bile acids. These results support a mechanism of ligand binding and release controlled by the onset of a bile salt concentration gradient within the polarized cell. The location of a specific protein region interacting with liposomes is highlighted. PMID:19836400

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

    PubMed

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

    2009-12-18

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

  8. A Sialic Acid Binding Site in a Human Picornavirus

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2010-04-01

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

  10. Selection of 2′-deoxy-2′-fluoroarabinonucleotide (FANA) aptamers that bind HIV-1 reverse transcriptase with picomolar affinity

    PubMed Central

    Alves Ferreira-Bravo, Irani; Cozens, Christopher; Holliger, Philipp; DeStefano, Jeffrey J.

    2015-01-01

    Using a Systematic Evolution of Ligands by Exponential Enrichment (SELEX) protocol capable of selecting xeno-nucleic acid (XNA) aptamers, a 2′-deoxy-2′-fluoroarabinonucleotide (FANA) aptamer (referred to as FA1) to HIV-1 reverse transcriptase (HIV-1 RT) was selected. FA1 bound HIV-1 RT with KD,app values in the low pM range under different ionic conditions. Comparisons to published HIV-1 RT RNA and DNA aptamers indicated that FA1 bound at least as well as these aptamers. FA1 contained a 20 nucleotide 5′ DNA sequence followed by a 57 nucleotide region of FANA nucleotides. Removal of the fourteen 5′ DNA nucleotides did not affect binding. FA1's predicted structure was composed of four stems and four loops. All stem nucleotides could be modified to G-C base pairs (14 total changes) with a small effect on binding. Eliminating or altering most loop sequences reduced or abolished tight binding. Overall, results suggested that the structure and the sequence of FA1 were important for binding. FA1 showed strong inhibition of HIV-1 RT in extension assays while no specific binding to avian myeloblastosis or Moloney murine leukemia RTs was detected. A complete DNA version of FA1 showed low binding to HIV-1 RT, emphasizing the unique properties of FANA in HIV-1 RT binding. PMID:26476448

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

    Hanus, Martin; Zhorov, Eugene

    2006-12-01

    It has been previously reported that anions in common beverages may bind to bile acid sequestrants (BAS), reducing their capacity for binding bile acid salts. This study examined the ability of the novel BAS colesevelam hydrochloride (HCl), in vitro, to bind bile acid sodium salts following suspension in common beverages. Equilibrium binding was evaluated under conditions of constant time and varying concentrations of bile acid salts in simulated intestinal fluid (SIF). A stock solution of sodium salts of glycochenodeoxycholic acid (GCDC), taurodeoxycholic acid (TDC), and glycocholic acid (GC), was added to each prepared sample of colesevelam HCl. Bile acid salt binding was calculated by high-performance liquid chromatography (HPLC) analysis. Kinetics experiments were conducted using constant initial bile acid salt concentrations and varying binding times. The affinity, capacity, and kinetics of colesevelam HCl binding for GCDC, TDC, and GC were not significantly altered after suspension in water, carbonated water, Coca-Cola, Sprite, grape juice, orange juice, tomato juice, or Gatorade. The amount of bile acid sodium salt bound as a function of time was unchanged by pretreatment with any beverage tested. The in vitro binding characteristics of colesevelam HCl are unchanged by suspension in common beverages. PMID:16937334

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

    USGS Publications Warehouse

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

    1998-01-01

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

  14. Long-term fertilization modifies the structures of soil fulvic acids and their binding capability with Al.

    PubMed

    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. PMID:25137372

  15. Long-Term Fertilization Modifies the Structures of Soil Fulvic Acids and Their Binding Capability with Al

    PubMed Central

    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. PMID:25137372

  16. Long-term fertilization modifies the structures of soil fulvic acids and their binding capability with Al.

    PubMed

    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.

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

    PubMed

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

    2005-10-01

    GST fusion proteins of the six gene products from RNAs 2,3 and 4 of the tenuivirus, Rice stripe virus (RSV), were used to study the nucleic acid binding activities in vitro. Three of the proteins, p3, pc3 and pc4, bound both single- and double-stranded cDNA of RSV RNA4 and also RNA3 transcribed from its cDNA clone, while p2, pc2-N (the N-terminal part of pc2) nor p4 bound the cDNA or RNA transcript. The binding activity of p3 is located in the carboxyl-terminus amino acid 154-194, which contains basic amino acid rich beta-sheets. The acidic amino acid-rich amino-terminus (amino acids 1-100) of p3 did not have nucleic acid binding activity. The related analogous gene product of the tenuivirus, Rice hoja blanca virus, is a suppressor of gene silencing and the possibility of the nucleic acid binding ability of RSV p3 being associated with this property is discussed. The C-terminal part of the RSV nucleocapsid protein, which also contains a basic region, binds nucleic acids, which is consistent with its function. The central and C-terminal regions of pc4 bind nucleic acid. It has been suggested that this protein is a cell-to-cell movement protein and nucleic acid binding would be in accord with this function. PMID:16025246

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

    PubMed

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

    2015-01-01

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

  19. A single-molecule approach to explore binding, uptake and transport of cancer cell targeting nanotubes

    NASA Astrophysics Data System (ADS)

    Lamprecht, C.; Plochberger, B.; Ruprecht, V.; Wieser, S.; Rankl, C.; Heister, E.; Unterauer, B.; Brameshuber, M.; Danzberger, J.; Lukanov, P.; Flahaut, E.; Schütz, G.; Hinterdorfer, P.; Ebner, A.

    2014-03-01

    In the past decade carbon nanotubes (CNTs) have been widely studied as a potential drug-delivery system, especially with functionality for cellular targeting. Yet, little is known about the actual process of docking to cell receptors and transport dynamics after internalization. Here we performed single-particle studies of folic acid (FA) mediated CNT binding to human carcinoma cells and their transport inside the cytosol. In particular, we employed molecular recognition force spectroscopy, an atomic force microscopy based method, to visualize and quantify docking of FA functionalized CNTs to FA binding receptors in terms of binding probability and binding force. We then traced individual fluorescently labeled, FA functionalized CNTs after specific uptake, and created a dynamic ‘roadmap’ that clearly showed trajectories of directed diffusion and areas of nanotube confinement in the cytosol. Our results demonstrate the potential of a single-molecule approach for investigation of drug-delivery vehicles and their targeting capacity.

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

    PubMed Central

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

    1994-01-01

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

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

    SciTech Connect

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

    1985-10-01

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

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

    PubMed

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

    2014-05-01

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

  3. High-resolution neutron and X-ray diffraction room-temperature studies of an H-FABP–oleic acid complex: study of the internal water cluster and ligand binding by a transferred multipolar electron-density distribution

    PubMed Central

    Howard, E. I.; Guillot, B.; Blakeley, M. P.; Haertlein, M.; Moulin, M.; Mitschler, A.; Cousido-Siah, A.; Fadel, F.; Valsecchi, W. M.; Tomizaki, Takashi; Petrova, T.; Claudot, J.; Podjarny, A.

    2016-01-01

    Crystal diffraction data of heart fatty acid binding protein (H-FABP) in complex with oleic acid were measured at room temperature with high-resolution X-ray and neutron protein crystallography (0.98 and 1.90 Å resolution, respectively). These data provided very detailed information about the cluster of water molecules and the bound oleic acid in the H-FABP large internal cavity. The jointly refined X-ray/neutron structure of H-FABP was complemented by a transferred multipolar electron-density distribution using the parameters of the ELMAMII library. The resulting electron density allowed a precise determination of the electrostatic potential in the fatty acid (FA) binding pocket. Bader’s quantum theory of atoms in molecules was then used to study interactions involving the internal water molecules, the FA and the protein. This approach showed H⋯H contacts of the FA with highly conserved hydrophobic residues known to play a role in the stabilization of long-chain FAs in the binding cavity. The determination of water hydrogen (deuterium) positions allowed the analysis of the orientation and electrostatic properties of the water molecules in the very ordered cluster. As a result, a significant alignment of the permanent dipoles of the water molecules with the protein electrostatic field was observed. This can be related to the dielectric properties of hydration layers around proteins, where the shielding of electrostatic interactions depends directly on the rotational degrees of freedom of the water molecules in the interface. PMID:27006775

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

    PubMed

    Shao, Qing; Hall, Carol K

    2016-08-01

    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.

  5. Sources of variability in fatty acid (FA) biomarkers in the application of compound-specific stable isotopes (CSSIs) to soil and sediment fingerprinting and tracing: A review.

    PubMed

    Reiffarth, D G; Petticrew, E L; Owens, P N; Lobb, D A

    2016-09-15

    Determining soil redistribution and sediment budgets in watersheds is often challenging. One of the methods for making such determinations employs soil and sediment fingerprinting techniques, using sediment properties such as geochemistry, fallout radionuclides, and mineral magnetism. These methods greatly improve the estimation of erosion and deposition within a watershed, but are limited when determining land use-based soil and sediment movement. Recently, compound-specific stable isotopes (CSSIs), which employ fatty acids naturally occurring in the vegetative cover of soils, offer the possibility of refining fingerprinting techniques based on land use, complementing other methods that are currently in use. The CSSI method has been met with some success; however, challenges still remain with respect to scale and resolution due to a potentially large degree of biological, environmental and analytical uncertainty. By better understanding the source of tracers used in CSSI work and the inherent biochemical variability in those tracers, improvement in sample design and tracer selection is possible. Furthermore, an understanding of environmental and analytical factors affecting the CSSI signal will lead to refinement of the approach and the ability to generate more robust data. This review focuses on sources of biological, environmental and analytical variability in applying CSSI to soil and sediment fingerprinting, and presents recommendations based on past work and current research in this area for improving the CSSI technique. A recommendation, based on current information available in the literature, is to use very-long chain saturated fatty acids and to avoid the use of the ubiquitous saturated fatty acids, C16 and C18. PMID:27155260

  6. Sources of variability in fatty acid (FA) biomarkers in the application of compound-specific stable isotopes (CSSIs) to soil and sediment fingerprinting and tracing: A review.

    PubMed

    Reiffarth, D G; Petticrew, E L; Owens, P N; Lobb, D A

    2016-09-15

    Determining soil redistribution and sediment budgets in watersheds is often challenging. One of the methods for making such determinations employs soil and sediment fingerprinting techniques, using sediment properties such as geochemistry, fallout radionuclides, and mineral magnetism. These methods greatly improve the estimation of erosion and deposition within a watershed, but are limited when determining land use-based soil and sediment movement. Recently, compound-specific stable isotopes (CSSIs), which employ fatty acids naturally occurring in the vegetative cover of soils, offer the possibility of refining fingerprinting techniques based on land use, complementing other methods that are currently in use. The CSSI method has been met with some success; however, challenges still remain with respect to scale and resolution due to a potentially large degree of biological, environmental and analytical uncertainty. By better understanding the source of tracers used in CSSI work and the inherent biochemical variability in those tracers, improvement in sample design and tracer selection is possible. Furthermore, an understanding of environmental and analytical factors affecting the CSSI signal will lead to refinement of the approach and the ability to generate more robust data. This review focuses on sources of biological, environmental and analytical variability in applying CSSI to soil and sediment fingerprinting, and presents recommendations based on past work and current research in this area for improving the CSSI technique. A recommendation, based on current information available in the literature, is to use very-long chain saturated fatty acids and to avoid the use of the ubiquitous saturated fatty acids, C16 and C18.

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

    SciTech Connect

    Maita, Y.; Kawaguchi, S. )

    1989-09-01

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

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

    PubMed Central

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

    1977-01-01

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

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

    PubMed

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

    2015-07-01

    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.

  10. Characterization of binding and structural properties of rat liver fatty-acid-binding protein using tryptophan mutants.

    PubMed Central

    Thumser, A E; Wilton, D C

    1994-01-01

    Rat liver fatty-acid-binding protein (FABP) does not contain tryptophan. Three mutant proteins have been produced in which a single tryptophan residue has been inserted by site-directed mutagenesis at positions 3 (F3W), 18 (F18W) and 69 (C69W). These tryptophans have been strategically located in order to provide fluorescent reporter groups to study the binding and structural characteristics of rat liver FABP. Two fluorescent fatty acid analogues, DAUDA (11-[(5-dimethylaminonaphthalene-1- sulphonyl)amino]undecanoic acid) and 3-[p-(6-phenyl)-hexa-1,3,5-trienyl]phenylpropionic acid, showed no significant difference in binding affinities for the different mutant proteins, although maximum fluorescence values were decreased for F3W and increased for C69W. These findings were confirmed by studies of DAUDA displacement by oleate. Protein-denaturation studies in the presence of urea indicated subtle differences for the three mutants which could be explained by multiple unfolding pathways. Fatty acid binding increased tryptophan fluorescence emission in the case of the F18W protein, but had no effect on the F3W and C69W proteins. Fluorescence quenching studies with 2-bromopalmitate showed that a fatty acid carboxylate is close to the tryptophan in the F18W protein. Energy-transfer studies showed that the fluorescent moiety of DAUDA is equidistant from the three mutated amino acids and is bound within the beta-clam solvent cavity of liver FABP. This interpretation of the fluorescence quenching and energy-transfer data supports the difference in ligand orientation between intestinal and liver FABP observed in previous studies. PMID:8010966

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

    PubMed

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

    2015-12-01

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

  12. Polyphemin: a teichoic acid-binding lectin from the horseshoe crab, Limulus Polyphemus.

    PubMed

    Brandin, E R; Pistole, T G

    1983-06-15

    A Staphylococcus aureus-agglutinating lectin, capable of binding to N-acetyl-D-glucosamine, was isolated from the serum of Limulus polyphemus. The monosaccharide alone was incapable of inhibiting bacterial agglutination by this lectin. Quantitative precipitation studies with purified cell wall-derived teichoic acids, either devoid of or containing N-acetyl-D-glucosamine, confirmed the carbohydrate-binding specificity of the lectin and suggested that secondary, non-specific interactions contribute to binding biomolecules containing this sugar. The agglutination pattern with various S. aureus strains having N-acetyl-D-glucosamine-associated teichoic acid, teichoic acid without this sugar, and no teichoic acid indicated that this cell wall component is not the sole binding site for the lectin on intact S. aureus cells. Affinity gel chromatography, using N-acetyl-D-glucosamine-associated teichoic acid as the specific absorbent, has been used to isolate this lectin from Limulus serum.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  14. Heterozygous FA2H mutations in autism spectrum disorders

    PubMed Central

    2013-01-01

    Background Widespread abnormalities in white matter development are frequently reported in cases of autism spectrum disorders (ASD) and could be involved in the disconnectivity suggested in these disorders. Homozygous mutations in the gene coding for fatty-acid 2-hydroxylase (FA2H), an enzyme involved in myelin synthesis, are associated with complex leukodystrophies, but little is known about the functional impact of heterozygous FA2H mutations. We hypothesized that rare deleterious heterozygous mutations of FA2H might constitute risk factors for ASD. Methods We searched deleterious mutations affecting FA2H, by genotyping 1256 independent patients with ASD genotyped using Genome Wide SNP arrays, and also by sequencing in independent set of 186 subjects with ASD and 353 controls. We then explored the impact of the identified mutations by measuring FA2H enzymatic activity and expression, in transfected COS7 cells. Results One heterozygous deletion within 16q22.3-q23.1 including FA2H was observed in two siblings who share symptoms of autism and severe cognitive impairment, axial T2-FLAIR weighted MRI posterior periventricular white matter lesions. Also, two rare non-synonymous mutations (R113W and R113Q) were reported. Although predictive models suggested that R113W should be a deleterious, we did not find that FA2H activity was affected by expression of the R113W mutation in cultured COS cells. Conclusions While our results do not support a major role for FA2H coding variants in ASD, a screening of other genes related to myelin synthesis would allow us to better understand the role of non-neuronal elements in ASD susceptibility. PMID:24299421

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

    PubMed Central

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

    2015-01-01

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

  16. Modeling Fatty Acid Transfer from Artery to Cardiomyocyte.

    PubMed

    Arts, Theo; Reneman, Robert S; Bassingthwaighte, James B; van der Vusse, Ger J

    2015-12-01

    Despite the importance of oxidation of blood-borne long-chain fatty acids (Fa) in the cardiomyocytes for contractile energy of the heart, the mechanisms underlying the transfer of Fa from the coronary plasma to the cardiomyocyte is still incompletely understood. To obtain detailed insight into this transfer process, we designed a novel model of Fa transfer dynamics from coronary plasma through the endothelial cells and interstitium to the cardiomyocyte, applying standard physicochemical principles on diffusion and on the chemical equilibrium of Fa binding to carrier proteins Cp, like albumin in plasma and interstitium and Fatty Acid-Binding Proteins within endothelium and cardiomyocytes. Applying these principles, the present model strongly suggests that in the heart, binding and release of Fa to and from Cp in the aqueous border zones on both sides of the cell membranes form the major hindrance to Fa transfer. Although often considered, the membrane itself appears not to be a significant hindrance to diffusion of Fa. Proteins, residing in the cellular membrane, may facilitate transfer of Fa between Cp and membrane. The model is suited to simulate multiple tracer dilution experiments performed on isolated rabbit hearts administrating albumin and Fa as tracer substances into the coronary arterial perfusion line. Using parameter values on myocardial ultrastructure and physicochemical properties of Fa and Cp as reported in literature, simulated washout curves appear to be similar to the experimentally determined ones. We conclude therefore that the model is realistic and, hence, can be considered as a useful tool to better understand Fa transfer by evaluation of experimentally determined tracer washout curves.

  17. Modeling Fatty Acid Transfer from Artery to Cardiomyocyte

    PubMed Central

    Arts, Theo; Reneman, Robert S.; Bassingthwaighte, James B.; van der Vusse, Ger J.

    2015-01-01

    Despite the importance of oxidation of blood-borne long-chain fatty acids (Fa) in the cardiomyocytes for contractile energy of the heart, the mechanisms underlying the transfer of Fa from the coronary plasma to the cardiomyocyte is still incompletely understood. To obtain detailed insight into this transfer process, we designed a novel model of Fa transfer dynamics from coronary plasma through the endothelial cells and interstitium to the cardiomyocyte, applying standard physicochemical principles on diffusion and on the chemical equilibrium of Fa binding to carrier proteins Cp, like albumin in plasma and interstitium and Fatty Acid-Binding Proteins within endothelium and cardiomyocytes. Applying these principles, the present model strongly suggests that in the heart, binding and release of Fa to and from Cp in the aqueous border zones on both sides of the cell membranes form the major hindrance to Fa transfer. Although often considered, the membrane itself appears not to be a significant hindrance to diffusion of Fa. Proteins, residing in the cellular membrane, may facilitate transfer of Fa between Cp and membrane. The model is suited to simulate multiple tracer dilution experiments performed on isolated rabbit hearts administrating albumin and Fa as tracer substances into the coronary arterial perfusion line. Using parameter values on myocardial ultrastructure and physicochemical properties of Fa and Cp as reported in literature, simulated washout curves appear to be similar to the experimentally determined ones. We conclude therefore that the model is realistic and, hence, can be considered as a useful tool to better understand Fa transfer by evaluation of experimentally determined tracer washout curves. PMID:26675003

  18. Requirement for the heart-type fatty acid binding protein in cardiac fatty acid utilization.

    PubMed

    Binas, B; Danneberg, H; McWhir, J; Mullins, L; Clark, A J

    1999-05-01

    Nonenzymatic cytosolic fatty acid binding proteins (FABPs) are abundantly expressed in many animal tissues with high rates of fatty acid metabolism. No physiological role has been demonstrated for any FABP, although these proteins have been implicated in transport of free long-chain fatty acids (LCFAs) and protection against LCFA toxicity. We report here that mice lacking heart-type FABP (H-FABP) exhibit a severe defect of peripheral (nonhepatic, non-fat) LCFA utilization. In these mice, the heart is unable to efficiently take up plasma LCFAs, which are normally its main fuel, and switches to glucose usage. Altered plasma levels of LCFAs, glucose, lactate and beta-hydroxybutyrate are consistent with depressed peripheral LCFA utilization, intensified carbohydrate usage, and increased hepatic LCFA oxidation; these changes are most pronounced under conditions favoring LCFA oxidation. H-FABP deficiency is only incompletely compensated, however, causing acute exercise intolerance and, at old age, a localized cardiac hypertrophy. These data establish a requirement for H-FABP in cardiac intracellular lipid transport and fuel selection and a major role in metabolic homeostasis. This new animal model should be particularly useful for investigating the significance of peripheral LCFA utilization for heart function, insulin sensitivity, and blood pressure.

  19. Three amino acid residues of an odorant-binding protein are involved in binding odours in Loxostege sticticalis L.

    PubMed

    Yin, J; Zhuang, X; Wang, Q; Cao, Y; Zhang, S; Xiao, C; Li, K

    2015-10-01

    Odorant-binding proteins (OBPs) play an important role in insect olfactory processes and are thought to be responsible for the transport of pheromones and other semiochemicals across the sensillum lymph to the olfactory receptors within the antennal sensilla. As an important general odorant binding protein in the process of olfactory recognition, LstiGOBP1 of Loxostege sticticalis L. has been shown to have good affinity to various plant volatiles. However, the binding specificity of LstiGOBP1 should be further explored in order to better understand the olfactory recognition mechanism of L. sticticalis. In this study, real-time PCR experiments indicated that LstiGOBP1 was expressed primarily in adult antennae. Homology modelling and molecular docking were then conducted on the interactions between LstiGOBP1 and 1-heptanol to understand the interactions between LstiGOBP1 and their ligands. Hydrogen bonds formed by amino acid residues might be crucial for the ligand-binding specificity on molecular docking, a hypothesis that was tested by site-directed mutagenesis. As predicted binding sites for LstiGOBP1, Thr15, Trp43 and Val14 were replaced by alanine to determine the changes in binding affinity. Finally, fluorescence assays revealed that the mutants Thr15 and Trp43 had significantly decreased binding affinity to most odours; in mutants that had two-site mutations, the binding to the six odours that were tested was completely abolished. This result indicates that Thr15 and Trp43 were involved in binding these compounds, possibly by forming multiple hydrogen bonds with the functional groups of the ligands. These results provide new insights into the detailed chemistry of odours' interactions with proteins. PMID:26152502

  20. Ascorbic acid reduction of compound I of mammalian catalases proceeds via specific binding to the NADPH binding pocket.

    PubMed

    Korth, Hans-Gert; Meier, Ann-Cathérine; Auferkamp, Oliver; Sicking, Willi; de Groot, Herbert; Sustmann, Reiner; Kirsch, Michael

    2012-06-12

    Mammalian (Clade 3) catalases utilize NADPH as a protective cofactor to prevent one-electron reduction of the central reactive intermediate Compound I (Cpd I) to the catalytically inactive Compound II (Cpd II) species by re-reduction of Cpd I to the enzyme's resting state (ferricatalase). It has long been known that ascorbate/ascorbic acid is capable of reducing Cpd I of NADPH-binding catalases to Cpd II, but the mode of this one-electron reduction had hitherto not been explored. We here demonstrate that ascorbate-mediated reduction of Cpd I, generated by addition of peroxoacetic acid to NADPH-free bovine liver catalase (BLC), requires specific binding of the ascorbate anion to the NADPH binding pocket. Ascorbate-mediated Cpd II formation was found to be suppressed by added NADPH in a concentration-dependent manner, for the achievement of complete suppression at a stoichiometric 1:1 NADPH:heme concentration ratio. Cpd I → Cpd II reduction by ascorbate was similarly inhibited by addition of NADH, NADP(+), thio-NADP(+), or NAD(+), though with 0.5-, 0.1-, 0.1-, and 0.01-fold reduced efficiencies, respectively, in agreement with the relative binding affinities of these dinucleotides. Unexpected was the observation that although Cpd II formation is not observed in the presence of NADP(+), the decay of Cpd I is slightly accelerated by ascorbate rather than retarded, leading to direct regeneration of ferricatalase. The experimental findings are supported by molecular mechanics docking computations, which show a similar binding of NADPH, NADP(+), and NADH, but not NAD(+), as found in the X-ray structure of NADPH-loaded human erythrocyte catalase. The computations suggest that two ascorbate molecules may occupy the empty NADPH pocket, preferably binding to the adenine binding site. The biological relevance of these findings is discussed. PMID:22616883

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

    PubMed Central

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

    2015-01-01

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

  2. FaPYR1 is involved in strawberry fruit ripening.

    PubMed

    Chai, Ye-Mao; Jia, Hai-Feng; Li, Chun-Li; Dong, Qing-Hua; Shen, Yuan-Yue

    2011-10-01

    Although the plant hormone abscisic acid (ABA) has been suggested to play a role in the ripening of non-climatic fruit, direct genetic/molecular evidence is lacking. In the present study, a strawberry gene homologous to the Arabidopsis ABA receptor gene PYR1, named FaPYR1, was isolated and characterized. The 627 bp cDNA includes an intact open reading frame that encodes a deduced protein of 208 amino acids, in which putative conserved domains were detected by homology analysis. Using tobacco rattle virus-induced gene silencing (VIGS), the FaPYR1 gene was silenced in strawberry fruit. Down-regulation of the FaPYR1 gene not only significantly delayed fruit ripening, but also markedly altered ABA content, ABA sensitivity, and a set of ABA-responsive gene transcripts, including ABI1 and SnRK2. Furthermore, the loss of red colouring in FaPYR1 RNAi (RNA interference) fruits could not be rescued by exogenously applied ABA, which could promote the ripening of wild-type fruits. Collectively, these results demonstrate that the putative ABA receptor FaPYR1 acts as a positive regulator in strawberry fruit ripening. It was also revealed that the application of the VIGS technique in strawberry fruit could be used as a novel tool for studying strawberry fruit development.

  3. Specific binding of gibberellic acid by cytokinin-specific binding proteins: a new aspect of plant hormone-binding proteins with the PR-10 fold.

    PubMed

    Ruszkowski, Milosz; Sliwiak, Joanna; Ciesielska, Agnieszka; Barciszewski, Jakub; Sikorski, Michal; Jaskolski, Mariusz

    2014-07-01

    Pathogenesis-related proteins of class 10 (PR-10) are a family of plant proteins with the same fold characterized by a large hydrophobic cavity that allows them to bind various ligands, such as phytohormones. A subfamily with only ~20% sequence identity but with a conserved canonical PR-10 fold have previously been recognized as Cytokinin-Specific Binding Proteins (CSBPs), although structurally the binding mode of trans-zeatin (a cytokinin phytohormone) was found to be quite diversified. Here, it is shown that two CSBP orthologues from Medicago truncatula and Vigna radiata bind gibberellic acid (GA3), which is an entirely different phytohormone, in a conserved and highly specific manner. In both cases a single GA3 molecule is found in the internal cavity of the protein. The structural data derived from high-resolution crystal structures are corroborated by isothermal titration calorimetry (ITC), which reveals a much stronger interaction with GA3 than with trans-zeatin and pH dependence of the binding profile. As a conclusion, it is postulated that the CSBP subfamily of plant PR-10 proteins should be more properly linked with general phytohormone-binding properties and termed phytohormone-binding proteins (PhBP).

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  5. Acid Gas Capture Using CO2-Binding Organic Liquids

    SciTech Connect

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

    2010-11-10

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

  6. An amino acid substitution in the human intestinal fatty acid binding protein is associated with increased fatty acid binding, increased fat oxidation, and insulin resistance.

    PubMed

    Baier, L J; Sacchettini, J C; Knowler, W C; Eads, J; Paolisso, G; Tataranni, P A; Mochizuki, H; Bennett, P H; Bogardus, C; Prochazka, M

    1995-03-01

    The intestinal fatty acid binding protein locus (FABP2) was investigated as a possible genetic factor in determining insulin action in the Pima Indian population. A polymorphism at codon 54 of FABP2 was identified that results in an alanine-encoding allele (frequency 0.71) and a threonine-encoding allele (frequency 0.29). Pimas who were homozygous or heterozygous for the threonine-encoding allele were found to have a higher mean fasting plasma insulin concentration, a lower mean insulin-stimulated glucose uptake rate, a higher mean insulin response to oral glucose and a mixed meal, and a higher mean fat oxidation rate compared with Pimas who were homozygous for the alanine-encoding allele. Since the FABP2 threonine-encoding allele was found to be associated with insulin resistance and increased fat oxidation in vivo, we further analyzed the FABP2 gene products for potential functional differences. Titration microcalorimetry studies with purified recombinant protein showed that the threonine-containing protein had a twofold greater affinity for long-chain fatty acids than the alanine-containing protein. We conclude that the threonine-containing protein may increase absorption and/or processing of dietary fatty acids by the intestine and thereby increase fat oxidation, which has been shown to reduce insulin action. PMID:7883976

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

    NASA Astrophysics Data System (ADS)

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

    2015-02-01

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

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

    PubMed Central

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

    1980-01-01

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

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

    USGS Publications Warehouse

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

    1999-01-01

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

  10. FaGAST2, a strawberry ripening-related gene, acts together with FaGAST1 to determine cell size of the fruit receptacle.

    PubMed

    Moyano-Cañete, Enriqueta; Bellido, María L; García-Caparrós, Nicolás; Medina-Puche, Laura; Amil-Ruiz, Francisco; González-Reyes, José A; Caballero, José L; Muñoz-Blanco, Juan; Blanco-Portales, Rosario

    2013-02-01

    Numerous GAST-like genes have been reported in higher plants, but only one GAST-like gene (FaGAST1) has been described in strawberry so far. Herein, we have identified a novel strawberry FaGAST gene (FaGAST2) whose expression showed an increase throughout fruit receptacle development and ripening, coinciding with those stages where a decrease in fruit expansion processes (G3-W and R-OR stages) occurs. FaGAST2 only shares 31% and 15.7% amino acid and nucleotide sequence homology, respectively, with the previously reported FaGAST1 gene, but both genes contain a signal peptide and a highly conserved GASA domain (cysteine-rich domain) in the C-terminal region. FaGAST2 expression is mainly confined to the fruit receptacle and is not regulated by auxins, GA(3) or ABA, but is regulated by ethephon, an intracellular generator of ethylene. In addition, the expression of the FaGAST2 gene also increased under oxidative stress conditions (H(2)O(2) or Colletotrichum acutatum infection), suggesting a direct role for FaGAST2 protein in reactive oxygen species scavenging during fruit growth and ripening and during fungal infection. On the other hand, the overexpression of the FaGAST2 gene in different transgenic lines analyzed caused a delay in the growth of strawberry plants and a reduction in the size of the transgenic fruits. The histological studies performed in these fruits showed that their parenchymal cells were smaller than those of the controls, supporting a relationship between FaGAST2 gene expression, strawberry fruit cell elongation and fruit size. However, transitory silencing of FaGAST2 gene expression through RNA interference approaches revealed an increase in FaGAST1 expression, but no changes in fruit cell size were observed. These results support the hypothesis that both genes must act synergistically to determine fruit cell size during fruit development and ripening.

  11. FaGAST2, a strawberry ripening-related gene, acts together with FaGAST1 to determine cell size of the fruit receptacle.

    PubMed

    Moyano-Cañete, Enriqueta; Bellido, María L; García-Caparrós, Nicolás; Medina-Puche, Laura; Amil-Ruiz, Francisco; González-Reyes, José A; Caballero, José L; Muñoz-Blanco, Juan; Blanco-Portales, Rosario

    2013-02-01

    Numerous GAST-like genes have been reported in higher plants, but only one GAST-like gene (FaGAST1) has been described in strawberry so far. Herein, we have identified a novel strawberry FaGAST gene (FaGAST2) whose expression showed an increase throughout fruit receptacle development and ripening, coinciding with those stages where a decrease in fruit expansion processes (G3-W and R-OR stages) occurs. FaGAST2 only shares 31% and 15.7% amino acid and nucleotide sequence homology, respectively, with the previously reported FaGAST1 gene, but both genes contain a signal peptide and a highly conserved GASA domain (cysteine-rich domain) in the C-terminal region. FaGAST2 expression is mainly confined to the fruit receptacle and is not regulated by auxins, GA(3) or ABA, but is regulated by ethephon, an intracellular generator of ethylene. In addition, the expression of the FaGAST2 gene also increased under oxidative stress conditions (H(2)O(2) or Colletotrichum acutatum infection), suggesting a direct role for FaGAST2 protein in reactive oxygen species scavenging during fruit growth and ripening and during fungal infection. On the other hand, the overexpression of the FaGAST2 gene in different transgenic lines analyzed caused a delay in the growth of strawberry plants and a reduction in the size of the transgenic fruits. The histological studies performed in these fruits showed that their parenchymal cells were smaller than those of the controls, supporting a relationship between FaGAST2 gene expression, strawberry fruit cell elongation and fruit size. However, transitory silencing of FaGAST2 gene expression through RNA interference approaches revealed an increase in FaGAST1 expression, but no changes in fruit cell size were observed. These results support the hypothesis that both genes must act synergistically to determine fruit cell size during fruit development and ripening. PMID:23231876

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

    SciTech Connect

    Heldebrant, David J.; Yonker, Clement R.

    2010-09-21

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

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

    USGS Publications Warehouse

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

    2001-01-01

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

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

    PubMed

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

    2015-08-01

    The gypsy moth utilizes a pheromone, (7R,8S)-2-methyl-7,8-epoxyoctadecane, for mate location. The pheromone is detected by sensory hairs (sensilla) on the antennae of adult males. Sensilla contain the dendrites of olfactory neurons bathed in lymph, which contains pheromone binding proteins (PBPs). We have extracted and identified free fatty acids from lymph of sensory hairs, and we demonstrate that these function as endogenous ligands for gypsy moth PBP1 and PBP2. Homology modeling of both PBPs, and docking of fatty acids reveal multiple binding sites: one internal, the others external. Pheromone binding assays suggest that these fatty acids increase PBP-pheromone binding affinity. We show that fatty acid binding causes an increase in α-helix content in the N-terminal domain, but not in the C-terminal peptide of both proteins. The C-terminal peptide was shown to form a α-helix in a hydrophobic, homogeneous environment, but not in the presence of fatty acid micelles. Through partition assays we show that the fatty acids prevent adsorption of the pheromone on hydrophobic surfaces and facilitate pheromone partition into an aqueous phase. We propose that lymph is an emulsion of fatty acids and PBP that influence each other and thereby control the partition equilibria of hydrophobic odorants. PMID:26032337

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

    PubMed Central

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

    2014-01-01

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

  16. In vitro enantioselective displacement of propranolol from protein binding sites by acetyl salicylic acid and salicylic acid.

    PubMed

    Rezaei, Z; Khabnadideh, S; Hemmateenejad, B; Dehghani, Z

    2007-09-01

    The influences of acetyl salicylic acid (ASA) and salicylic acid (SA) on the enantioselective binding of propranolol (PL) and its enantiomers to plasma proteins and human serum albumin (HSA) were investigated. The equilibrium dialysis was employed for protein binding studies. We observed statistically significant displacement of racemic-PL, (+)-(R)-PL, and (-)-(S)-PL (0.1-10 microM) from their protein binding sites by ASA (200 microg/ml) and SA (100 microg/ml). ASA and SA displaced PL stereoselectivly from its binding sites. We concluded that ASA and its metabolite SA could change R/S ratio of PL unbound fractions and they might affect pharmacokinetic properties of PL.

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

    PubMed

    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. Crystal Structure of Species D Adenovirus Fiber Knobs and Their Sialic Acid Binding Sites

    PubMed Central

    Burmeister, Wim P.; Guilligay, Delphine; Cusack, Stephen; Wadell, Göran; Arnberg, Niklas

    2004-01-01

    Adenovirus serotype 37 (Ad37) belongs to species D and can cause epidemic keratoconjunctivitis, whereas the closely related Ad19p does not. Primary cell attachment by adenoviruses is mediated through receptor binding of the knob domain of the fiber protein. The knobs of Ad37 and Ad19p differ at only two positions, Lys240Glu and Asn340Asp. We report the high-resolution crystal structures of the Ad37 and Ad19p knobs, both native and in complex with sialic acid, which has been proposed as a receptor for Ad37. Overall, the Ad37 and Ad19p knobs are very similar to previously reported knob structures, especially to that of Ad5, which binds the coxsackievirus-adenovirus receptor (CAR). Ad37 and Ad19p knobs are structurally identical with the exception of the changed side chains and are structurally most similar to CAR-binding knobs (e.g., that of Ad5) rather than non-CAR-binding knobs (e.g., that of Ad3). The two mutations in Ad19p result in a partial loss of the exceptionally high positive surface charge of the Ad37 knob but do not affect sialic acid binding. This site is located on the top of the trimer and binds both α(2,3) and α(2,6)-linked sialyl-lactose, although only the sialic acid residue makes direct contact. Amino acid alignment suggests that the sialic acid binding site is conserved in several species D serotypes. Our results show that the altered viral tropism and cell binding of Ad19p relative to those of Ad37 are not explained by a different binding ability toward sialyl-lactose. PMID:15220447

  19. Conformational and nucleic acid binding studies on the synthetic nucleocapsid protein of HIV-1.

    PubMed

    Surovoy, A; Dannull, J; Moelling, K; Jung, G

    1993-01-01

    A 55 residue peptide corresponding to the nucleocapsid protein of HIV-1 (NCp7) containing two zinc binding domains as well as three truncated peptides were synthesized by Fmoc-based solid phase synthesis using the fragment condensation approach. Circular dichroism (CD) data support a conformational model in trifluoroethanol/buffer solution consisting of two helical segments at the chain ends with two Zn-modules in the center of the molecule. CD titration experiments show that the synthetic protein binds two equivalents of Zn2+ stoichiometrically, and the Zn2+ induced conformational changes are completely reversible by addition of EDTA. NCp7 and its S-acetamidomethylated analog (NCp7-Acm), devoid of the zinc co-ordination centers, exhibit preferential binding to RNA with a Kd = approximately 10(-9) M irrespective of the cysteine modification as determined by filter binding assays. The binding affinity of the NCp7 protein to single-stranded DNA is lower than to RNA. Binding to double-stranded DNA is lower than to ssDNA. The NCp7-Acm protein exhibits reduced single-stranded DNA binding affinity compared to the unmodified protein. Nucleic acid binding analyses with the fragments of NCp7 protein suggest that two basic amino acid stretches are involved in RNA binding of the NCp7.

  20. Conjugates of folic acids with BSA-coated quantum dots for cancer cell targeting and imaging by single-photon and two-photon excitation.

    PubMed

    Meng, He; Chen, Ji-Yao; Mi, Lan; Wang, Pei-Nan; Ge, Mei-Ying; Yue, Yang; Dai, Ning

    2011-01-01

    Bovine serum albumin (BSA)-coated CdTe/ZnS quantum dots (BSA-QDs) were selected to conjugate with folic acid (FA), forming FA-BSA-QDs. This study aims to develop these small FA-BSA-QDs (less than 10 nm) for the diagnosis of cancers in which the FA receptor (FR) is overexpressed. The enhancement of cellular uptake in FR-positive human nasopharyngeal carcinoma cells (KB cells) for FA-BSA-QDs was found by means of confocal fluorescence microscopy under single-photon and two-photon excitation. The uptake enhancement for FA-BSA-QDs was further evaluated by flow-cytometric analysis in 10(4) KB cells, and was about 3 times higher than for BSA-QDs on average. The uptake enhancement was suppressed when KB cells had been pretreated with excess FA, reflecting that the enhancement was mediated by the association of FR at cell membranes with FA-BSA-QDs. When human embryonic kidney cells (293T) (FR-negative cells) and KB cells, respectively, were incubated with FA-BSA-QDs (1 μM) for 40 min, the FA-BSA-QD uptake by 293T cells was much weaker than that by KB cells, demonstrating that FA-BSA-QDs could undergo preferential binding on FR-positive cancer cells. These characteristics suggest that FA-BSA-QDs are potential candidates for cancer diagnosis.

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

    USGS Publications Warehouse

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

    1995-01-01

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

  2. 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.

    PubMed

    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.

  3. Involvement of fertilization antigen (FA-1) in involuntary immunoinfertility in humans.

    PubMed Central

    Naz, R K

    1987-01-01

    Sera from immunoinfertile patients (n = 32) and fertile controls (n = 20) were analyzed for cross-reaction with a purified and characterized sperm-specific glycoprotein, the fertilization antigen (FA-1), employing an enzyme-linked immunosorbent assay. The immunoinfertile sera demonstrated a strong reaction with FA-1 when compared with fertile control sera. There was no correlation between the reaction of sera with FA-1 and the titers obtained through the sperm agglutination technique and the sperm immobilization technique. Immunoinfertile sera showed binding with the protein bands in the regions corresponding to FA-1 on Western blots involving sodium deoxycholate-solubilized human sperm. Antigens isolated with immunoaffinity chromatography involving immunoinfertile sera also demonstrated antigen bands corresponding to FA-1 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Of the seven immunoinfertile couples, three that had antibodies to FA-1 in the male as well as female partners demonstrated a block of fertilization (IVF) due to antibodies bound on the sperm surface. The anti-FA-1 antibody activity was detected in serum as well as in follicular fluid and seminal plasma. Immunoinfertile sera that showed an inhibition of human sperm penetration of zona-free hamster ova showed a significant (P less than 0.001) increase in penetration rates after absorption with FA-1. These results indicate that sera from immunoinfertile patients had antibodies reacting with FA-1, and these antibodies are involved in the fertilization process. Images PMID:3316276

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

    DOEpatents

    Tabor, Stanley; Richardson, Charles C.

    1996-01-01

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

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

    PubMed Central

    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

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

    PubMed

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

    2016-01-01

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

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

    USGS Publications Warehouse

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

    1999-01-01

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

  8. A glycoprotein binding retinoids and fatty acids is present in Drosophila.

    PubMed

    Duncan, T; Kutty, G; Chader, G J; Wiggert, B

    1994-07-01

    In the search for a possible Drosophila melanogaster homolog of interphotoreceptor retinoid-binding protein (IRBP), a approximately 140-kDa retinoid- and fatty acid-binding glycoprotein found in vertebrates, the 110,000 g supernatant fraction prepared from homogenates of fly heads was analyzed for the presence of proteins capable of binding radiolabeled retinol and palmitic acid. A soluble protein, which binds concanavalin A and has a retention time on size-exclusion high-performance liquid chromatography identical to that of purified bovine IRBP, was identified as binding both ligands. As assessed by fluorescence titration, the protein fraction obtained by concanavalin A-Sepharose affinity chromatography and size-exclusion chromatography of fly head supernatant had apparent dissociation constants of 2.9 x 10(-7) +/- 0.6 M for all-trans retinol, with the number (n) of independent ligand binding sites per protein molecule = 2, and 3.5 x 10(-7) +/- 0.1 M for 16-[9-anthroyloxy] palmitic acid with n = 7. High-performance liquid chromatography of hexane extracts of this protein fraction resolved several peaks with polarity and relative retention times similar, but not identical to all-trans retinol and retinal and their 9-, 11-, and 13-cis isomers. Gas chromatography/mass spectrometry analysis of fatty acid methyl esters prepared following lipid extraction of the protein identified lauric, myristic, palmitic, palmitoleic, and oleic acids as being covalently bound. Laurate, myristate, palmitate, and stearate were noncovalently bound. The apparent molecular mass of the Drosophila protein as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of the retinoid- and fatty acid-binding peak obtained by hydrophobic interaction chromatography of the size-exclusion fraction was approximately 70 kDa. PMID:8031123

  9. Analysis of a nucleotide-binding site of 5-lipoxygenase by affinity labelling: binding characteristics and amino acid sequences.

    PubMed Central

    Zhang, Y Y; Hammarberg, T; Radmark, O; Samuelsson, B; Ng, C F; Funk, C D; Loscalzo, J

    2000-01-01

    5-Lipoxygenase (5LO) catalyses the first two steps in the biosynthesis of leukotrienes, which are inflammatory mediators derived from arachidonic acid. 5LO activity is stimulated by ATP; however, a consensus ATP-binding site or nucleotide-binding site has not been found in its protein sequence. In the present study, affinity and photoaffinity labelling of 5LO with 5'-p-fluorosulphonylbenzoyladenosine (FSBA) and 2-azido-ATP showed that 5LO bound to the ATP analogues quantitatively and specifically and that the incorporation of either analogue inhibited ATP stimulation of 5LO activity. The stoichiometry of the labelling was 1.4 mol of FSBA/mol of 5LO (of which ATP competed with 1 mol/mol) or 0.94 mol of 2-azido-ATP/mol of 5LO (of which ATP competed with 0.77 mol/mol). Labelling with FSBA prevented further labelling with 2-azido-ATP, indicating that the same binding site was occupied by both analogues. Other nucleotides (ADP, AMP, GTP, CTP and UTP) also competed with 2-azido-ATP labelling, suggesting that the site was a general nucleotide-binding site rather than a strict ATP-binding site. Ca(2+), which also stimulates 5LO activity, had no effect on the labelling of the nucleotide-binding site. Digestion with trypsin and peptide sequencing showed that two fragments of 5LO were labelled by 2-azido-ATP. These fragments correspond to residues 73-83 (KYWLNDDWYLK, in single-letter amino acid code) and 193-209 (FMHMFQSSWNDFADFEK) in the 5LO sequence. Trp-75 and Trp-201 in these peptides were modified by the labelling, suggesting that they were immediately adjacent to the C-2 position of the adenine ring of ATP. Given the stoichiometry of the labelling, the two peptide sequences of 5LO were probably near each other in the enzyme's tertiary structure, composing or surrounding the ATP-binding site of 5LO. PMID:11042125

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

    PubMed Central

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

    2016-01-01

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

  11. Cold shock domain protein from Philosamia ricini prefers single-stranded nucleic acids binding.

    PubMed

    Mani, Ashutosh; Yadava, P K; Gupta, Dwijendra K

    2012-01-01

    The cold shock proteins are evolutionarily conserved nucleic acid-binding proteins. Their eukaryotic homologs are present as cold shock domain (CSD) in Y-box proteins. CSDs too share striking similarity among different organisms and show nucleic acid binding properties. The purpose of the study was to investigate the preferential binding affinity of CSD protein for nucleic acids in Philosamia ricini. We have cloned and sequenced the first cDNA coding for Y-box protein in P. ricini; the sequence has been deposited in GenBank. Comparative genomics and phylogenetic analytics further confirmed that the deduced amino acid sequence belongs to the CSD protein family. A comparative study employing molecular docking was performed with P. ricini CSD, human CSD, and bacterial cold shock protein with a range of nucleic acid entities. The results indicate that CSD per se exhibits preferential binding affinity for single-stranded RNA and DNA. Possibly, the flanking N- and C-terminal domains are additionally involved in interactions with dsDNA or in conferring extra stability to CSD for improved binding.

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

    PubMed

    Chowrira, B M; Burke, J M

    1991-09-01

    The "hairpin" ribozyme derived from the minus strand of tobacco ringspot virus satellite RNA [(-)sTRSV] efficiently catalyzes sequence-specific RNA hydrolysis in trans (Feldstein et al., 1989; Hampel & Triz, 1989; Haseloff & Gerlach, 1989). The ribozyme does not cleave DNA. An RNA substrate analogue containing a single deoxyribonucleotide residue 5' to the cleavage site (A-1) binds to the ribozyme efficiently but cannot be cleaved. A DNA substrate analogue with a ribonucleotide at A-1 is cleaved; thus A-1 provides the only 2'-OH required for cleavage. These results support cleavage via a transphosphorylation mechanism initiated by attack of the 2'-OH of A-1 on the scissile phosphodiester. The ribozyme discriminates between DNA and RNA in both binding and cleavage. Results indicate that the 2'-OH of A-1 functions in complex stabilization as well as cleavage. The ribozyme efficiently cleaves a phosphorothioate diester linkage, suggesting that the pro-Rp oxygen at the scissile phosphodiester does not coordinate Mg2+. PMID:1909564

  13. Spectroscopic and microcalorimetric studies on the molecular binding of food colorant acid red 27 with deoxyribonucleic acid.

    PubMed

    Basu, Anirban; Kumar, Gopinatha Suresh

    2016-08-01

    Interaction of the food colorant acid red 27 with double stranded DNA was investigated using spectroscopic and calorimetric methods. Absorbance and fluorescence studies suggested an intimate binding interaction between the dye and DNA. The quantum efficiency value testified an effective energy transfer from the DNA base pairs to the dye molecules. Minor groove displacement assay with Hoechst 33258 revealed that the binding occurs in the minor groove of DNA. Circular dichroism studies revealed that acid red 27 induces moderate conformational perturbations in DNA. Results of calorimetric studies suggested that the complexation process was driven largely by positive entropic contribution with a smaller favorable enthalpy contribution. The equilibrium constant of the binding was calculated to be (3.04 ± 0.09) × 10(4)  M(-1) at 298.15 K. Negative heat capacity value along with the enthalpy-entropy compensation phenomenon established the involvement of dominant hydrophobic forces in the binding process. Differential scanning calorimetry studies presented evidence for an increased thermal stability of DNA on binding of acid red 27. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26846192

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

    SciTech Connect

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

    2010-01-01

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

  15. Identification of Apolipoprotein A-I as a Retinoic Acid-binding Protein in the Eye.

    PubMed

    Summers, Jody A; Harper, Angelica R; Feasley, Christa L; Van-Der-Wel, Hanke; Byrum, Jennifer N; Hermann, Marcela; West, Christopher M

    2016-09-01

    All-trans-retinoic acid may be an important molecular signal in the postnatal control of eye size. The goal of this study was to identify retinoic acid-binding proteins secreted by the choroid and sclera during visually guided ocular growth. Following photoaffinity labeling with all-trans-[11,12-(3)H]retinoic acid, the most abundant labeled protein detected in the conditioned medium of choroid or sclera had an apparent Mr of 27,000 Da. Following purification and mass spectrometry, the Mr 27,000 band was identified as apolipoprotein A-I. Affinity capture of the radioactive Mr 27,000 band by anti-chick apolipoprotein A-I antibodies confirmed its identity as apolipoprotein A-I. Photoaffinity labeling and fluorescence quenching experiments demonstrated that binding of retinoic acid to apolipoprotein A-I is 1) concentration-dependent, 2) selective for all-trans-retinoic acid, and 3) requires the presence of apolipoprotein A-I-associated lipids for retinoid binding. Expression of apolipoprotein A-I mRNA and protein synthesis were markedly up-regulated in choroids of chick eyes during the recovery from induced myopia, and apolipoprotein A-I mRNA was significantly increased in choroids following retinoic acid treatment. Together, these data suggest that apolipoprotein A-I may participate in a regulatory feedback mechanism with retinoic acid to control the action of retinoic acid on ocular targets during postnatal ocular growth.

  16. Identification of Apolipoprotein A-I as a Retinoic Acid-binding Protein in the Eye.

    PubMed

    Summers, Jody A; Harper, Angelica R; Feasley, Christa L; Van-Der-Wel, Hanke; Byrum, Jennifer N; Hermann, Marcela; West, Christopher M

    2016-09-01

    All-trans-retinoic acid may be an important molecular signal in the postnatal control of eye size. The goal of this study was to identify retinoic acid-binding proteins secreted by the choroid and sclera during visually guided ocular growth. Following photoaffinity labeling with all-trans-[11,12-(3)H]retinoic acid, the most abundant labeled protein detected in the conditioned medium of choroid or sclera had an apparent Mr of 27,000 Da. Following purification and mass spectrometry, the Mr 27,000 band was identified as apolipoprotein A-I. Affinity capture of the radioactive Mr 27,000 band by anti-chick apolipoprotein A-I antibodies confirmed its identity as apolipoprotein A-I. Photoaffinity labeling and fluorescence quenching experiments demonstrated that binding of retinoic acid to apolipoprotein A-I is 1) concentration-dependent, 2) selective for all-trans-retinoic acid, and 3) requires the presence of apolipoprotein A-I-associated lipids for retinoid binding. Expression of apolipoprotein A-I mRNA and protein synthesis were markedly up-regulated in choroids of chick eyes during the recovery from induced myopia, and apolipoprotein A-I mRNA was significantly increased in choroids following retinoic acid treatment. Together, these data suggest that apolipoprotein A-I may participate in a regulatory feedback mechanism with retinoic acid to control the action of retinoic acid on ocular targets during postnatal ocular growth. PMID:27402828

  17. Clarithromycin and Tetracycline Binding to Soil Humic Acid in the Absence and Presence of Calcium.

    PubMed

    Christl, Iso; Ruiz, Mercedes; Schmidt, J R; Pedersen, Joel A

    2016-09-20

    Numerous ionizable organic micropollutants contain positively charged moieties at pH values typical of environmental systems. Describing organic cation and zwitterion interaction with dissolved natural organic matter requires explicit consideration of the pH-dependent speciation of both sorbate and sorbent. We studied the pH-, ionic strength-, and concentration-dependent binding of relatively large, organic cations and zwitterions (viz., the antibiotics clarithromycin and tetracycline) to dissolved humic acid in the absence and presence of Ca(2+) and evaluated the ability of the NICA-Donnan model to describe the data. Clarithromycin interaction with dissolved humic acid was well described by the model including the competitive effect of Ca(2+) on clarithromycin binding over a wide range of solution conditions by considering only the binding of the cationic species to low proton-affinity sites in humic acid. Tetracycline possesses multiple ionizable moieties and forms complexes with Ca(2+). An excellent fit to experimental data was achieved by considering tetracycline cation interaction with both low and high proton-affinity sites of humic acid and zwitterion interaction with high proton-affinity sites. In contrast to clarithromycin, tetracycline binding to humic acid increased in the presence of Ca(2+), especially under alkaline conditions. Model calculations indicate that this increase is due to electrostatic interaction of positively charged tetracycline-Ca complexes with humic acid rather than due to the formation of ternary complexes, except at very low TC concentrations. PMID:27438991

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

    PubMed

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

    2015-01-01

    Long chain fatty acids (LCFA) serve as energy sources, components of cell membranes and precursors for signalling molecules. Here we show that these biological compounds also regulate gene expression and that they do so by controlling the transcriptional activities of the retinoic acid (RA)-activated nuclear receptors RAR and PPARβ/δ. The data indicate that these activities of LCFA are mediated by FABP5, which delivers ligands from the cytosol to nuclear PPARβ/δ. Both saturated and unsaturated LCFA (SLCFA, ULCFA) bind to FABP5, thereby displacing RA and diverting it to RAR. However, while SLCFA inhibit, ULCFA activate the FABP5/PPARβ/δ pathway. We show further that, by concomitantly promoting 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. PMID:26592976

  19. Binding of small basic peptides to membranes containing acidic lipids: theoretical models and experimental results.

    PubMed Central

    Ben-Tal, N; Honig, B; Peitzsch, R M; Denisov, G; McLaughlin, S

    1996-01-01

    We measured directly the binding of Lys3, Lys5, and Lys7 to vesicles containing acidic phospholipids. When the vesicles contain 33% acidic lipids and the aqueous solution contains 100 mM monovalent salt, the standard Gibbs free energy for the binding of these peptides is 3, 5, and 7 kcal/mol, respectively. The binding energies decrease as the mol% of acidic lipids in the membrane decreases and/or as the salt concentration increases. Several lines of evidence suggest that these hydrophilic peptides do not penetrate the polar headgroup region of the membrane and that the binding is mainly due to electrostatic interactions. To calculate the binding energies from classical electrostatics, we applied the nonlinear Poisson-Boltzmann equation to atomic models of the phospholipid bilayers and the basic peptides in aqueous solution. The electrostatic free energy of interaction, which arises from both a long-range coulombic attraction between the positively charged peptide and the negatively charged lipid bilayer, and a short-range Born or image charge repulsion, is a minimum when approximately 2.5 A (i.e., one layer of water) exists between the van der Waals surfaces of the peptide and the lipid bilayer. The calculated molar association constants, K, agree well with the measured values: K is typically about 10-fold smaller than the experimental value (i.e., a difference of about 1.5 kcal/mol in the free energy of binding). The predicted dependence of K (or the binding free energies) on the ionic strength of the solution, the mol% of acidic lipids in the membrane, and the number of basic residues in the peptide agree very well with the experimental measurements. These calculations are relevant to the membrane binding of a number of important proteins that contain clusters of basic residues. Images FIGURE 2 FIGURE 3 PMID:8842196

  20. Chromatographic characterisation, under highly aqueous conditions, of a molecularly imprinted polymer binding the herbicide 2,4-dichlorophenoxyacetic acid.

    PubMed

    Legido-Quigley, C; Oxelbark, J; De Lorenzi, E; Zurutuza-Elorza, A; Cormack, P A G

    2007-05-15

    The affinity of a 2,4-dichlorophenoxyacetic acid (2,4-D) molecularly imprinted polymer (MIP), which was synthesised directly in an aqueous organic solvent, for its template (2,4-D) was studied and compared with the affinity exhibited by two other reference (control) polymers, NIPA and NIPB, for the same analyte. Zonal chromatography was performed to establish the optimal selectivity, expressed as imprinting factor (IF), under chromatographic conditions more aqueous than those described so far in the literature. Frontal analysis (FA) was performed on columns packed with these polymers, using an optimized mobile phase composed of methanol/phosphate buffer (50/50, v/v), to extract adsorption isotherm data and retrieve binding parameters from the best isotherm model. Surprisingly, the template had comparable and strong affinity for both MIP (K = 3.8x10(4) M(-1)) and NIPA (K = 1.9x10(4) M(-1)), although there was a marked difference in the saturation capacities of selective and non-selective sites, as one would expect for an imprinted polymer. NIPB acts as a true control polymer in the sense that it has relatively low affinity for the template (K = 8.0x10(2) M(-1)). This work provides the first frontal chromatographic characterization of such a polymer in a water-rich environment over a wide concentration range. The significance of this work stems from the fact that the chromatographic approach used is generic and can be applied readily to other analytes, but also because there is an increasing demand for well-characterised imprinted materials that function effectively in aqueous media and are thus well-suited for analytical science applications involving, for example, biofluids and environmental water samples.

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

    PubMed Central

    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

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

    PubMed Central

    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 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. PMID:26681179

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

    PubMed

    Miao, Zhichao; Westhof, Eric

    2015-12-01

    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 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. PMID:26681179

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

    PubMed

    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. PMID:24498422

  5. Polar solvent effects on tartaric acid binding by aromatic oligoamide foldamer capsules.

    PubMed

    Chandramouli, Nagula; El-Behairy, Mohammed Farrag; Lautrette, Guillaume; Ferrand, Yann; Huc, Ivan

    2016-02-28

    Aromatic oligoamide sequences able to fold into single helical capsules were functionalized with two types of side chains to make them soluble in various solvents such as chloroform, methanol or water and their propensity to recognize tartaric acid was evaluated. The binding affinities to tartaric acid and binding thermodynamics in different media were investigated by variable temperature (1)H NMR and ITC experiments, the two methods giving consistent results. We show that tartaric acid binding mainly rests on enthalpically favourable polar interactions that were found to be sufficiently strong to be effective in the presence of a polar aprotic solvent (DMSO) and even in pure methanol. Binding in water was very weak. The stronger binding interactions were found to be more susceptible to the effect of competitive solvents and compensated by unfavourable entropic effects. Thus, the best host in a less polar medium eventually was found to be the worst host in protic solvents. An interesting case of entropically driven binding was evidenced in methanol.

  6. Medium-chain fatty acid binding to albumin and transfer to phospholipid bilayers

    SciTech Connect

    Hamilton, J.A. )

    1989-04-01

    Temperature-dependent (5-42{degree}C) {sup 13}C NMR spectra of albumin complexes with 90% isotopically substituted (1-{sup 13}C)octanoic or (1-{sup 13}C)decanoic acids showed a single peak at >30{degree}C but three peaks at lower temperatures. The chemical-shift differences result from different ionic and/or hydrogen-bonding interactions between amino acid side chains and the fatty acid carboxyl carbon. Rapid exchange of fatty acid among binding sites obscures these sites at temperatures >30{degree}C. Rate constants for exchange at 33{degree}C were 350 sec{sup {minus}1} for octanoate and 20 sec {sup {minus}1} for decanoate. Temperature-dependent data for octanoate showed an activation energy of 2 kcal/mol for exchange. Spectra of albumin complexes with the 12-carbon saturated fatty acid, lauric acid, had several narrow laurate carboxyl peaks at 35{degree}C, indicating longer lifetimes in the different binding sites. Fatty acid exchange between albumin and model membranes (phosphatidylcholine bilayers) occurred on a time scale comparable to that for exchange among albumin binding sites, following the order octanoate > decanoate > laurate. The equilibrium distribution of fatty acid between lipid bilayers and protein was measured directly from NMR spectra. Decreasing pH increased the relative affinity of fatty acid for the lipid bilayer. The results predict that the relative affinity of octanoic acid for albumin and membranes will be similar to that of long-chain fatty acids, but the rate of equilibration will be {approx} 10{sup 4} faster for octanoic acid.

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

    PubMed

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

    1998-05-15

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

  8. Effects of iron deficiency on iron binding and internalization into acidic vacuoles in Dunaliella salina.

    PubMed

    Paz, Yakov; Shimoni, Eyal; Weiss, Meira; Pick, Uri

    2007-07-01

    Uptake of iron in the halotolerant alga Dunaliella salina is mediated by a transferrin-like protein (TTf), which binds and internalizes Fe(3+) ions. Recently, we found that iron deficiency induces a large enhancement of iron binding, which is associated with accumulation of three other plasma membrane proteins that associate with TTf. In this study, we characterized the kinetic properties of iron binding and internalization and identified the site of iron internalization. Iron deficiency induces a 4-fold increase in Fe binding, but only 50% enhancement in the rate of iron uptake and also increases the affinity for iron and bicarbonate, a coligand for iron binding. These results indicate that iron deprivation leads to accumulation and modification of iron-binding sites. Iron uptake in iron-sufficient cells is preceded by an apparent time lag, resulting from prebound iron, which can be eliminated by unloading iron-binding sites. Iron is tightly bound to surface-exposed sites and hardly exchanges with medium iron. All bound iron is subsequently internalized. Accumulation of iron inhibits further iron binding and internalization. The vacuolar inhibitor bafilomycin inhibits iron uptake and internalization. Internalized iron was localized by electron microscopy within vacuolar structures that were identified as acidic vacuoles. Iron internalization is accompanied by endocytosis of surface proteins into these acidic vacuoles. A novel kinetic mechanism for iron uptake is proposed, which includes two pools of bound/compartmentalized iron separated by a rate-limiting internalization stage. The major parameter that is modulated by iron deficiency is the iron-binding capacity. We propose that excessive iron binding in iron-deficient cells serves as a temporary reservoir for iron that is subsequently internalized. This mechanism is particularly suitable for organisms that are exposed to large fluctuations in iron availability. PMID:17513481

  9. Na+ Inhibits the Epithelial Na+ Channel by Binding to a Site in an Extracellular Acidic Cleft*

    PubMed Central

    Kashlan, Ossama B.; Blobner, Brandon M.; Zuzek, Zachary; Tolino, Michael; Kleyman, Thomas R.

    2015-01-01

    The epithelial Na+ channel (ENaC) has a key role in the regulation of extracellular fluid volume and blood pressure. ENaC belongs to a family of ion channels that sense the external environment. These channels have large extracellular regions that are thought to interact with environmental cues, such as Na+, Cl−, protons, proteases, and shear stress, which modulate gating behavior. We sought to determine the molecular mechanism by which ENaC senses high external Na+ concentrations, resulting in an inhibition of channel activity. Both our structural model of an ENaC α subunit and the resolved structure of an acid-sensing ion channel (ASIC1) have conserved acidic pockets in the periphery of the extracellular region of the channel. We hypothesized that these acidic pockets host inhibitory allosteric Na+ binding sites. Through site-directed mutagenesis targeting the acidic pocket, we modified the inhibitory response to external Na+. Mutations at selected sites altered the cation inhibitory preference to favor Li+ or K+ rather than Na+. Channel activity was reduced in response to restraining movement within this region by cross-linking structures across the acidic pocket. Our results suggest that residues within the acidic pocket form an allosteric effector binding site for Na+. Our study supports the hypothesis that an acidic cleft is a key ligand binding locus for ENaC and perhaps other members of the ENaC/degenerin family. PMID:25389295

  10. Na+ inhibits the epithelial Na+ channel by binding to a site in an extracellular acidic cleft.

    PubMed

    Kashlan, Ossama B; Blobner, Brandon M; Zuzek, Zachary; Tolino, Michael; Kleyman, Thomas R

    2015-01-01

    The epithelial Na(+) channel (ENaC) has a key role in the regulation of extracellular fluid volume and blood pressure. ENaC belongs to a family of ion channels that sense the external environment. These channels have large extracellular regions that are thought to interact with environmental cues, such as Na(+), Cl(-), protons, proteases, and shear stress, which modulate gating behavior. We sought to determine the molecular mechanism by which ENaC senses high external Na(+) concentrations, resulting in an inhibition of channel activity. Both our structural model of an ENaC α subunit and the resolved structure of an acid-sensing ion channel (ASIC1) have conserved acidic pockets in the periphery of the extracellular region of the channel. We hypothesized that these acidic pockets host inhibitory allosteric Na(+) binding sites. Through site-directed mutagenesis targeting the acidic pocket, we modified the inhibitory response to external Na(+). Mutations at selected sites altered the cation inhibitory preference to favor Li(+) or K(+) rather than Na(+). Channel activity was reduced in response to restraining movement within this region by cross-linking structures across the acidic pocket. Our results suggest that residues within the acidic pocket form an allosteric effector binding site for Na(+). Our study supports the hypothesis that an acidic cleft is a key ligand binding locus for ENaC and perhaps other members of the ENaC/degenerin family. PMID:25389295

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

    NASA Astrophysics Data System (ADS)

    Zhao, Haiyan; Gabbaï, François P.

    2010-11-01

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

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

    PubMed

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

    2009-11-15

    Bile acid binding proteins (BABPs) are cytosolic lipid chaperones contributing to the maintenance of bile acid homeostasis and functional distribution within the cell. Liver BABPs act in parallel with ileal transporters to ensure vectorial transport of bile salts in hepatocytes and enterocytes, respectively. We describe the investigation of ligand binding to liver BABP, an essential step in the understanding of intracellular bile salt transport. Binding site occupancies were monitored in NMR titration experiments using (15)N-labelled ligand, while the relative populations of differently bound BABP forms were assessed by mass spectrometry. This site-specific information allowed the determination of intrinsic thermodynamic parameters and the identification of an extremely high cooperativity between two binding sites. Protein-observed NMR experiments revealed a global structural rearrangement which suggests an allosteric mechanism at the basis of the observed cooperativity. The view of a molecular tool capable of buffering against significant concentrations of free bile salts in a large range of solution conditions emerges from the observed pH-dependence of binding. We set to determine the molecular determinants of cooperativity by analysing the binding properties of a protein containing a mutated internal histidine. Both mass spectrometry and NMR experiments are consistent with an overall decreased binding affinity of the mutant, while the measured diffusion coefficients of ligand species reveal that the affinity loss concerns essentially one of the two binding sites. We therefore identified a mutation able to disrupt energetic communication functional to efficient binding and conclude that the buried histidine establishes contacts that stabilize the ternary complex. PMID:19603488

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

    PubMed

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

    2009-11-15

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

  14. GPM Video of In-fa

    NASA Video Gallery

    On Nov. 19 GPM saw a few towering storms in In-fa's eye wall were reaching heights of up to 17.3 km (10.7 miles). The most intense precipitation was measured in In-fa's eye wall by DPR where it was...

  15. Probing the general time scale question of boronic acid binding with sugars in aqueous solution at physiological pH.

    PubMed

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

    2012-05-01

    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 pairs examined, reactions were complete within seconds. The k(on) 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.

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

    PubMed

    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.

  17. Stacking interaction and its role in kynurenic acid binding to glutamate ionotropic receptors.

    PubMed

    Zhuravlev, Alexander V; Zakharov, Gennady A; Shchegolev, Boris F; Savvateeva-Popova, Elena V

    2012-05-01

    Stacking interaction is known to play an important role in protein folding, enzyme-substrate and ligand-receptor complex formation. It has been shown to make a contribution into the aromatic antagonists binding with glutamate ionotropic receptors (iGluRs), in particular, the complex of NMDA receptor NR1 subunit with the kynurenic acid (KYNA) derivatives. The specificity of KYNA binding to the glutamate receptors subtypes might partially result from the differences in stacking interaction. We have calculated the optimal geometry and binding energy of KYNA dimers with the four types of aromatic amino acid residues in Rattus and Drosophila ionotropic iGluR subunits. All ab initio quantum chemical calculations were performed taking into account electron correlations at MP2 and MP4 perturbation theory levels. We have also investigated the potential energy surfaces (PES) of stacking and hydrogen bonds (HBs) within the receptor binding site and calculated the free energy of the ligand-receptor complex formation. The energy of stacking interaction depends both on the size of aromatic moieties and the electrostatic effects. The distribution of charges was shown to determine the geometry of polar aromatic ring dimers. Presumably, stacking interaction is important at the first stage of ligand binding when HBs are weak. The freedom of ligand movements and rotation within receptor site provides the precise tuning of the HBs pattern, while the incorrect stacking binding prohibits the ligand-receptor complex formation. PMID:21833825

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

    SciTech Connect

    J Fleming; J Wojciak; M Campbell; T Huxford

    2011-12-31

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

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

    PubMed

    Tang, Ning; Skibsted, Leif H

    2016-06-01

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

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

    SciTech Connect

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

    2009-04-15

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

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

    PubMed

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

    2015-03-01

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

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

    PubMed

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

    2014-10-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-10-01

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

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

    SciTech Connect

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

    2015-09-18

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

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

    DOE PAGES

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

    2015-09-18

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

  6. Modulation of FadR binding capacity for acyl-CoA fatty acids through structure-guided mutagenesis.

    PubMed

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

    2015-10-01

    FadR is a versatile global regulator in Escherichia coli that controls fatty acid metabolism and thereby modulates the ability of this bacterium to grow using fatty acids or acetate as the sole carbon source. FadR regulates fatty acid metabolism in response to intra-cellular concentrations of acyl-CoA lipids. The ability of FadR to bind acyl-CoA fatty acids is thus of significant interest for the engineering of biosynthetic pathways for the production of lipid-based biofuels and commodity chemicals. Based on the available crystal structure of E. coli bound to myristoyl-CoA, we predicted amino acid positions within the effector binding pocket that would alter the ability of FadR to bind acyl-CoA fatty acids without affecting DNA binding. We utilized fluorescence polarization to characterize the in vitro binding properties of wild type and mutant FadR. We found that a Leu102Ala mutant enhanced binding of the effector, likely by increasing the size of the binding pocket for the acyl moiety of the molecule. Conversely, the elimination of the guanidine side chain (Arg213Ala and Arg213Met mutants) of the CoA moiety binding site severely diminished the ability of FadR to bind the acyl-CoA effector. These results demonstrate the ability to fine tune FadR binding capacity. The validation of an efficient method to fully characterize all the binding events involved in the specific activity (effector and DNA operator binding) of FadR has allowed us to increase our understanding of the role of specific amino acids in the binding and recognition of acyl-CoA fatty acids and will greatly facilitate efforts aimed at engineering tunable FadR regulators for synthetic biology. PMID:26385696

  7. Identification of a nucleic acid-binding region within the largest subunit of Drosophila melanogaster RNA polymerase II.

    PubMed Central

    Kontermann, R. E.; Kobor, M.; Bautz, E. K.

    1993-01-01

    The largest and the second-largest subunit of the multisubunit eukaryotic RNA polymerases are involved in interaction with the DNA template and the nascent RNA chain. Using Southwestern DNA-binding techniques and nitrocellulose filter binding assays of bacterially expressed fusion proteins, we have identified a region of the largest, 215-kDa, subunit of Drosophila RNA polymerase II that has the potential to bind nucleic acids nonspecifically. This nucleic acid-binding region is located between amino acid residues 309-384 and is highly conserved within the largest subunits of eukaryotic and bacterial RNA polymerases. A homology to a region of the DNA-binding cleft of Escherichia coli DNA polymerase I involved in binding of the newly synthesized DNA duplex provides indirect evidence that the nucleic acid-binding region of the largest subunit participates in interaction with double-stranded nucleic acids during transcription. The nonspecific DNA-binding behavior of the region is similar to that observed for the native enzyme in nitrocellulose filter binding assays and that of the separated largest subunit in Southwestern assays. A high content of basic amino acid residues is consistent with the electrostatic nature of nonspecific DNA binding by RNA polymerases. PMID:8443600

  8. Effects on sialic acid recognition of amino acid mutations in the carbohydrate-binding cleft of the rotavirus spike protein.

    PubMed

    Kraschnefski, Mark J; Bugarcic, Andrea; Fleming, Fiona E; Yu, Xing; von Itzstein, Mark; Coulson, Barbara S; Blanchard, Helen

    2009-03-01

    The rotavirus spike protein VP4 mediates attachment to host cells and subsequent membrane penetration. The VP8(*) domain of VP4 forms the spike tips and is proposed to recognize host-cell surface glycans. For sialidase-sensitive rotaviruses such as rhesus (RRV), this recognition involves terminal sialic acids. We show here that the RRV VP8(*)(64-224) protein competes with RRV infection of host cells, demonstrating its relevance to infection. In addition, we observe that the amino acids revealed by X-ray crystallography to be in direct contact with the bound sialic acid derivative methyl alpha-D-N-acetylneuraminide, and that are highly conserved amongst sialidase-sensitive rotaviruses, are residues that are also important in interactions with host-cell carbohydrates. Residues Arg101 and Ser190 of the RRV VP8(*) carbohydrate-binding site were mutated to assess their importance for binding to the sialic acid derivative and their competition with RRV infection of host cells. The crystallographic structure of the Arg(101)Ala mutant crystallized in the presence of the sialic acid derivative was determined at 295 K to a resolution of 1.9 A. Our multidisciplinary study using X-ray crystallography, saturation transfer difference nuclear magnetic resonance spectroscopy, isothermal titration calorimetry, and competitive virus infectivity assays to investigate RRV wild-type and mutant VP8(*) proteins has provided the first evidence that the carbohydrate-binding cavity in RRV VP8(*) is used for host-cell recognition, and this interaction is not only with the sialic acid portion but also with other parts of the glycan structure.

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

    PubMed

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

    2009-12-01

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

  10. Structural aspects of catalytic mechanisms of endonucleases and their binding to nucleic acids

    SciTech Connect

    Zhukhlistova, N. E.; Balaev, V. V.; Lyashenko, A. V.; Lashkov, A. A.

    2012-05-15

    Endonucleases (EC 3.1) are enzymes of the hydrolase class that catalyze the hydrolytic cleavage of deoxyribonucleic and ribonucleic acids at any region of the polynucleotide chain. Endonucleases are widely used both in biotechnological processes and in veterinary medicine as antiviral agents. Medical applications of endonucleases in human cancer therapy hold promise. The results of X-ray diffraction studies of the spatial organization of endonucleases and their complexes and the mechanism of their action are analyzed and generalized. An analysis of the structural studies of this class of enzymes showed that the specific binding of enzymes to nucleic acids is characterized by interactions with nitrogen bases and the nucleotide backbone, whereas the nonspecific binding of enzymes is generally characterized by interactions only with the nucleic-acid backbone. It should be taken into account that the specificity can be modulated by metal ions and certain low-molecular-weight organic compounds. To test the hypotheses about specific and nonspecific nucleic-acid-binding proteins, it is necessary to perform additional studies of atomic-resolution three-dimensional structures of enzyme-nucleic-acid complexes by methods of structural biology.

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

    PubMed Central

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

    2009-01-01

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

  12. Structural aspects of catalytic mechanisms of endonucleases and their binding to nucleic acids

    NASA Astrophysics Data System (ADS)

    Zhukhlistova, N. E.; Balaev, V. V.; Lyashenko, A. V.; Lashkov, A. A.

    2012-05-01

    Endonucleases (EC 3.1) are enzymes of the hydrolase class that catalyze the hydrolytic cleavage of deoxyribonucleic and ribonucleic acids at any region of the polynucleotide chain. Endonucleases are widely used both in biotechnological processes and in veterinary medicine as antiviral agents. Medical applications of endonucleases in human cancer therapy hold promise. The results of X-ray diffraction studies of the spatial organization of endonucleases and their complexes and the mechanism of their action are analyzed and generalized. An analysis of the structural studies of this class of enzymes showed that the specific binding of enzymes to nucleic acids is characterized by interactions with nitrogen bases and the nucleotide backbone, whereas the nonspecific binding of enzymes is generally characterized by interactions only with the nucleic-acid backbone. It should be taken into account that the specificity can be modulated by metal ions and certain low-molecular-weight organic compounds. To test the hypotheses about specific and nonspecific nucleic-acid-binding proteins, it is necessary to perform additional studies of atomic-resolution three-dimensional structures of enzyme-nucleic-acid complexes by methods of structural biology.

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

    PubMed Central

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

    1996-01-01

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

  14. Acid-base and copper-binding properties of three organic matter fractions isolated from a forest floor soil solution

    NASA Astrophysics Data System (ADS)

    van Schaik, Joris W. J.; Kleja, Dan B.; Gustafsson, Jon Petter

    2010-02-01

    Vast amounts of knowledge about the proton- and metal-binding properties of dissolved organic matter (DOM) in natural waters have been obtained in studies on isolated humic and fulvic (hydrophobic) acids. Although macromolecular hydrophilic acids normally make up about one-third of DOM, their proton- and metal-binding properties are poorly known. Here, we investigated the acid-base and Cu-binding properties of the hydrophobic (fulvic) acid fraction and two hydrophilic fractions isolated from a soil solution. Proton titrations revealed a higher total charge for the hydrophilic acid fractions than for the hydrophobic acid fraction. The most hydrophilic fraction appeared to be dominated by weak acid sites, as evidenced by increased slope of the curve of surface charge versus pH at pH values above 6. The titration curves were poorly predicted by both Stockholm Humic Model (SHM) and NICA-Donnan model calculations using generic parameter values, but could be modelled accurately after optimisation of the proton-binding parameters (pH ⩽ 9). Cu-binding isotherms for the three fractions were determined at pH values of 4, 6 and 9. With the optimised proton-binding parameters, the SHM model predictions for Cu binding improved, whereas the NICA-Donnan predictions deteriorated. After optimisation of Cu-binding parameters, both models described the experimental data satisfactorily. Iron(III) and aluminium competed strongly with Cu for binding sites at both pH 4 and pH 6. The SHM model predicted this competition reasonably well, but the NICA-Donnan model underestimated the effects significantly at pH 6. Overall, the Cu-binding behaviour of the two hydrophilic acid fractions was very similar to that of the hydrophobic acid fraction, despite the differences observed in proton-binding characteristics. These results show that for modelling purposes, it is essential to include the hydrophilic acid fraction in the pool of 'active' humic substances.

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

    PubMed

    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.

  16. Thermodynamics of the enantiomers of amino acid and monosaccharide binding to fullerenol used as an artificial sweet taste receptor model.

    PubMed

    Dong, Wen-Rui; Chen, Guo; Chen, Zhong-Xiu; Deng, Shao-Ping

    2013-12-01

    Fullerenol was used as sweet taste receptor model to investigate the binding affinities of structurally related pairs of enantiomers by isothermal titration calorimetry (ITC). It reveals that amino acid binding with fullerenol are enthalpy-cost and entropically-driven processes, whereas enthalpy contributes to monosaccharide binding to fullerenol. Spontaneous binding of amino acids was found through two sequential steps in which the sweeter enantiomer displays larger binding constants. Association of the d-form of fructose and l-form galactose with fullerenol suggested that, the higher the perceived sweetness intensity of the enantiomer, the larger was the binding constant with respect to their antipodes. Further investigation by molecular dynamic simulation showed that the binding energy and the perceived sweetness intensity were well correlated. The preliminary results of this biomimetic research cover the lack of information about the thermodynamic basis of sweet sensation and the underlying principles of sweetness differences between the enantiomers of amino acids and monosaccharides.

  17. Observation of multiple, identical binding sites in the exchange of carboxylic acid ligands with CdS nanocrystals.

    PubMed

    Li, Xin; Nichols, Valerie M; Zhou, Dapeng; Lim, Cynthia; Pau, George Shu Heng; Bardeen, Christopher J; Tang, Ming L

    2014-06-11

    We study ligand exchange between the carboxylic acid group and 5.0 nm oleic-acid capped CdS nanocrystals (NCs) using fluorescence resonance energy transfer (FRET). This is the first measurement of the initial binding events between cadmium chalcogenide NCs and carboxylic acid groups. The binding behavior can be described as an interaction between a ligand with single binding group and a substrate with multiple, identical binding sites. Assuming Poissonian binding statistics, our model fits both steady-state and time-resolved photoluminescence (SSPL and TRPL, respectively) data well. A modified Langmuir isotherm reveals that a CdS nanoparticle has an average of 3.0 new carboxylic acid ligands and binding constant, Ka, of 3.4 × 10(5) M(-1).

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bile acid binding potential of foods and food fractions has been related to lowering the risk of heart disease and that of cancer. Sautéing or steam cooking has been observed to significantly improve bile acid binding of green/leafy vegetables. It was hypothesized that microwave cooking could impr...

  19. Ferulic Acid Administered at Various Time Points Protects against Cerebral Infarction by Activating p38 MAPK/p90RSK/CREB/Bcl-2 Anti-Apoptotic Signaling in the Subacute Phase of Cerebral Ischemia-Reperfusion Injury in Rats

    PubMed Central

    Cheng, Chin-Yi; Tang, Nou-Ying; Kao, Shung-Te; Hsieh, Ching-Liang

    2016-01-01

    Objectives This study aimed to evaluate the effects of ferulic acid (FA) administered at various time points before or after 30 min of middle cerebral artery occlusion (MCAo) followed by 7 d of reperfusion and to examine the involvement of mitogen-activated protein kinase (MAPK) signaling pathways in the cortical penumbra. Methods FA was intravenously administered to rats at a dose of 100 mg/kg 24 h before ischemia (B-FA), 2 h before ischemia (P-FA), immediately after ischemic insult (I-FA), 2 h after reperfusion (R-FA), or 24 h after reperfusion (D-FA). Results Our study results indicated that P-FA, I-FA, and R-FA effectively reduced cerebral infarct areas and neurological deficits. P-FA, I-FA, and R-FA significantly downregulated glial fibrillary acidic protein (GFAP), mitochondrial Bax, cytochrome c, and cleaved caspase-3 expression, and effectively restored the phospho-p38 MAPK (p-p38 MAPK)/p38 MAPK ratio, phospho-90 kDa ribosomal S6 kinase (p-p90RSK) expression, phospho-Bad (p-Bad) expression, the phospho-cAMP response element-binding protein (p-CREB)/CREB ratio, the cytosolic and mitochondrial Bcl-2/Bax ratios, and the cytosolic Bcl-xL/Bax ratio in the cortical penumbra 7 d after reperfusion. SB203580, a specific inhibitor of p38 MAPK, administered 30 min prior to ischemia abrogated the downregulating effects of I-FA on cerebral infarction, and mitochondrial Bax and cleaved caspase-3 expression, and the upregulating effects of I-FA on the p-p38 MAPK/p38 MAPK ratio, p-p90RSK expression, p-Bad expression, and the p-CREB/CREB, and cytosolic and mitochondrial Bcl-2/Bax ratios. Conclusions Our study results thus indicate that P-FA, I-FA, and R-FA effectively suppress reactive astrocytosis and exert neuroprotective effects against cerebral infarction by activating p38 MAPK signaling. The regulating effects of P-FA, I-FA, and R-FA on Bax-induced apoptosis result from activation of the p38 MAPK/p90RSK/CREB/Bcl-2 signaling pathway, and eventually contribute to

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

    PubMed

    An, Jieun; Kwon, Hyeji; Kim, Eunjung; Lee, Young Mi; Ko, Hyeok Jin; Park, Hongjae; Choi, In-Geol; Kim, Sooah; Kim, Kyoung Heon; Kim, Wankee; Choi, Wonja

    2015-03-01

    Screening a library of overexpressing mutant alleles of the TATA-binding gene SPT15 yielded two Saccharomyces cerevisiae strains (MRRC 3252 and 3253) with enhanced tolerance to acetic acid. They were also tolerant to propionic acid and hydrogen peroxide. Transcriptome profile analysis identified 58 upregulated genes and 106 downregulated genes in MRRC 3252. Stress- and protein synthesis-related transcription factors were predominantly enriched in the upregulated and downregulated genes respectively. Eight deletion mutants for some of the highly downregulated genes were acetic acid-tolerant. The level of intracellular reactive oxygen species was considerably lessened in MRRC 3252 and 3253 upon exposure to acetic acid. Metabolome profile analysis revealed that intracellular concentrations of 5 and 102 metabolites were increased and decreased, respectively, in MRRC 3252, featuring a large increase of urea and a significant decrease of amino acids. The dur1/2Δmutant, in which the urea degradation gene DUR1/2 is deleted, displayed enhanced tolerance to acetic acid. Enhanced tolerance to acetic acid was also observed on the medium containing a low concentration of amino acids. Taken together, this study identified two SPT15 alleles, nine gene deletions and low concentration of amino acids in the medium that confer enhanced tolerance to acetic acid.

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

    PubMed

    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 (P<0.001), asthenozoospermic (P<0.001) and oligoasthenozoospermic (P<0.001) groups. Disomy of sex 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 (P<0.001) between sperm concentration and HA binding (r=0.658), between sperm concentration and estimated numerical chromosome 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

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

    PubMed Central

    Petukh, Marharyta; Dai, Luogeng; Alexov, Emil

    2016-01-01

    Predicting the effect of amino acid substitutions on protein–protein affinity (typically evaluated via the change of protein binding free energy) is important for both understanding the disease-causing mechanism of missense mutations and guiding protein engineering. In addition, researchers are also interested in understanding which energy components are mostly affected by the mutation and how the mutation affects the overall structure of the corresponding protein. Here we report a webserver, the Single Amino Acid Mutation based change in Binding free Energy (SAAMBE) webserver, which addresses the demand for tools for predicting the change of protein binding free energy. SAAMBE is an easy to use webserver, which only requires that a coordinate file be inputted and the user is provided with various, but easy to navigate, options. The user specifies the mutation position, wild type residue and type of mutation to be made. The server predicts the binding free energy change, the changes of the corresponding energy components and provides the energy minimized 3D structure of the wild type and mutant proteins for download. The SAAMBE protocol performance was tested by benchmarking the predictions against over 1300 experimentally determined changes of binding free energy and a Pearson correlation coefficient of 0.62 was obtained. How the predictions can be used for discriminating disease-causing from harmless mutations is discussed. The webserver can be accessed via http://compbio.clemson.edu/saambe_webserver/. PMID:27077847

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

    PubMed

    Petukh, Marharyta; Dai, Luogeng; Alexov, Emil

    2016-04-12

    Predicting the effect of amino acid substitutions on protein-protein affinity (typically evaluated via the change of protein binding free energy) is important for both understanding the disease-causing mechanism of missense mutations and guiding protein engineering. In addition, researchers are also interested in understanding which energy components are mostly affected by the mutation and how the mutation affects the overall structure of the corresponding protein. Here we report a webserver, the Single Amino Acid Mutation based change in Binding free Energy (SAAMBE) webserver, which addresses the demand for tools for predicting the change of protein binding free energy. SAAMBE is an easy to use webserver, which only requires that a coordinate file be inputted and the user is provided with various, but easy to navigate, options. The user specifies the mutation position, wild type residue and type of mutation to be made. The server predicts the binding free energy change, the changes of the corresponding energy components and provides the energy minimized 3D structure of the wild type and mutant proteins for download. The SAAMBE protocol performance was tested by benchmarking the predictions against over 1300 experimentally determined changes of binding free energy and a Pearson correlation coefficient of 0.62 was obtained. How the predictions can be used for discriminating disease-causing from harmless mutations is discussed. The webserver can be accessed via http://compbio.clemson.edu/saambe_webserver/.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    D-amino acids (D-aas) are reported to bind to IgE antibodies from people with allergy and asthma. The objectives of this study were to determine if D-aas bind or inhibit IgE binding to peanut allergens, and if they are more effective than L-amino acids (L-aas) in this respect. Several D-aa cocktails...

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

    PubMed

    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)

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

    PubMed

    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.

  7. Solution structure of the single-stranded DNA binding protein of the filamentous Pseudomonas phage Pf3: similarity to other proteins binding to single-stranded nucleic acids.

    PubMed Central

    Folmer, R H; Nilges, M; Konings, R N; Hilbers, C W

    1995-01-01

    The three-dimensional structure of the homodimeric single-stranded DNA binding protein encoded by the filamentous Pseudomonas bacteriophage Pf3 has been determined using heteronuclear multidimensional NMR techniques and restrained molecular dynamics. NMR experiments and structure calculations have been performed on a mutant protein (Phe36 --> His) that was successfully designed to reduce the tendency of the protein to aggregate. The protein monomer is composed of a five-stranded antiparallel beta-sheet from which two beta-hairpins and a large loop protrude. The structure is compared with the single-stranded DNA binding protein encoded by the filamentous Escherichia coli phage Ff, a protein with a similar biological function and DNA binding properties, yet quite different amino acid sequence, and with the major cold shock protein of Escherichia coli, a single-stranded DNA binding protein with an entirely different sequence, biological function and binding characteristics. The amino acid sequence of the latter is highly homologous to the nucleic acid binding domain (i.e. the cold shock domain) of proteins belonging to the Y-box family. Despite their differences in amino acid sequence and function, the folds of the three proteins are remarkably similar, suggesting that this is a preferred folding pattern shared by many single-stranded DNA binding proteins. Images PMID:7556054

  8. Fatty acid composition and desaturase gene expression in flax (Linum usitatissimum L.).

    PubMed

    Thambugala, Dinushika; Cloutier, Sylvie

    2014-11-01

    Little is known about the relationship between expression levels of fatty acid desaturase genes during seed development and fatty acid (FA) composition in flax. In the present study, we looked at promoter structural variations of six FA desaturase genes and their relative expression throughout seed development. Computational analysis of the nucleotide sequences of the sad1, sad2, fad2a, fad2b, fad3a and fad3b promoters showed several basic transcriptional elements including CAAT and TATA boxes, and several putative target-binding sites for transcription factors, which have been reported to be involved in the regulation of lipid metabolism. Using semi-quantitative reverse transcriptase PCR, the expression patterns throughout seed development of the six FA desaturase genes were measured in six flax genotypes that differed for FA composition but that carried the same desaturase isoforms. FA composition data were determined by phenotyping the field grown genotypes over four years in two environments. All six genes displayed a bell-shaped pattern of expression peaking at 20 or 24 days after anthesis. Sad2 was the most highly expressed. The expression of all six desaturase genes did not differ significantly between genotypes (P = 0.1400), hence there were no correlations between FA desaturase gene expression and variations in FA composition in relatively low, intermediate and high linolenic acid genotypes expressing identical isoforms for all six desaturases. These results provide further clues towards understanding the genetic factors responsible for FA composition in flax.

  9. Locations of the three primary binding sites for long-chain fatty acids on bovine serum albumin

    SciTech Connect

    Hamilton, J.A.; Era, S.; Bhamidipati, S.P. ); Reed, R.G. )

    1991-03-15

    Binding of {sup 13}C-enriched oleic acid to bovine serum albumin and to three large proteolytic fragments of albumin - two complementary fragments corresponding to the two halved of albumin and one fragment corresponding to the carboxyl-terminal domain - yielded unique patterns of NMR resonances (chemical shifts and relative intensities) that were used to identify the locations of binding of the first 5 mol of oleic acid to the multidomain albumin molecule. The first 3 mol of oleic acid added to intact albumin generated three distinct NMR resonances as a result of simultaneous binding of oleic acid to three heterogeneous sites (primary sites). This distribution suggests albumin to be a less symmetrical binding molecule than theoretical models predict. This work also demonstrates the power of NMR for the study of microenvironments of individual fatty acid binding sites in specific domain.

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

    PubMed

    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.

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

    PubMed

    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. PMID:26774272

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

    PubMed

    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.

  13. Structural determinants of human APOBEC3A enzymatic and nucleic acid binding properties

    PubMed Central

    Mitra, Mithun; Hercík, Kamil; Byeon, In-Ja L.; Ahn, Jinwoo; Hill, Shawn; Hinchee-Rodriguez, Kathyrn; Singer, Dustin; Byeon, Chang-Hyeock; Charlton, Lisa M.; Nam, Gabriel; Heidecker, Gisela; Gronenborn, Angela M.; Levin, Judith G.

    2014-01-01

    Human APOBEC3A (A3A) is a single-domain cytidine deaminase that converts deoxycytidine residues to deoxyuridine in single-stranded DNA (ssDNA). It inhibits a wide range of viruses and endogenous retroelements such as LINE-1, but it can also edit genomic DNA, which may play a role in carcinogenesis. Here, we extend our recent findings on the NMR structure of A3A and report structural, biochemical and cell-based mutagenesis studies to further characterize A3A’s deaminase and nucleic acid binding activities. We find that A3A binds ssRNA, but the RNA and DNA binding interfaces differ and no deamination of ssRNA is detected. Surprisingly, with only one exception (G105A), alanine substitution mutants with changes in residues affected by specific ssDNA binding retain deaminase activity. Furthermore, A3A binds and deaminates ssDNA in a length-dependent manner. Using catalytically active and inactive A3A mutants, we show that the determinants of A3A deaminase activity and anti-LINE-1 activity are not the same. Finally, we demonstrate A3A’s potential to mutate genomic DNA during transient strand separation and show that this process could be counteracted by ssDNA binding proteins. Taken together, our studies provide new insights into the molecular properties of A3A and its role in multiple cellular and antiviral functions. PMID:24163103

  14. Amino-terminal basic residues of Src mediate membrane binding through electrostatic interaction with acidic phospholipids.

    PubMed Central

    Sigal, C T; Zhou, W; Buser, C A; McLaughlin, S; Resh, M D

    1994-01-01

    Membrane targeting of pp60src (Src) is mediated by its myristoylated amino terminus. We demonstrate that, in addition to myristate, six basic residues in the amino terminus are essential for high-affinity binding to the lipid bilayer via electrostatic interaction with acidic phospholipids. Specifically, c-Src was shown to bind 2500-fold more strongly to vesicles composed of the physiological ratio of 2:1 phosphatidylcholine (PC)/phosphatidylserine (PS) than to neutral PC bilayer vesicles. The apparent Kd for binding of c-Src to the PC/PS bilayer was 6 x 10(-7) M. This interaction is sufficiently strong to account for c-Src membrane targeting. Mutants of c-Src in which the amino-terminal basic residues were replaced by neutral asparagine residues exhibited binding isotherms approaching that of wild-type binding to neutral bilayers (apparent Kd of 2 x 10(-3) M). The transforming v-Src and activated c-Src (Y527F) proteins also bound more strongly to PC/PS bilayers (apparent Kd of approximately 1 x 10(-5) M) than to neutral PC bilayers. In vivo experiments with Src mutants confirmed the role of positive charge in mediating membrane binding and cellular transformation. Images PMID:7527558

  15. Polymerization and nucleic acid-binding properties of human L1 ORF1 protein.

    PubMed

    Callahan, Kathryn E; Hickman, Alison B; Jones, Charles E; Ghirlando, Rodolfo; Furano, Anthony V

    2012-01-01

    The L1 (LINE 1) retrotransposable element encodes two proteins, ORF1p and ORF2p. ORF2p is the L1 replicase, but the role of ORF1p is unknown. Mouse ORF1p, a coiled-coil-mediated trimer of ∼42-kDa monomers, binds nucleic acids and has nucleic acid chaperone activity. We purified human L1 ORF1p expressed in insect cells and made two findings that significantly advance our knowledge of the protein. First, in the absence of nucleic acids, the protein polymerizes under the very conditions (0.05 M NaCl) that are optimal for high (∼1 nM)-affinity nucleic acid binding. The non-coiled-coil C-terminal half mediates formation of the polymer, an active conformer that is instantly resolved to trimers, or multimers thereof, by nucleic acid. Second, the protein has a biphasic effect on mismatched double-stranded DNA, a proxy chaperone substrate. It protects the duplex from dissociation at 37°C before eventually melting it when largely polymeric. Therefore, polymerization of ORF1p seemingly affects its interaction with nucleic acids. Additionally, polymerization of ORF1p at its translation site could explain the heretofore-inexplicable phenomenon of cis preference-the favored retrotransposition of the actively translated L1 transcript, which is essential for L1 survival. PMID:21937507

  16. Molecular dynamics simulation of ligand dissociation from liver fatty acid binding protein.

    PubMed

    Long, Dong; Mu, Yuguang; Yang, Daiwen

    2009-06-30

    The mechanisms of how ligands enter and leave the binding cavity of fatty acid binding proteins (FABPs) have been a puzzling question over decades. Liver fatty acid binding protein (LFABP) is a unique family member which accommodates two molecules of fatty acids in its cavity and exhibits the capability of interacting with a variety of ligands with different chemical structures and properties. Investigating the ligand dissociation processes of LFABP is thus a quite interesting topic, which however is rather difficult for both experimental approaches and ordinary simulation strategies. In the current study, random expulsion molecular dynamics simulation, which accelerates ligand motions for rapid dissociation, was used to explore the potential egress routes of ligands from LFABP. The results showed that the previously hypothesized "portal region" could be readily used for the dissociation of ligands at both the low affinity site and the high affinity site. Besides, one alternative portal was shown to be highly favorable for ligand egress from the high affinity site and be related to the unique structural feature of LFABP. This result lends strong support to the hypothesis from the previous NMR exchange studies, which in turn indicates an important role for this alternative portal. Another less favored potential portal located near the N-terminal end was also identified. Identification of the dissociation pathways will allow further mechanistic understanding of fatty acid uptake and release by computational and/or experimental techniques.

  17. Binding of /sup 14/C-5-aminolevulinic acid to a stromal protein from developing pea chloroplasts

    SciTech Connect

    Thayer, S.S.; Castelfranco, P.A.; Wilkinson, J.; Benson, G.

    1987-04-01

    /sup 14/-5-Aminolevulinic acid (/sup 14/C-ALA) binds to a stromal protein with an apparent molecular weight of 42-43 KD on LDS and non-denaturing gels. The reaction is rapid. Binding is inhibited by sulfhydryl reagents, mM concentrations of levulinic, dihydroxy heptanoic acids and gabaculine, 10 ..mu..M N-methylprotoporphyrin. Dicarboxilic acids, such as deltaKG, Glu, OAA, do not inhibit. Chloramphenicol, ATP, protoporphyrin, anoxia, light, darkness have no effect. The product, once formed, is stable to treatment with 5% conc. HCl in cold acetone. It can be chased in a second incubation with unlabeled ALA, but not with levulinic acid. No activity was detected in the subplastidic membrane fractions. Western blot analysis failed to reveal any homology between the labeled protein and either cytochrome for ALA dehydratase. This ALA-binding protein was not formed in chloroplasts isolated from fully expanded pea leaves. Therefore, it is deemed likely to participate in ALA metabolism during chloroplast development.

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

    PubMed

    Bhowmik, Debipreeta; Kumar, Gopinatha Suresh

    2016-01-01

    Berberine is one of the most widely known alkaloids belonging to the protoberberine group exhibiting myriad therapeutic properties. The anticancer potency of berberine appears to derive from its multiple actions including strong interaction with nucleic acids exhibiting adenine-thymine base pair specificity, inhibition of the enzymes topoisomerases and telomerases, and stabilizing the quadruplex structures. It was realized that the development of berberine as a potential anticancer agent necessitates enhancing its nucleic acid binding efficacy through appropriate structural modifications. More recently a number of such approaches have been attempted in various laboratories with great success. Several derivatives have been synthesized mostly with substitutions at the 8, 9 and 13 positions of the isoquinoline chromophore, and studied for enhanced nucleic acid binding activity. In this article, we present an up to date review of the details of the interaction of berberine and several of its important synthetic 8, 9 and 13 substituted derivatives with various nucleic acid structures reported recently. These studies provide interesting knowledge on the mode, mechanism, sequence and structural specificity of the binding of berberine derivatives and correlate structural and energetic aspects of the interaction providing better understanding of the structure- activity relations for designing and development of berberine based therapeutic agents with higher efficacy and therapeutic potential.

  19. Tannic acid inhibited norovirus binding to HBGA receptors, a study of 50 Chinese medicinal herbs.

    PubMed

    Zhang, Xu-Fu; Dai, Ying-Chun; Zhong, Weiming; Tan, Ming; Lv, Zhi-Ping; Zhou, Ying-Chun; Jiang, Xi

    2012-02-15

    Noroviruses (NoVs) are the leading cause of viral acute gastroenteritis affecting people of all ages worldwide. The disease is difficult to control due to its widespread nature and lack of an antiviral or vaccine. NoV infection relies on the interaction of the viruses with histo-blood group antigens (HBGAs) as host receptors. Here we investigated inhibition effects of Chinese medicinal herbs against NoVs binding to HBGAs for potential antivirals against NoVs. Blocking assays was performed using the NoV protrusion (P) protein as NoV surrogate and saliva as HBGAs. Among 50 clinically effective Chinese medicinal herbs against gastroenteritis diseases, two herbs were found highly effective. Chinese Gall blocked NoV P dimer binding to type A saliva at IC(50)=5.35 μg/ml and to B saliva at IC(50)=21.7 μg/ml. Similarly, Pomegranate blocked binding of NoV P dimer to type A saliva at IC(50)=15.59 μg/ml and B saliva at IC(50)=66.67 μg/ml. Literature data on preliminary biochemistry analysis showed that tannic acid is a common composition in the extracts of the two herbs, so we speculate that it might be the effective compound and further studies using commercially available, highly purified tannic acid confirmed the tannic acid as a strong inhibitor in the binding of NoV P protein to both A and B saliva (IC(50)≈0.1 μM). In addition, we tested different forms of hydrolysable tannins with different alkyl esters, including gallic acid, ethyl gallate, lauryl gallate, octyl gallate and propyl gallate. However, none of these tannins-derivatives revealed detectable inhibiting activities. Our data suggested that tannic acid is a promising candidate antiviral against NoVs.

  20. The cathepsin B inhibitor, z-FA-CMK is toxic and readily induced cell death in human T lymphocytes

    SciTech Connect

    Liow, K.Y.; Chow, S.C.

    2013-11-01

    The cathepsin B inhibitor, benzyloxycarbonyl-phenylalanine-alanine-chloromethylketone (z-FA-CMK) was found to be toxic and readily induced cell death in the human T cell line, Jurkat, whereas two other analogs benzyloxycarbonyl-phenylalanine-alanine-fluoromethylketone (z-FA-FMK) and benzyloxycarbonyl-phenylalanine-alanine-diazomethylketone (z-FA-DMK) were not toxic. The toxicity of z-FA-CMK requires not only the CMK group, but also the presence of alanine in the P1 position and the benzyloxycarbonyl group at the N-terminal. Dose–response studies showed that lower concentrations of z-FA-CMK induced apoptosis in Jurkat T cells whereas higher concentrations induced necrosis. In z-FA-CMK-induced apoptosis, both initiator caspases (-8 and -9) and effector caspases (-3, -6 and -7) were processed to their respective subunits in Jurkat T cells. However, only the pro-form of the initiator caspases were reduced in z-FA-CMK-induced necrosis and no respective subunits were apparent. The caspase inihibitor benzyloxycarbonyl-valine-alanine-aspartic acid-(O-methyl)-fluoromehylketone (z-VAD-FMK) inhibits apoptosis and caspase processing in Jurkat T cells treated with low concentration of z-FA-CMK but has no effect on z-FA-CMK-induced necrosis and the loss of initiator caspases. This suggests that the loss of initiator caspases in Jurkat T cells during z-FA-CMK-induced necrosis is not a caspase-dependent process. Taken together, we have demonstrated that z-FA-CMK is toxic to Jurkat T cells and induces apoptosis at low concentrations, while at higher concentrations the cells die of necrosis. - Highlights: • z-FA-CMK is toxic and induce cell death in the human T cells. • z-FA-CMK toxicity requires the CMK group, alanine and the benzyloxycarbonyl group. • z-FA-CMK induced apoptosis at low concentration and necrosis at high concentration.

  1. Purification and characterization of a proteinase from pineapple fruit, fruit bromelain FA2.

    PubMed

    Yamada, F; Takahashi, N; Murachi, T

    1976-06-01

    Fruit bromelain FA2, the main proteinase component of the juice of pineapple fruit, has been purified and characterized. 1. Efficient extraction of this enzyme from the crude material was possible using "Cellulosin AP," a microbial polysaccharidase preparation containing cellulase, hemicellulase, and pectinase. The enzyme was purified mainly by successive applications of anion-exchange chromatography, yielding an apparently homogeneous protein as judged by several physical, chemical, and immunochemical criteria. Properties of FA2 include: molecular weight, 31,000; isoelectric point, pH 4.6; absorbance at 280 nm of a 1% solution at pH 7.0 per cm, 19.2. 2. FA2 gave only alanine phenylthiohydantoin upon amino-terminal group analysis by the Edman procedure. Stepwise degradation yielded the amino-terminal sequence Ala-Val-Pro-Gln-Ser-Ile-Asp-Trp-Arg-Asp-Tyr-Gly-Ala. The amino acid composition of FA2 was not markedly different from that of stem bromelain, except for a much smaller lysine content and a smaller alanine content relative to glycine in FA2. FA2 contained neither amino sugars nor neutral carbohydrates as determined by several methods, so FA2 is not a glycoprotein. 3. By labeling the reactive cysteine residue (CYS) with [14C]iodoacetate, the following partial amino acid sequence has been determined. Asn-Glx-Asn-Pro-Cys-Gly-Ala-CYS.

  2. Displacement of specific serotonin and lysergic acid diethylamide binding by Ergalgin, a new antiserotonin drug.

    PubMed

    Oelszner, W

    1980-01-01

    [3H]-serotonin and [3H]-lysergic acid diethylamide (LSD) bind with a high affinity, KD = 12 nM and 6 nM, respectively, to distinct receptors of rat caudate membranes in vitro. Displacement experiments with unlabeled serotonin and LSD support the hypothesis of serotonin receptors existing in an agonist and antagonist state. Methysergide and Ergalgin display quite similar potencies in displacing [3H]-serotonin and [3H]-LSD from their specific binding sites (Ki = 46,7 and 53,4 nM; 22,3 and 36,5 nM, respectively). Contrary to pharmacological findings these binding results are in favour of mixed agonist/antagonist properties of these compounds.

  3. A report on emergent uranyl binding phenomena by an amidoxime phosphonic acid co-polymer.

    PubMed

    Abney, C W; Das, S; Mayes, R T; Kuo, L-J; Wood, J; Gill, G; Piechowicz, M; Lin, Z; Lin, W; Dai, S

    2016-09-14

    The development of technology to harvest the uranium dissolved in seawater would enable access to vast quantities of this critical metal for nuclear power generation. Amidoxime polymers are the most promising platforms for achieving this separation, yet the design of advanced adsorbents is hindered by uncertainty regarding the uranium binding mode. In this work we use XAFS to investigate the uranium coordination environment in an amidoxime-phosphonic acid copolymer adsorbent. In contrast to the binding mode predicted computationally and from small molecule studies, a cooperative chelating model is favoured, attributable to emergent behavior resulting from inclusion of amidoxime in the polymer. Samples exposed to seawater also display a feature consistent with a μ(2)-oxo-bridged transition metal, suggesting the formation of an in situ specific binding site. These findings challenge long held assumptions and provide new opportunities for the design of advanced adsorbent materials. PMID:27507226

  4. A report on emergent uranyl binding phenomena by an amidoxime phosphonic acid co-polymer

    DOE PAGES

    Abney, C. W.; Das, S.; Mayes, R. T.; Kuo, L. -J.; Wood, J.; Gill, G.; Piechowicz, M.; Lin, Z.; Lin, W.; Dai, S.

    2016-08-01

    Development of technology to harvest the uranium dissolved in seawater would enable access to vast quantities of this critical metal for nuclear power generation. Amidoxime polymers are the most promising platform for achieving this separation, yet design of advanced adsorbents is hindered by uncertainty regarding the uranium binding mode. In this work we use XAFS to investigate the uranium coordination environment in an amidoxime-phosphonic acid copolymer adsorbent. In contrast to the binding mode predicted computationally and from small molecule studies, a cooperative chelating model is favoured, attributable to emergent behavior resulting from inclusion of amidoxime in a polymer. Samples exposedmore » to seawater also display a feature consistent with a 2-oxo-bridged transition metal, suggesting formation of an in situ specific binding site. As a result, these findings challenge long held assumptions and provide new opportunities for the design of advanced adsorbent materials.« less

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

    PubMed

    Kiani, Amir; Almasi, Khadijeh; Shokoohinia, Yalda; Sadrjavadi, Komail; Nowroozi, Amin; Shahlaei, Mohsen

    2015-11-01

    The molecular mechanism of galbanic acid (GBA) binding to matrix metalloproteinase 9 (MMP9) was investigated by fluorescence quenching, absorption spectroscopy, FT-IR, molecular docking and molecular dynamics (MD) simulation procedures. The fluorescence emission of MMP9 was quenched by GBA. The titration of MMP9 by various amount of GBA was also followed by UV-Vis absorption spectroscopy. The results revealed that GBA, as a biologically active sesquiterpene coumarin derivative, has an ability to bind strongly to MMP9. Molecular docking results indicated that the main active binding site for GBA has been located in a hydrophobic cavity in the vicinity of Zn atom. Moreover, MD simulation results suggested that GBA as a coumarin derivative can interact with MMP9, without affecting the secondary structure of MMP9. MD simulations, molecular docking as computational methods from one hand and experimental data from other hand reciprocally supported each other.

  6. BEDAM Binding Free Energy Predictions for the SAMPL4 Octa-Acid Host Challenge

    PubMed Central

    Gallicchio, Emilio; Chen, Haoyuan; Chen, He; Fitzgerald, Michael; Gao, Yang; He, Peng; Kalyanikar, Malathi; Kao, Chuan; Lu, Beidi; Niu, Yijie; Pethe, Manasi; Zhu, Jie; Levy, Ronald M.

    2015-01-01

    The Binding Energy Distribution Analysis Method (BEDAM) protocol has been employed as part of the SAMPL4 blind challenge to predict the binding free energies of a set of octa-acid host-guest complexes. The resulting predictions were consistently judged as some of the most accurate predictions in this category of the SAMPL4 challenge in terms of quantitative accuracy and statistical correlation relative to the experimental values, which were not known at the time the predictions were made. The work has been conducted as part of a hands-on graduate class laboratory session. Collectively the students, aided by automated setup and analysis tools, performed the bulk of the calculations and the numerical and structural analysis. The success of the experiment confirms the reliability of the BEDAM methodology and it shows that physics-based atomistic binding free energy estimation models, when properly streamlined and automated, can be successfully employed by non-specialists. PMID:25726024

  7. Zinc-Mediated Binding of Nucleic Acids to Amyloid-β Aggregates: Role of Histidine Residues.

    PubMed

    Khmeleva, Svetlana A; Radko, Sergey P; Kozin, Sergey A; Kiseleva, Yana Y; Mezentsev, Yuri V; Mitkevich, Vladimir A; Kurbatov, Leonid K; Ivanov, Alexis S; Makarov, Alexander A

    2016-09-01

    Amyloid-β peptide (Aβ) plays a central role in Alzheimer's disease (AD) pathogenesis. Besides extracellular Aβ, intraneuronal Aβ (iAβ) has been suggested to contribute to AD onset and development. Based on reported in vitro Aβ-DNA interactions and nuclear localization of iAβ, the interference of iAβ with the normal DNA expression has recently been proposed as a plausible pathway by which Aβ can exert neurotoxicity. Employing the sedimentation assay, thioflavin T fluorescence, and dynamic light scattering we have studied effects of zinc ions on binding of RNA and single- and double-stranded DNA molecules to Aβ42 aggregates. It has been found that zinc ions significantly enhance the binding of RNA and DNA molecules to pre-formed β-sheet rich Aβ42 aggregates. Another type of Aβ42 aggregates, the zinc-induced amorphous aggregates, was demonstrated to also bind all types of nucleic acids tested. To evaluate the role of the Aβ metal-binding domain's histidine residues in Aβ-nucleic acid interactions mediated by zinc, Aβ16 mutants with substitutions H6R and H6A-H13A and rat Aβ16 lacking histidine residue 13 were used. The zinc-induced interaction of Aβ16 with DNA was shown to critically depend on histidine residues 6 and 13. However, the inclusion of H6R mutation in Aβ42 peptide did not affect DNA binding to Aβ42 aggregates. Since oxidative and/or nitrosative stresses implicated in AD pathogenesis are known to release zinc ions from metallothioneins in cytoplasm and cell nuclei, our findings suggest that intracellular zinc can be an important player in iAβ-nucleic acid interactions. PMID:27567853

  8. Binding of polyphenols to plant cell wall analogues - Part 2: Phenolic acids.

    PubMed

    Padayachee, A; Netzel, G; Netzel, M; Day, L; Zabaras, D; Mikkelsen, D; Gidley, M J

    2012-12-15

    Bacterial cellulose and cellulose-pectin composites were used as well-defined model plant cell wall (PCW) systems to study the interaction between phenolic acids (PA) derived from purple carrot juice concentrate (PCJC) and PCW components. Significant PA depletion from solution occurred, with pure cellulose initially (30s-1h) absorbing more than cellulose-pectin composites in the first hour (ca 20% cf 10-15%), but with all composites absorbing similar levels (ca 30%) after several days. Individual PAs bound to different relative extents with caffeic acid>chlorogenic acid>ferulic acid. Extrapolation of data for these model systems to carrot puree suggests that nutritionally-significant amounts of PAs could bind to cell walls, potentially restricting bioavailability in the small intestine and, as a consequence, delivering PAs to the large intestine for fermentation and metabolism by gut bacteria.

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

    PubMed

    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. PMID:26345909

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

    PubMed

    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.

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

    PubMed Central

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

    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 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. PMID:22247133

  12. Deciphering the binding patterns and conformation changes upon the bovine serum albumin-rosmarinic acid complex.

    PubMed

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

    2015-08-01

    Rosmarinic acid (RA) is an importantly and naturally occurring polyphenol from plants of the mint family with potent biological activities. Here, the in vitro interaction of RA with bovine serum albumin (BSA) has been investigated using various biophysical approaches as well as molecular modeling methods, to ascertain its binding mechanism and conformational changes. The fluorescence results demonstrated that the fluorescence quenching of BSA by RA was mainly the result of the formation of a ground state BSA-RA complex, and BSA had one high affinity RA binding site with a binding constant of 4.18 × 10(4) mol L(-1) at 298 K. Analysis of thermodynamic parameters revealed that hydrophobic and hydrogen bond interactions were the dominant intermolecular force in the complex formation. The primary binding site of RA in BSA (site I) had been identified by site marker competitive experiments. The distance between RA and the tryptophan residue of BSA was evaluated at 3.12 nm based on Förster's theory of non-radiation energy transfer. The UV-vis absorption, synchronous fluorescence, three-dimensional fluorescence, 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescence, circular dichroism (CD), and Fourier transform infrared (FT-IR) spectra confirmed that the conformation and structure of BSA were altered in the presence of RA. Moreover, the nuclear magnetic spectroscopy showed that the aromatic groups of RA took part in the binding reaction during the BSA-RA complexation. In addition, the molecular picture of the interaction mechanism between BSA and RA at the atomic level was well examined by molecular docking and dynamics studies. In brief, RA can bind to BSA with noncovalent bonds in a relatively stable way, and these findings will be beneficial to the functional food research of RA.

  13. Deciphering the binding patterns and conformation changes upon the bovine serum albumin-rosmarinic acid complex.

    PubMed

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

    2015-08-01

    Rosmarinic acid (RA) is an importantly and naturally occurring polyphenol from plants of the mint family with potent biological activities. Here, the in vitro interaction of RA with bovine serum albumin (BSA) has been investigated using various biophysical approaches as well as molecular modeling methods, to ascertain its binding mechanism and conformational changes. The fluorescence results demonstrated that the fluorescence quenching of BSA by RA was mainly the result of the formation of a ground state BSA-RA complex, and BSA had one high affinity RA binding site with a binding constant of 4.18 × 10(4) mol L(-1) at 298 K. Analysis of thermodynamic parameters revealed that hydrophobic and hydrogen bond interactions were the dominant intermolecular force in the complex formation. The primary binding site of RA in BSA (site I) had been identified by site marker competitive experiments. The distance between RA and the tryptophan residue of BSA was evaluated at 3.12 nm based on Förster's theory of non-radiation energy transfer. The UV-vis absorption, synchronous fluorescence, three-dimensional fluorescence, 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescence, circular dichroism (CD), and Fourier transform infrared (FT-IR) spectra confirmed that the conformation and structure of BSA were altered in the presence of RA. Moreover, the nuclear magnetic spectroscopy showed that the aromatic groups of RA took part in the binding reaction during the BSA-RA complexation. In addition, the molecular picture of the interaction mechanism between BSA and RA at the atomic level was well examined by molecular docking and dynamics studies. In brief, RA can bind to BSA with noncovalent bonds in a relatively stable way, and these findings will be beneficial to the functional food research of RA. PMID:26146359

  14. Cry1Aa binding to the cadherin receptor does not require conserved amino acid sequences in the domain II loops

    PubMed Central

    Fujii, Yuki; Tanaka, Shiho; Otsuki, Manami; Hoshino, Yasushi; Morimoto, Chinatsu; Kotani, Takuya; Harashima, Yuko; Endo, Haruka; Yoshizawa, Yasutaka; Sato, Ryoichi

    2012-01-01

    Characterizing the binding mechanism of Bt (Bacillus thuringiensis) Cry toxin to the cadherin receptor is indispensable to understanding the specific insecticidal activity of this toxin. To this end, we constructed 30 loop mutants by randomly inserting four serial amino acids covering all four receptor binding loops (loops α8, 1, 2 and 3) and analysed their binding affinities for Bombyx mori cadherin receptors via Biacore. High binding affinities were confirmed for all 30 mutants containing loop sequences that differed from those of wild-type. Insecticidal activities were confirmed in at least one mutant from loops 1, 2 and 3, suggesting that there is no critical amino acid sequence for the binding of the four loops to BtR175. When two mutations at different loops were integrated into one molecule, no reduction in binding affinity was observed compared with wild-type sequences. Based on these results, we discussed the binding mechanism of Cry toxin to cadherin protein. PMID:23145814

  15. RNA-Binding Efficacy of N-Phenylbenzohydroxamic Acid: An Invitro and Insilico Approach.

    PubMed

    Khilari, Rubi; Thakur, Yamini; Pardhi, Manish; Pande, Rama

    2015-01-01

    RNA has attracted recent attention for its key role in gene expression and hence targeting by small molecules for therapeutic intervention. This study is aimed to elucidate the specificity of RNA binding affinity of parent compound of N-arylhydroxamic acids series, N-phenylbenzohydroxamic acid trivially named as PBHA,C6H5NOH.C6H5C˭O. The binding behavior was examined by various biophysical methods such as absorption, fluorescence, and viscosity measurements. Molecular docking was also done. The value of affinity constant and overall binding constant was calculated 5.79±0.03×10(4) M(-1) and K'=1.09±0.03×10(5) M(-1), respectively. The Stern-Volmer constant Ksv obtained was 2.28±0.04×10(4) M(-1). The compound (PBHA) shows a concentration-based enhancement of fluorescence intensity with increasing RNA concentration. Fluorescence quenching of PBHA-RNA complex in presence of K4 [Fe(CN)6] was also observed. Viscometric studies complimented the UV results where a continuous increase in relative viscosity of the RNA solution was observed with added optimal PBHA concentration. All the experimental evidences indicate that PBHA can strongly bind to RNA through an intercalative mode. PMID:25874942

  16. G-CSF receptor-binding cyclic peptides designed with artificial amino-acid linkers

    SciTech Connect

    Shibata, Kenji . E-mail: kshibata@kyowa.co.jp; Maruyama-Takahashi, Kumiko; Yamasaki, Motoo; Hirayama, Noriaki . E-mail: hirayama@is.icc.u-tokai.ac.jp

    2006-03-10

    Designing small molecules that mimic the receptor-binding local surface structure of large proteins such as cytokines or growth factors is fascinating and challenging. In this study, we designed cyclic peptides that reproduce the receptor-binding loop structures of G-CSF. We found it is important to select a suitable linker to join two or more discontinuous sequences and both termini of the peptide corresponding to the receptor-binding loop. Structural simulations based on the crystallographic structure of KW-2228, a stable and potent analog of human G-CSF, led us to choose 4-aminobenzoic acid (Abz) as a part of the linker. A combination of 4-Abz with {beta}-alanine or glycine, and disulfide bridges between cysteins or homocysteins, gave a structure suitable for receptor binding. In this structure, the side-chains of several amino acids important for the interactions with the receptor are protruding from one side of the peptide ring. This artificial peptide showed G-CSF antagonistic activity in a cell proliferation assay.

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

    PubMed

    Maltas, Esra; Ertekin, Betul

    2015-01-01

    Thioglycolic acid modified superparamagnetic iron oxide nanoparticles (TG-APTS-SPION) were synthesized by using (3-aminopropyl) triethoxysilane (APTS) and thioglycolic acid (TG). Actin was immobilized on the nanoparticle surfaces. Binding amount of the actin (Act) on TG-APTS-SPIONs was determined by using a calibration curve equation that was drawn using fluorescence spectra at 280 and 342 nm of excitation and emission wavelengths. Anti-Actin (anti-Act) was interacted with the actin immobilized TG-APTS-SPIONs as primary antibody. Horse radish peroxidase (HRP) was also interacted with antibody conjugated nanoparticles as secondary antibody. The binding capacity of primary and secondary antibodies was also estimated by fluorescence spectroscopy. Scanning electron microscopy (SEM), Infrared spectroscopy (FTIR) and energy dispersive X-ray (EDX) analysis were also clarified binding of the protein and antibodies to the nanoparticles' surfaces. Western blot analysis was also done for actin conjunction with anti Act antibody to confirm binding of the antibody to the protein. PMID:25451750

  18. Evolution of the chalcone-isomerase fold from fatty-acid binding to stereospecific catalysis.

    PubMed

    Ngaki, Micheline N; Louie, Gordon V; Philippe, Ryan N; Manning, Gerard; Pojer, Florence; Bowman, Marianne E; Li, Ling; Larsen, Elise; Wurtele, Eve Syrkin; Noel, Joseph P

    2012-05-24

    Specialized metabolic enzymes biosynthesize chemicals of ecological importance, often sharing a pedigree with primary metabolic enzymes. However, the lineage of the enzyme chalcone isomerase (CHI) remained unknown. In vascular plants, CHI-catalysed conversion of chalcones to chiral (S)-flavanones is a committed step in the production of plant flavonoids, compounds that contribute to attraction, defence and development. CHI operates near the diffusion limit with stereospecific control. Although associated primarily with plants, the CHI fold occurs in several other eukaryotic lineages and in some bacteria. Here we report crystal structures, ligand-binding properties and in vivo functional characterization of a non-catalytic CHI-fold family from plants. Arabidopsis thaliana contains five actively transcribed genes encoding CHI-fold proteins, three of which additionally encode amino-terminal chloroplast-transit sequences. These three CHI-fold proteins localize to plastids, the site of de novo fatty-acid biosynthesis in plant cells. Furthermore, their expression profiles correlate with those of core fatty-acid biosynthetic enzymes, with maximal expression occurring in seeds and coinciding with increased fatty-acid storage in the developing embryo. In vitro, these proteins are fatty-acid-binding proteins (FAPs). FAP knockout A. thaliana plants show elevated α-linolenic acid levels and marked reproductive defects, including aberrant seed formation. Notably, the FAP discovery defines the adaptive evolution of a stereospecific and catalytically 'perfected' enzyme from a non-enzymatic ancestor over a defined period of plant evolution.

  19. Antioxidant and bile acid binding activity of buckwheat protein in vitro digests.

    PubMed

    Ma, Yuanyuan; Xiong, Youling L

    2009-05-27

    The objective of the study was to assess the antioxidant and bile acid removing potential of buckwheat protein (BWP) during a two-stage in vitro digestion (1 h of pepsin followed by 2 h of pancreatin). Antioxidant activity of the digests was analyzed by determining: (1) Fe(2+) chelation, (2) reducing power, (3) 2,2'-azinobis (3-ethylbenzothiszoline-6-sulfonic acid) (ABTS(+•)) radical scavenging capacity, and (4) TBARS formation in a liposome system. The initial pepsin digestion decreased the BWP antioxidant activity; however, subsequent pancreatin digestion fully recovered the reducing power and increased (P < 0.05) the ability to chelate Fe(2+) (45%), scavenge ABTS(+•) (87%), and curtail lipid peroxidation (45%) when compared with intact BWP. The final BWP digest exhibited a 67% increase (P < 0.05) in cholic acid binding capability over that of the nondigested BWP control but was comparable to the control in binding chenodeoxycholic and deoxycholic acids. Digestion-resistant peptides were largely responsible for bile acid elimination. PMID:19320435

  20. Nucleomorphin. A novel, acidic, nuclear calmodulin-binding protein from dictyostelium that regulates nuclear number.

    PubMed

    Myre, Michael A; O'Day, Danton H

    2002-05-31

    Probing of Dictyostelium discoideum cell extracts after SDS-PAGE using (35)S-recombinant calmodulin (CaM) as a probe has revealed approximately three-dozen Ca(2+)-dependent calmodulin binding proteins. Here, we report the molecular cloning, expression, and subcellular localization of a gene encoding a novel calmodulin-binding protein (CaMBP); we have called nucleomorphin, from D. discoideum. A lambdaZAP cDNA expression library of cells from multicellular development was screened using a recombinant calmodulin probe ((35)S-VU1-CaM). The open reading frame of 1119 nucleotides encodes a polypeptide of 340 amino acids with a calculated molecular mass of 38.7 kDa and is constitutively expressed throughout the Dictyostelium life cycle. Nucleomorphin contains a highly acidic glutamic/aspartic acid inverted repeat (DEED) with significant similarity to the conserved nucleoplasmin domain and a putative transmembrane domain in the carboxyl-terminal region. Southern blotting reveals that nucleomorphin exists as a single copy gene. Using gel overlay assays and CaM-agarose we show that bacterially expressed nucleomorphin binds to bovine CaM in a Ca(2+)-dependent manner. Amino-terminal fusion to the green fluorescence protein (GFP) showed that GFP-NumA localized to the nucleus as distinct arc-like patterns similar to heterochromatin regions. GFP-NumA lacking the acidic DEED repeat still showed arc-like accumulations at the nuclear periphery, but the number of nuclei in these cells was increased markedly compared with control cells. Cells expressing GFP-NumA lacking the transmembrane domain localized to the nuclear periphery but did not affect nuclear number or gross morphology. Nucleomorphin is the first nuclear CaMBP to be identified in Dictyostelium. Furthermore, these data present the first identification of a member of the nucleoplasmin family as a calmodulin-binding protein and suggest nucleomorphin has a role in nuclear structure in Dictyostelium. PMID:11919178

  1. Ebselen Inhibits Hepatitis C Virus NS3 Helicase Binding to Nucleic Acid and Prevents Viral Replication

    PubMed Central

    2015-01-01

    The hepatitis C virus (HCV) nonstructural protein 3 (NS3) is both a protease, which cleaves viral and host proteins, and a helicase that separates nucleic acid strands, using ATP hydrolysis to fuel the reaction. Many antiviral drugs, and compounds in clinical trials, target the NS3 protease, but few helicase inhibitors that function as antivirals have been reported. This study focuses on the analysis of the mechanism by which ebselen (2-phenyl-1,2-benzisoselenazol-3-one), a compound previously shown to be a HCV antiviral agent, inhibits the NS3 helicase. Ebselen inhibited the abilities of NS3 to unwind nucleic acids, to bind nucleic acids, and to hydrolyze ATP, and about 1 μM ebselen was sufficient to inhibit each of these activities by 50%. However, ebselen had no effect on the activity of the NS3 protease, even at 100 times higher ebselen concentrations. At concentrations below 10 μM, the ability of ebselen to inhibit HCV helicase was reversible, but prolonged incubation of HCV helicase with higher ebselen concentrations led to irreversible inhibition and the formation of covalent adducts between ebselen and all 14 cysteines present in HCV helicase. Ebselen analogues with sulfur replacing the selenium were just as potent HCV helicase inhibitors as ebselen, but the length of the linker between the phenyl and benzisoselenazol rings was critical. Modifications of the phenyl ring also affected compound potency over 30-fold, and ebselen was a far more potent helicase inhibitor than other, structurally unrelated, thiol-modifying agents. Ebselen analogues were also more effective antiviral agents, and they were less toxic to hepatocytes than ebselen. Although the above structure–activity relationship studies suggest that ebselen targets a specific site on NS3, we were unable to confirm binding to either the NS3 ATP binding site or nucleic acid binding cleft by examining the effects of ebselen on NS3 proteins lacking key cysteines. PMID:25126694

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

    PubMed

    Fu, Wen-Jin; Wang, Du-Juan; Deng, Ren-Tang; Huang, Zhi-Hong; Chen, Mei-Lian; Jang, You-Ming; Wen, Shu; Yang, Hong-Ling; Huang, Xian-zhang

    2015-09-01

    We compared urinary liver-type fatty acid-binding protein (L-FABP) among non-pregnant and pregnant women with and without gestational diabetes mellitus (GDM). Higher urinary L-FABP was found in pregnant with and without GDM, and considerably higher urinary L-FABP was found in the GDM group compared with the non-GDM group. Hyperglycemia and anemia were related with high urinary L-FABP expression. PMID:26254248

  3. 1-Aminocyclopropane-1-carboxylic acid oxidase: insight into cofactor binding from experimental and theoretical studies.

    PubMed

    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. PMID:22711330

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

    PubMed

    Fu, Wen-Jin; Wang, Du-Juan; Deng, Ren-Tang; Huang, Zhi-Hong; Chen, Mei-Lian; Jang, You-Ming; Wen, Shu; Yang, Hong-Ling; Huang, Xian-zhang

    2015-09-01

    We compared urinary liver-type fatty acid-binding protein (L-FABP) among non-pregnant and pregnant women with and without gestational diabetes mellitus (GDM). Higher urinary L-FABP was found in pregnant with and without GDM, and considerably higher urinary L-FABP was found in the GDM group compared with the non-GDM group. Hyperglycemia and anemia were related with high urinary L-FABP expression.

  5. Binding of α,α-disubstituted amino acids to arginase suggests new avenues for inhibitor design.

    PubMed

    Ilies, Monica; Di Costanzo, Luigi; Dowling, Daniel P; Thorn, Katherine J; Christianson, David W

    2011-08-11

    Arginase is a binuclear manganese metalloenzyme that hydrolyzes L-arginine to form L-ornithine and urea, and aberrant arginase activity is implicated in various diseases such as erectile dysfunction, asthma, atherosclerosis, and cerebral malaria. Accordingly, arginase inhibitors may be therapeutically useful. Continuing our efforts to expand the chemical space of arginase inhibitor design and inspired by the binding of 2-(difluoromethyl)-L-ornithine to human arginase I, we now report the first study of the binding of α,α-disubstituted amino acids to arginase. Specifically, we report the design, synthesis, and assay of racemic 2-amino-6-borono-2-methylhexanoic acid and racemic 2-amino-6-borono-2-(difluoromethyl)hexanoic acid. X-ray crystal structures of human arginase I and Plasmodium falciparum arginase complexed with these inhibitors reveal the exclusive binding of the L-stereoisomer; the additional α-substituent of each inhibitor is readily accommodated and makes new intermolecular interactions in the outer active site of each enzyme. Therefore, this work highlights a new region of the protein surface that can be targeted for additional affinity interactions, as well as the first comparative structural insights on inhibitor discrimination between a human and a parasitic arginase.

  6. Zebrafish cellular nucleic acid-binding protein: gene structure and developmental behaviour.

    PubMed

    Armas, Pablo; Cachero, Sebastián; Lombardo, Verónica A; Weiner, Andrea; Allende, Miguel L; Calcaterra, Nora B

    2004-08-01

    Here we analyse the structural organisation and expression of the zebrafish cellular nucleic acid-binding protein (zCNBP) gene and protein. The gene is organised in five exons and four introns. A noteworthy feature of the gene is the absence of a predicted promoter region. The coding region encodes a 163-amino acid polypeptide with the highly conserved general structural organisation of seven CCHC Zn knuckle domains and an RGG box between the first and the second Zn knuckles. Although theoretical alternative splicing is possible, only one form of zCNBP is actually detected. This form is able to bind to single-stranded DNA and RNA probes in vitro. The analysis of zCNBP developmental expression shows a high amount of CNBP-mRNA in ovary and during the first developmental stages. CNBP-mRNA levels decrease while early development progresses until the midblastula transition (MBT) stage and increases again thereafter. The protein is localised in the cytoplasm of blastomeres whereas it is mainly nuclear in developmental stages after the MBT. These findings suggest that CNBP is a strikingly conserved single-stranded nucleic acid-binding protein which might interact with maternal mRNA during its storage in the embryo cell cytoplasm. It becomes nuclear once MBT takes place possibly in order to modulate zygotic transcription and/or to associate with newly synthesised transcripts.

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

    SciTech Connect

    Ilies, Monica; Di Costanzo, Luigi; Dowling, Daniel P.; Thorn, Katherine J.; Christianson, David W.

    2011-10-21

    Arginase is a binuclear manganese metalloenzyme that hydrolyzes L-arginine to form L-ornithine and urea, and aberrant arginase activity is implicated in various diseases such as erectile dysfunction, asthma, atherosclerosis, and cerebral malaria. Accordingly, arginase inhibitors may be therapeutically useful. Continuing our efforts to expand the chemical space of arginase inhibitor design and inspired by the binding of 2-(difluoromethyl)-L-ornithine to human arginase I, we now report the first study of the binding of {alpha},{alpha}-disubstituted amino acids to arginase. Specifically, we report the design, synthesis, and assay of racemic 2-amino-6-borono-2-methylhexanoic acid and racemic 2-amino-6-borono-2-(difluoromethyl)hexanoic acid. X-ray crystal structures of human arginase I and Plasmodium falciparum arginase complexed with these inhibitors reveal the exclusive binding of the L-stereoisomer; the additional {alpha}-substituent of each inhibitor is readily accommodated and makes new intermolecular interactions in the outer active site of each enzyme. Therefore, this work highlights a new region of the protein surface that can be targeted for additional affinity interactions, as well as the first comparative structural insights on inhibitor discrimination between a human and a parasitic arginase.

  8. Development and validation of a cholate binding capacity method for DMP 504, a bile acid sequestrant.

    PubMed

    Schreiber, M A; Moyer, K L; Mueller, B J; Ramos, M A; Green, J S; White, L; Hedgepeth, W; Juliano, K; Scull, J R; Hovsepian, P K

    2001-06-01

    DMP 504, a highly cross-linked insoluble polymer, is a bile acid sequestrant developed by the DuPont Pharmaceuticals Company for serum cholesterol reduction. Since DMP 504 is insoluble, it was necessary to develop unique specific analytical methods to measure and control the quality of different lots of the drug. Since the mechanism of action of DMP 504 is believed to be by sequestration of bile acids, the in-vitro binding capacity of the polymer for cholic acid was chosen as a surrogate of in-vivo performance and used to assess potency of the compound. In this method, individual aliquots of DMP 504 at three different levels were incubated with a cholate solution of known concentration. The residual cholate solution was filtered and analyzed by a reversed-phase HPLC method using refractive index detection. When the bound cholate was plotted versus the mass of DMP 504, the resulting curve was linear. The slope of this curve is the cholate binding capacity of DMP 504. This method has been shown to be precise and robust. Precision of the method was shown to have an RSD of 2.0% with injection precision of 0.4% and stability of cholate solutions up to 73 h. It is also a unique binding capacity method due to its multi-point determination, and it has been shown to be a suitable quality control method for ensuring lot-to-lot consistency of drug substance.

  9. Acidic Residues in the Hfq Chaperone Increase the Selectivity of sRNA Binding and Annealing.

    PubMed

    Panja, Subrata; Santiago-Frangos, Andrew; Schu, Daniel J; Gottesman, Susan; Woodson, Sarah A

    2015-11-01

    Hfq facilitates gene regulation by small non-coding RNAs (sRNAs), thereby affecting bacterial attributes such as biofilm formation and virulence. Escherichia coli Hfq recognizes specific U-rich and AAN motifs in sRNAs and target mRNAs, after which an arginine patch on the rim promotes base pairing between their complementary sequences. In the cell, Hfq must discriminate between many similar RNAs. Here, we report that acidic amino acids lining the sRNA binding channel between the inner pore and rim of the Hfq hexamer contribute to the selectivity of Hfq's chaperone activity. RNase footprinting, in vitro binding and stopped-flow fluorescence annealing assays showed that alanine substitution of D9, E18 or E37 strengthened RNA interactions with the rim of Hfq and increased annealing of non-specific or U-tailed RNA oligomers. Although the mutants were less able than wild-type Hfq to anneal sRNAs with wild-type rpoS mRNA, the D9A mutation bypassed recruitment of Hfq to an (AAN)4 motif in rpoS, both in vitro and in vivo. These results suggest that acidic residues normally modulate access of RNAs to the arginine patch. We propose that this selectivity limits indiscriminate target selection by E. coli Hfq and enforces binding modes that favor genuine sRNA and mRNA pairs.

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

    PubMed

    Tamilmani, Poonkodi; Pandey, Mohan Chandra

    2016-04-15

    Dietary polyphenols are markedly studied for their antioxidant activity. They also have a negative impact on nutrition whereby they interfere with iron absorption. Common dietary polyphenols include: catechins, flavonols, flavanols, flavones, anthocyanins, proanthocyanidins and phenolic acids. Ascorbic acid (AA) and Ethylenediaminetetraacetic acid (EDTA) are commonly used to counter act this reaction and increase iron bioavailability. This study was aimed at determining the effect of AA and EDTA on the catechol or galloyl iron binding ability of pure phenolics, coffee and tea. Phenolic concentrations of 40, 80, 610, 240, 320, 400, 520 and 900 μg/ml were tested against six levels of AA and EDTA. These effects were studied in detail using Multivariate Analysis of Variance (MANOVA) with the hypothesis that there would be one or more mean differences between the ratio of enhancer and the different concentrations of samples tested. AA was found to be more efficient than EDTA in a way that lesser quantity is required for completely overcoming negative iron binding effects of polyphenols and similar samples.

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

    PubMed

    Kalescky, Robert; Kraka, Elfi; Cremer, Dieter

    2014-02-28

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

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

    SciTech Connect

    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.

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

    SciTech Connect

    McGary, C.T.

    1988-01-01

    The binding, endocytosis, and degradation of {sup 125}I-hyaluronic acid ({sup 125}I-HA) by liver endothelial cells (LEC) was studied under several conditions. The dissociation of receptor-bound {sup 125}I-HA was rapid, with a half time of {approx}31 min and a K{sub off} of 6.3 {times} 10{sup {minus}4}/sec. A large reversible increase in {sup 125}I-HA binding to LEC at pH 5.0 was due to an increase in the observed affinity of the binding interaction. Pronase digestion suggested the protein nature of the receptor and the intracellular location of the digitonin exposed binding activity. Binding and endocytosis occur in the presence of 10 mM EGTA indicating that divalent cations are not required for receptor function. To study the degradation of {sup 125}I-HA by LEC, a cetylpyridinium chloride (CPC) precipitation assay was characterized. The minimum HA length required for precipitation was elucidated. The fate of the LEC HA receptor after endocytosis was examined.

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

    PubMed

    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.

  16. Sensing the neuronal glycocalyx by glial sialic acid binding immunoglobulin-like lectins.

    PubMed

    Linnartz-Gerlach, B; Mathews, M; Neumann, H

    2014-09-01

    Sialic acid binding immunoglobulin-like lectins (Siglecs) are cell surface receptors of microglia and oligodendrocytes that recognize the sialic acid cap of healthy neurons and neighboring glial cells. Upon ligand binding, Siglecs typically signal through an immunoreceptor tyrosine-based inhibition motif (ITIM) to keep the cell in a homeostatic status and support healthy neighboring cells. Siglecs can be divided into two groups; the first, being conserved among different species. The conserved Siglec-4/myelin-associated glycoprotein is expressed on oligodendrocytes and Schwann cells. Siglec-4 protects neurons from acute toxicity via interaction with sialic acids bound to neuronal gangliosides. The second group of Siglecs, named CD33-related Siglecs, is almost exclusively expressed on immune cells and is highly variable among different species. Microglial expression of Siglec-11 is human lineage-specific and prevents neurotoxicity via interaction with α2.8-linked sialic acid oligomers exposed on the neuronal glycocalyx. Microglial Siglec-E is a mouse CD33-related Siglec member that prevents microglial phagocytosis and the associated oxidative burst. Mouse Siglec-E of microglia binds to α2.8- and α2.3-linked sialic acid residues of the healthy glycocalyx of neuronal and glial cells. Recently, polymorphisms of the human Siglec-3/CD33 were linked to late onset Alzheimer's disease by genome-wide association studies. Human Siglec-3 is expressed on microglia and produces inhibitory signaling that decreases uptake of particular molecules such as amyloid-β aggregates. Thus, glial ITIM-signaling Siglecs recognize the intact glycocalyx of neurons and are involved in the modulation of neuron-glia interaction in healthy and diseased brain.

  17. Mechanistic Studies on the Photoallergy Mediated by Fenofibric Acid: Photoreactivity with Serum Albumins.

    PubMed

    Vayá, Ignacio; Andreu, Inmaculada; Monje, Vicente T; Jiménez, M Consuelo; Miranda, Miguel A

    2016-01-19

    The photoreactivity of fenofibric acid (FA) in the presence of human and bovine serum albumins (HSA and BSA, respectively) has been investigated by steady-state irradiation, fluorescence, and laser flash photolysis (LFP). Spectroscopic measurements allowed for the determination of a 1:1 stoichiometry for the FA/SA complexes and pointed to a moderate binding of FA to the proteins; by contrast, the FA photoproducts were complexed more efficiently with SAs. Covalent photobinding to the protein, which is directly related to the photoallergic properties of the drug, was detected after long irradiation times and was found to be significantly higher in the case of BSA. Intermolecular FA-amino acid and FA-albumin irradiations resulted in the formation of photoproducts arising from coupling between both moieties, as indicated by mass spectrometric analysis. Mechanistic studies using model drug-amino acid linked systems indicated that the key photochemical step involved in photoallergy is formal hydrogen atom transfer from an amino acid residue to the excited benzophenone chromophore of FA or (more likely) its photoproducts. This results in the formation of caged radical pairs followed by C-C coupling to give covalent photoaducts.

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

    PubMed

    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

  19. Cooperative and noncooperative binding of protein ligands to nucleic acid lattices: experimental approaches to the determination of thermodynamic parameters.

    PubMed

    Kowalczykowski, S C; Paul, L S; Lonberg, N; Newport, J W; McSwiggen, J A; von Hippel, P H

    1986-03-25

    Many biologically important proteins bind nonspecifically, and often cooperatively, to single-or double-stranded nucleic acid lattices in discharging their physiological functions. This binding can generally be described in thermodynamic terms by three parameters: n, the binding site size; K, the intrinsic binding constant; omega, the binding cooperativity parameter. The experimental determination of these parameters often appears to be straightforward but can be fraught with conceptual and methodological difficulties that may not be readily apparent. In this paper we describe and analyze a number of approaches that can be used to measure these protein-nucleic acid interaction parameters and illustrate these methods with experiments on the binding of T4-coded gene 32 (single-stranded DNA binding) protein to various nucleic acid lattices. We consider the following procedures: (i) the titration of a fixed amount of lattice (nucleic acid) with added ligand (protein); (ii) the titration of a fixed amount of ligand with added lattice; (iii) the determination of ligand binding affinities at very low levels of lattice saturation; (iv) the analysis of ligand cluster size distribution on the lattice; (v) the analysis of ligand binding to lattices of finite length. The applicability and limitations of each approach are considered and discussed, and potential pitfalls are explicitly pointed out.

  20. Characterization of a Single-Stranded DNA-Binding-Like Protein from Nanoarchaeum equitans—A Nucleic Acid Binding Protein with Broad Substrate Specificity

    PubMed Central

    Olszewski, Marcin; Balsewicz, Jan; Nowak, Marta; Maciejewska, Natalia; Cyranka-Czaja, Anna; Zalewska-Piątek, Beata; Piątek, Rafał; Kur, Józef

    2015-01-01

    Background SSB (single-stranded DNA-binding) proteins play an essential role in all living cells and viruses, as they are involved in processes connected with ssDNA metabolism. There has recently been an increasing interest in SSBs, since they can be applied in molecular biology techniques and analytical methods. Nanoarchaeum equitans, the only known representative of Archaea phylum Nanoarchaeota, is a hyperthermophilic, nanosized, obligatory parasite/symbiont of Ignicoccus hospitalis. Results This paper reports on the ssb-like gene cloning, gene expression and characterization of a novel nucleic acid binding protein from Nanoarchaeum equitans archaeon (NeqSSB-like protein). This protein consists of 243 amino acid residues and one OB fold per monomer. It is biologically active as a monomer like as SSBs from some viruses. The NeqSSB-like protein displays a low sequence similarity to the Escherichia coli SSB, namely 10% identity and 29% similarity, and is the most similar to the Sulfolobus solfataricus SSB (14% identity and 32% similarity). The NeqSSB-like protein binds to ssDNA, although it can also bind mRNA and, surprisingly, various dsDNA forms, with no structure-dependent preferences as evidenced by gel mobility shift assays. The size of the ssDNA binding site, which was estimated using fluorescence spectroscopy, is 7±1 nt. No salt-dependent binding mode transition was observed. NeqSSB-like protein probably utilizes a different model for ssDNA binding than the SSB proteins studied so far. This protein is highly thermostable; the half-life of the ssDNA binding activity is 5 min at 100°C and melting temperature (Tm) is 100.2°C as shown by differential scanning calorimetry (DSC) analysis. Conclusion NeqSSB-like protein is a novel highly thermostable protein which possesses a unique broad substrate specificity and is able to bind all types of nucleic acids. PMID:25973760

  1. Binding free energy calculations between bovine β-lactoglobulin and four fatty acids using the MMGBSA method.

    PubMed

    Bello, Martiniano

    2014-10-01

    The bovine dairy protein β-lactoglobulin (βlg) is a promiscuous protein that has the ability to bind several hydrophobic ligands. In this study, based on known experimental data, the dynamic interaction mechanism between bovine βlg and four fatty acids was investigated by a protocol combining molecular dynamics (MD) simulations and molecular mechanics generalized Born surface area (MMGBSA) binding free energy calculations. Energetic analyses revealed binding free energy trends that corroborated known experimental findings; larger ligand size corresponded to greater binding affinity. Finally, binding free energy decomposition provided detailed information about the key residues stabilizing the complex.

  2. Both FA- and mPEG-conjugated chitosan nanoparticles for targeted cellular uptake and enhanced tumor tissue distribution

    NASA Astrophysics Data System (ADS)

    Hou, Zhenqing; Zhan, Chuanming; Jiang, Qiwei; Hu, Quan; Li, Le; Chang, Di; Yang, Xiangrui; Wang, Yixiao; Li, Yang; Ye, Shefang; Xie, Liya; Yi, Yunfeng; Zhang, Qiqing

    2011-10-01

    Both folic acid (FA)- and methoxypoly(ethylene glycol) (mPEG)-conjugated chitosan nanoparticles (NPs) had been designed for targeted and prolong anticancer drug delivery system. The chitosan NPs were prepared with combination of ionic gelation and chemical cross-linking method, followed by conjugation with both FA and mPEG, respectively. FA-mPEG-NPs were compared with either NPs or mPEG-/FA-NPs in terms of their size, targeting cellular efficiency and tumor tissue distribution. The specificity of the mPEG-FA-NPs targeting cancerous cells was demonstrated by comparative intracellular uptake of NPs and mPEG-/FA-NPs by human adenocarcinoma HeLa cells. Mitomycin C (MMC), as a model drug, was loaded to the mPEG-FA-NPs. Results show that the chitosan NPs presented a narrow-size distribution with an average diameter about 200 nm regardless of the type of functional group. In addition, MMC was easily loaded to the mPEG-FA-NPs with drug-loading content of 9.1%, and the drug releases were biphasic with an initial burst release, followed by a subsequent slower release. Laser confocal scanning imaging proved that both mPEG-FA-NPs and FA-NPs could greatly enhance uptake by HeLa cells. In vivo animal experiments, using a nude mice xenograft model, demonstrated that an increased amount of mPEG-FA-NPs or FA-NPs were accumulated in the tumor tissue relative to the mPEG-NPs or NPs alone. These results suggest that both FA- and mPEG-conjugated chitosan NPs are potentially prolonged drug delivery system for tumor cell-selective targeting treatments.

  3. Electrostatic binding and hydrophobic collapse of peptide-nucleic acid aggregates quantified using force spectroscopy.

    PubMed

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

    2013-06-25

    Knowledge of the mechanisms of interaction between self-aggregating peptides and nucleic acids or other polyanions is key to the understanding of many aggregation processes underlying several human diseases (e.g., Alzheimer's and Parkinson's diseases). Determining the affinity and kinetic steps of such interactions is challenging due to the competition between hydrophobic self-aggregating forces and electrostatic binding forces. Kahalalide F (KF) is an anticancer hydrophobic peptide that contains a single positive charge that confers strong aggregative properties with polyanions. This makes KF an ideal model to elucidate the mechanisms by which self-aggregation competes with binding to a strongly charged polyelectrolyte such as DNA. We use optical tweezers to apply mechanical forces to single DNA molecules and show that KF and DNA interact in a two-step kinetic process promoted by the electrostatic binding of DNA to the aggregate surface followed by the stabilization of the complex due to hydrophobic interactions. From the measured pulling curves we determine the spectrum of binding affinities, kinetic barriers, and lengths of DNA segments sequestered within the KF-DNA complex. We find there is a capture distance beyond which the complex collapses into compact aggregates stabilized by strong hydrophobic forces and discuss how the bending rigidity of the nucleic acid affects this process. We hypothesize that within an in vivo context, the enhanced electrostatic interaction of KF due to its aggregation might mediate the binding to other polyanions. The proposed methodology should be useful to quantitatively characterize other compounds or proteins in which the formation of aggregates is relevant. PMID:23706043

  4. Nucleic Acids Bind to Nanoparticulate iron (II) Monosulphide in Aqueous Solutions

    NASA Astrophysics Data System (ADS)

    Hatton, Bryan; Rickard, David

    2008-06-01

    In the hydrothermal FeS-world origin of life scenarios nucleic acids are suggested to bind to iron (II) monosulphide precipitated from the reaction between hydrothermal sulphidic vent solutions and iron-bearing oceanic water. In lower temperature systems, the first precipitate from this process is nanoparticulate, metastable FeSm with a mackinawite structure. Although the interactions between bulk crystalline iron sulphide minerals and nucleic acids have been reported, their reaction with nanoparticulate FeSm has not previously been investigated. We investigated the binding of different nucleic acids, and their constituents, to freshly precipitated, nanoparticulate FeSm. The degree to which the organic molecules interacted with FeSm is chromosomal DNA > RNA > oligomeric DNA > deoxadenosine monophosphate ≈ deoxyadenosine ≈ adenine. Although we found that FeSm does not fluoresce within the visible spectrum and there is no quantum confinement effect seen in the absorption, the mechanism of linkage of the FeSm to these biomolecules appears to be primarily electrostatic and similar to that found for the attachment of ZnS quantum dots. The results of a preliminary study of similar reactions with nanoparticulate CuS further supported the suggestion that the interaction mechanism was generic for nanoparticulate transition metal sulphides. In terms of the FeS-world hypothesis, the results of this study further support the idea that sulphide minerals precipitated at hydrothermal vents interact with biomolecules and could have assisted in the formation and polymerisation of nucleic acids.

  5. Autoradiographic localization and characterization of [125I]lysergic acid diethylamide binding to serotonin receptors in Aplysia.

    PubMed

    Kadan, M J; Hartig, P R

    1988-03-01

    The sensitive serotonergic radioligand 2-[125I]lysergic acid diethylamide was used to study the distribution and pharmacological binding properties of serotonin receptors in Aplysia californica. The high specific activity of this radioligand allowed us to develop a methodology for the investigation of receptor binding properties and receptor distribution in a single ganglion. [125I]Lysergic acid diethylamide labels a population of high-affinity serotonergic sites (Kd = 0.41 nM) in Aplysia ganglia whose regional distribution matches that expected from previous electrophysiological and immunohistochemical studies. The properties of [125I]lysergic acid diethylamide binding sites in Aplysia are in general agreement with previous studies on [3H]lysergic acid diethylamide binding in this system but these sites differ from the serotonergic receptor subtypes described in the mammalian brain. Guanine nucleotides were shown to modulate agonist but not antagonist affinity for the [125I]lysergic acid diethylamide binding site in Aplysia, suggesting that this site is coupled to a G-protein. Images of serotonin receptor distribution in the Aplysia nervous system were obtained from autoradiograms of [125I]lysergic acid diethylamide binding. Serotonin receptors in ganglia tissue sections are located primarily within the neuropil. In addition, a subset of neuronal soma are specifically labeled by [125I]lysergic acid diethylamide. These studies indicate that [125I]lysergic acid diethylamide binds to sites in the Aplysia nervous system which display a regional distribution, pharmacological binding properties and evidence of coupling to a G-protein consistent with labeling of a subset of functional serotonin receptors. In addition, the techniques used in this investigation provide a general approach for rapidly characterizing the pharmacological properties and anatomical distribution of receptor binding sites in single invertebrate ganglia. Individual neurons containing these receptor

  6. A Mollusk Retinoic Acid Receptor (RAR) Ortholog Sheds Light on the Evolution of Ligand Binding

    PubMed Central

    Gutierrez-Mazariegos, Juliana; Nadendla, Eswar Kumar; Lima, Daniela; Pierzchalski, Keely; Jones, Jace W.; Kane, Maureen; Nishikawa, Jun-Ichi; Hiromori, Youhei; Nakanishi, Tsuyoshi; Santos, Miguel M.; Castro, L. Filipe C.; Bourguet, William

    2014-01-01

    Nuclear receptors are transcription factors that regulate networks of target genes in response to small molecules. There is a strong bias in our knowledge of these receptors because they were mainly characterized in classical model organisms, mostly vertebrates. Therefore, the evolutionary origins of specific ligand-receptor couples still remain elusive. Here we present the identification and characterization of a retinoic acid receptor (RAR) from the mollusk Nucella lapillus (NlRAR). We show that this receptor specifically binds to DNA response elements organized in direct repeats as a heterodimer with retinoid X receptor. Surprisingly, we also find that NlRAR does not bind all-trans retinoic acid or any other retinoid we tested. Furthermore, NlRAR is unable to activate the transcription of reporter genes in response to stimulation by retinoids and to recruit coactivators in the presence of these compounds. Three-dimensional modeling of the ligand-binding domain of NlRAR reveals an overall structure that is similar to vertebrate RARs. However, in the ligand-binding pocket (LBP) of the mollusk receptor, the alteration of several residues interacting with the ligand has apparently led to an overall decrease in the strength of the interaction with the ligand. Accordingly, mutations of NlRAR at key positions within the LBP generate receptors that are responsive to retinoids. Altogether our data suggest that, in mollusks, RAR has lost its affinity for all-trans retinoic acid, highlighting the evolutionary plasticity of its LBP. When put in an evolutionary context, our results reveal new structural and functional features of nuclear receptors validated by millions of years of evolution that were impossible to reveal in model organisms. PMID:25116705

  7. Thermodynamics of metal cation binding by a solid soil-derived humic acid: binding of Fe(III), Pb(II), and Cu(II).

    PubMed

    Ghabbour, Elham A; Shaker, Medhat; El-Toukhy, Ahmed; Abid, Ismail M; Davies, Geoffrey

    2006-04-01

    Metal binding and release by solid humic acids (HAs) in soils and sediments can affect metal mobility and bioavailability. Isotherms for tight binding of Fe(III), Pb(II) and Cu(II) by a solid humic acid at pH2.0 fit the Langmuir binding model. Low pH was chosen to protonate the HA carboxylate groups and avoid metal cation hydrolysis. Binding of Fe(III), Pb(II) and Cu(II) occurs in one detectable step labeled A. Site capacities nu(A) are temperature-independent from 10.0 to 40.0 degrees C and point to binding by charge-neutralization to form solid complexes M(OOC-R)(n)(s), where n appears to be 2 for Pb(II) and 3 for Fe(III). Thermodynamic data pairs (DeltaH(A), DeltaS(A)) for metal binding are linearly correlated with previous data for Ca(II), Co(II) and Mg(II) binding by solid HAs.

  8. Simian Virus 40 Deoxyribonucleic Acid Transcription In Vitro: Binding and Transcription Patterns with a Mammalian Ribonucleic Acid Polymerase 1

    PubMed Central

    Herzberg, Max; Winocour, Ernest

    1970-01-01

    The in vitro transcription pattern of simian virus 40 (SV40) deoxyribonucleic acid (DNA) by a mammalian ribonucleic acid (RNA) polymerase, was studied by electron microscopy and velocity sedimentation techniques. It was found that (i) the majority of supercoiled SV40 DNA molecules displayed a single binding site for the enzyme, (ii) the supercoiled structure of SV40 DNA was frequently retained during transcription, and (iii) the majority of RNA molecules synthesized from the supercoiled SV40 DNA template showed no self-complementarity and sedimented relatively homogeneously in the 15S to 16S region of a sucrose gradient (in contrast, the RNA product synthesized from the nicked-circular SV40 DNA template showed self-complementarity and sedimented heterogeneously). RNA polymerase preparations isolated from SV40-infected monkey cells were more active than those isolated from uninfected monkey cells. Images PMID:4320700

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

    PubMed Central

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

    1979-01-01

    Female sex and estrogen administration are associated with increased hepatic production of triglyceride-rich lipoproteins; the basis for this has not been fully elucidated. Inasmuch as hepatic lipoprotein production is also influenced by FFA availability and triglyceride biosynthesis, we investigated sex differences in FFA utilization in rat hepatocyte suspensions and in the components of the triglyceride biosynthetic pathway. Isolated adult rat hepatocyte suspensions were incubated with albumin-bound [14C]oleate for up to 15 min. At physiological and low oleate concentrations, cells from females incorporated significantly more 14C into glycerolipids, especially triglycerides, and into oxidation products than did male cells, per milligram cell protein. At 0.44 mM oleate, incorporation into triglycerides in female cells was approximately twice that in male cells. Comparable sex differences were observed in cells from fasted animals and when [14C]-glycerol incorporation was measured. At higher oleate concentrations, i.e., fatty acid:albumin mole ratios in excess of 2:1, these sex differences were no longer demonstrable, suggesting that maximal rates of fatty acid esterification and oxidation were similar in female and male cells. In female and male hepatic microsomes, specific activities of long chain acyl coenzyme A synthetase, phosphatidate phosphohydrolase, and diglyceride acyltransferase were similar, but glycerol-3-phosphate acyltransferase activity was slightly greater in females at certain substrate concentrations. Microsomal incorporation of [14C]oleate into total glycerolipids was not significantly greater in females. In further contrast to intact cells, microsomal incorporation of [14C]oleate into triglycerides, although significantly greater in female microsomes, accounted for only a small fraction of the fatty acid esterified. The binding affinity and stoichiometry of partially purified female hepatic fatty acid binding protein (FABP) were similar to

  10. Inhibition of Helicobacter pylori binding to gastrointestinal epithelial cells by sialic acid-containing oligosaccharides.

    PubMed Central

    Simon, P M; Goode, P L; Mobasseri, A; Zopf, D

    1997-01-01

    Helicobacterpylori, the ulcer pathogen residing in the human stomach, binds to epithelial cells of the gastric antrum. We have examined binding of 13 bacterial isolates to epithelial cell lines by use of a sensitive microtiter plate method in which measurement of bacterial urease activity provides the means for quantitation of bound organisms. Several established human gastrointestinal carcinoma cell lines grown as monolayers were compared for suitability in these assays, and the duodenum-derived cell line HuTu-80 was selected for testing bacterial binding inhibitors. When bacteria are pretreated with oligosaccharides, glycoproteins, and glycolipids, a complex picture of bacterial-epithelial adherence specificities emerges. Among the monovalent inhibitors tested, 3'-sialyllactose (NeuAc alpha2-3Gal beta1-4Glc; 3'SL) was the most active oligosaccharide, inhibiting adherence for recent clinical isolates of H. pylori with a millimolar 50% inhibitory concentration (IC50). Its alpha2-6 isomer (6'SL) was less active. Most of the recent clinical isolates examined were inhibited by sialyllactose, whereas long-passaged isolates were insensitive. Among the long-passaged bacterial strains whose binding was not inhibited by 3'SL was the strain ATCC 43504, also known as NCTC 11637 and CCUG 17874, in which the proposed sialyllactose adhesin was recently reported to lack surface expression (P. G. O'Toole, L. Janzon, P. Doig, J. Huang, M. Kostrzynska, and T. H. Trust, J. Bacteriol. 177:6049-6057, 1995). Pretreatment of the epithelial monolayer with neuraminidase reduced the extent of binding by those bacteria that are sensitive to inhibition by 3'SL. Other potent inhibitors of bacterial binding are the glycoproteins alpha1-acid glycoprotein, fetuin, porcine gastric and bovine submaxillary mucins, and the glycolipid sulfatide, all of which present multivalent sialylated and/or sulfated galactosyl residues under the conditions of the binding assay. Consistent with this pattern, a

  11. Matrix Domain Modulates HIV-1 Gag's Nucleic Acid Chaperone Activity via Inositol Phosphate Binding

    PubMed Central

    Jones, Christopher P.; Datta, Siddhartha A. K.; Rein, Alan; Rouzina, Ioulia; Musier-Forsyth, Karin

    2011-01-01

    Retroviruses replicate by reverse transcribing their single-stranded RNA genomes into double-stranded DNA using specific cellular tRNAs to prime cDNA synthesis. In HIV-1, human tRNA3Lys serves as the primer and is packaged into virions during assembly. The viral Gag protein is believed to chaperone tRNA3Lys placement onto the genomic RNA primer binding site; however, the timing and possible regulation of this event are currently unknown. Composed of the matrix (MA), capsid (CA), nucleocapsid (NC), and p6 domains, the multifunctional HIV-1 Gag polyprotein orchestrates the highly coordinated process of virion assembly, but the contribution of these domains to tRNA3Lys annealing is unclear. Here, we show that NC is absolutely essential for annealing and that the MA domain inhibits Gag's tRNA annealing capability. During assembly, MA specifically interacts with inositol phosphate (IP)-containing lipids in the plasma membrane (PM). Surprisingly, we find that IPs stimulate Gag-facilitated tRNA annealing but do not stimulate annealing in Gag variants lacking the MA domain or containing point mutations involved in PM binding. Moreover, we find that IPs prevent MA from binding to nucleic acids but have little effect on NC or Gag. We propose that Gag binds to RNA either with both NC and MA domains or with NC alone and that MA-IP interactions alter Gag's binding mode. We propose that MA's interactions with the PM trigger the switch between these two binding modes and stimulate Gag's chaperone function, which may be important for the regulation of events such as tRNA primer annealing. PMID:21123373

  12. Lipoteichoic acid-binding and biological properties of T protein of group A streptococcus.

    PubMed

    Johnson, R H; Simpson, W A; Dale, J B; Ofek, I; Beachey, E H

    1980-08-01

    T protein was extracted with trypsin from an avirulent, M protein-deficient, type 1 group A Streptococcus and purified by ammonium sulfate precipitation and anion-exchange chromatography. The latter procedure removed contaminating lipoteichoic acid (LTA) from the T protein, which consisted of a heterogeneous mixture of polypeptides resistant to digestion by trypsin and ranged in molecular size from 160,000 to 200,000 daltons. Threonine, aspartic acid, glutamic acid, lysine, and valine were the most predominant amino acids. The binding of LTA to an affinity column of T protein was reversible with increasing concentrations of ethanol but not with increasing ionic strength. T protein bound less palmitic acid and LTA than did fatty acid-free bovine albumin and did not stimulate human peripheral lymphocytes. Because the surface and cell wall distribution of the T proteins and LTA appear similar, the possibility exists that T proteins and LTA may interact in situ by weakly hydrophobic bonds. Such ligand-ligand interaction may be indirectly involved in the adherence of group A streptococci to host cell membranes that is known to be mediated by LTA.

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

    PubMed

    Wang, Yan-Su; Wu, Tian-Hao; Yang, Yao; Zhu, Cen-Ling; Ding, Cheng-Long; Dai, Chuan-Chao

    2016-01-01

    This investigation examined the reduction of pesticide residues on straw inoculated with lactic acid bacteria (LAB) during ensiling. Lactobacillus casei WYS3 was isolated from rice straw that contained pesticide residues. Non-sterilized rice straw, which was inoculated with L. casei WYS3, showed increased removal of chlorpyrifos after ensiling, compared with rice straw that was not inoculated with L. casei WYS3 or sterilized rice straw. In pure culture, these strains can bind chlorpyrifos as indicated by high-performance liquid chromatography analysis. Viable L. casei WYS3 was shown to bind 33.3-42% of exogenously added chlorpyrifos. These results are similar to those of acid-treated cells but less than those of heat-treated cells, which were found to bind 32.0% and 77.2% of the added chlorpyrifos respectively. Furthermore, gas chromatography-mass spectrometry analysis determined that L. casei WYS3 detoxified chlorpyrifos via P-O-C cleavage. Real-time polymerized chain reaction analysis determined that organophosphorus hydrolase gene expression tripled after the addition of chlorpyrifos to LAB cultures, compared with the control group (without chlorpyrifos). This paper highlights the potential use of LAB starter cultures for the detoxification and removal of chlorpyrifos residues in the environment.

  14. Formation of chiral morphologies through selective binding of amino acids to calcite surface steps

    NASA Astrophysics Data System (ADS)

    Orme, C. A.; Noy, A.; Wierzbicki, A.; McBride, M. T.; Grantham, M.; Teng, H. H.; Dove, P. M.; DeYoreo, J. J.

    2001-06-01

    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. But the general view that organic molecules affect mineralization through stereochemical recognition, where geometrical and chemical constraints dictate their binding to a mineral, seems difficult to reconcile with a mechanistic understanding, where crystallization is controlled by thermodynamic and kinetic factors. Indeed, traditional crystal growth models emphasize the inhibiting effect of so-called `modifiers' on surface-step growth, rather than stereochemical matching to newly expressed crystal facets. Here we report in situ atomic force microscope observations and molecular modelling studies of calcite growth in the presence of chiral amino acids that reconcile these two seemingly divergent views. We find that enantiomer-specific binding of the amino acids to those surface-step edges that offer the best geometric and chemical fit changes the step-edge free energies, which in turn results in macroscopic crystal shape modifications. Our results emphasize that the mechanism underlying crystal modification through organic molecules is best understood by considering both stereochemical recognition and the effects of binding on the interfacial energies of the growing crystal.

  15. DNA-binding properties of gene-5 protein encoded by bacteriophage M13. 2. Further characterization of the different binding modes for poly- and oligodeoxynucleic acids.

    PubMed

    Bulsink, H; Harmsen, B J; Hilbers, C W

    1988-10-01

    The binding of gene-5 protein, encoded by bacteriophage M13, to oligodeoxynucleic acids was studied by means of fluorescence binding experiments, fluorescence depolarization measurements and irreversible dissociation kinetics of the protein.nucleotide complexes with salt. The binding properties thus obtained are compared with those of the binding to polynucleotides, especially at very low salt concentration. It appears that the binding to oligonucleotides is always characterized by a stoichiometry (n) of 2-3 nucleotides/protein, and the absence of cooperativity. In contrast the protein can bind to polynucleotides in two different modes, one with a stoichiometry of n = 3 in the absence of salt and another with n = 4 at moderate salt concentrations. Both modes have a high intramode cooperativity (omega about 500) but are non-interacting and mutually exclusive. For deoxynucleic acids with a chain length of 25-30 residues a transition from oligonucleotide to polynucleotide binding is observed at increasing nucleotide/protein ratio in the solution. The n = 3 polynucleotide binding is very sensitive to the ionic strength and is only detectable at very low salt concentrations. The ionic strength dependency per nucleotide of the n = 4 binding is much less and is comparable with the salt dependency of the oligonucleotide binding. Furthermore it appears that the influence of the salt concentration on the oligonucleotide binding constant is to about the same degree determined by the effect of salt on the association and dissociation rate constants. Model calculations indicate that the fluorescence depolarization titration curves can only be explained by a model for oligonucleotide binding in which a protein dimer binds with its two dimer halves to the same strand. In addition it is only possible to explain the observed effect of the chain length of the oligonucleotide on both the apparent binding constant and the dissociation rate by assuming the existence of interactions

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

    PubMed

    Maximin, Elise; Langelier, Bénédicte; Aïoun, Josiane; Al-Gubory, Kaïs H; Bordat, Christian; Lavialle, Monique; Heberden, Christine

    2016-03-01

    Fatty acid binding protein 7 (FABP7), abundant in the embryonic brain, binds with the highest affinity to docosahexaenoic acid (DHA) and is expressed in the early stages of embryogenesis. Here, we have examined the consequences of the exposure to different DHA levels and of the in utero depletion of FABP7 on early rat brain development. Neurodevelopment was evaluated through the contents of two proteins, connexin 43 (Cx43) and cyclin-dependent kinase 5 (CDK5), both involved in neuroblast proliferation, differentiation, and migration. The dams were fed with diets presenting different DHA contents, from deficiency to supplementation. DHA brain embryos contents already differed at embryonic day 11.5 and the differences kept increasing with time. Cx43 and CDK5 contents were positively associated with the brain DHA levels. When FABP7 was depleted in vivo by injections of siRNA in the telencephalon, the enhancement of the contents of both proteins was lost in supplemented animals, but FABP7 depletion did not modify phospholipid compositions regardless of the diets. Thus, FABP7 is a necessary mediator of the effect of DHA on these proteins synthesis, but its role in DHA uptake is not critical, although FABP7 is localized in phospholipid-rich areas. Our study shows that high contents of DHA associated with FABP7 are necessary to promote early brain development, which prompted us to recommend DHA supplementation early in pregnancy.

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

    PubMed Central

    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. PMID:24497726

  18. Spectroscopic Evidence of a Bidentate-Binding of Meso-2,3-Dimercaptosuccinic Acid on Silver Nanoclusters

    SciTech Connect

    Zaluzhna, Oksana; Brightful, Lyndsey; Allison, Thomas C.; Tong, Yu ye J.

    2011-06-14

    New insight into the metal–ligand binding interaction in meso-2,3-dimercaptosuccinic acid (DMSA) protected silver nanoclusters (NCs) is presented in this work. IR, Raman and 13C NMR spectroscopic characterizations and DFT calculations suggest that DMSA forms a bidentate binding, rather than the originally- proposed monodentate binding, via two sulfur atoms with the underlying Ag7 NC, which is in agreement with recent ab initio calculations.

  19. Spectroscopic evidence of a bidentate-binding of meso-2,3-dimercaptosuccinic acid on silver nanoclusters

    NASA Astrophysics Data System (ADS)

    Zaluzhna, Oksana; Brightful, Lyndsey; Allison, Thomas C.; Tong, YuYe J.

    2011-06-01

    New insight into the metal-ligand binding interaction in meso-2,3-dimercaptosuccinic acid (DMSA) protected silver nanoclusters (NCs) is presented in this work. IR, Raman and 13C NMR spectroscopic characterizations and DFT calculations suggest that DMSA forms a bidentate binding, rather than the originally-proposed monodentate binding, via two sulfur atoms with the underlying Ag7 NC, which is in agreement with recent ab initio calculations.

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

    PubMed

    Lee, Chang Woo; Kim, Jung Eun; Do, Hackwon; Kim, Ryeo-Ok; Lee, Sung Gu; Park, Hyun Ho; Chang, Jeong Ho; Yim, Joung Han; Park, Hyun; Kim, Il-Chan; Lee, Jun Hyuck

    2015-09-11

    Fatty acid-binding proteins (FABPs) are involved in transporting hydrophobic fatty acids between various aqueous compartments of the cell by directly binding ligands inside their β-barrel cavities. Here, we report the crystal structures of ligand-unbound pFABP4, linoleate-bound pFABP4, and palmitate-bound pFABP5, obtained from gentoo penguin (Pygoscelis papua), at a resolution of 2.1 Å, 2.2 Å, and 2.3 Å, respectively. The pFABP4 and pFABP5 proteins have a canonical β-barrel structure with two short α-helices that form a cap region and fatty acid ligand binding sites in the hydrophobic cavity within the β-barrel structure. Linoleate-bound pFABP4 and palmitate-bound pFABP5 possess different ligand-binding modes and a unique ligand-binding pocket due to several sequence dissimilarities (A76/L78, T30/M32, underlining indicates pFABP4 residues) between the two proteins. Structural comparison revealed significantly different conformational changes in the β3-β4 loop region (residues 57-62) as well as the flipped Phe60 residue of pFABP5 than that in pFABP4 (the corresponding residue is Phe58). A ligand-binding study using fluorophore displacement assays shows that pFABP4 has a relatively strong affinity for linoleate as compared to pFABP5. In contrast, pFABP5 exhibits higher affinity for palmitate than that for pFABP4. In conclusion, our high-resolution structures and ligand-binding studies provide useful insights into the ligand-binding preferences of pFABPs based on key protein-ligand interactions.

  1. Binding-Induced DNA Nanomachines Triggered by Proteins and Nucleic Acids.

    PubMed

    Zhang, Hongquan; Lai, Maode; Zuehlke, Albert; Peng, Hanyong; Li, Xing-Fang; Le, X Chris

    2015-11-23

    We introduce the concept and operation of a binding-induced DNA nanomachine that can be activated by proteins and nucleic acids. This new type of nanomachine harnesses specific target binding to trigger assembly of separate DNA components that are otherwise unable to spontaneously assemble. Three-dimensional DNA tracks of high density are constructed on gold nanoparticles functionalized with hundreds of single-stranded oligonucleotides and tens of an affinity ligand. A DNA swing arm, free in solution, is linked to a second affinity ligand. Binding of a target molecule to the two ligands brings the swing arm to AuNP and initiates autonomous, stepwise movement of the swing arm around the AuNP surface. The movement of the swing arm, powered by enzymatic cleavage of conjugated oligonucleotides, cleaves hundreds of oligonucleotides in response to a single binding event. We demonstrate three nanomachines that are specifically activated by streptavidin, platelet-derived growth factor, and the Smallpox gene. Substituting the ligands enables the nanomachine to respond to other molecules. The new nanomachines have several unique and advantageous features over DNA nanomachines that rely on DNA self-assembly.

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

    PubMed

    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.

  3. Spectroscopic study on binding of gentisic acid to bovine serum albumin.

    PubMed

    Garzón, Andrés; Bravo, Iván; Carrión-Jiménez, M Rosario; Rubio-Moraga, Ángela; Albaladejo, José

    2015-01-01

    The interaction of (gentisic acid) GA with (bovine serum albumin) BSA has been studied by different spectroscopic techniques. GA is a monoanionic specie at the working pH of 7.4, it was determined by combining UV-Vis absorption spectroscopy and theoretical calculations. A set of fluorescence quenching experiments at different temperatures was carried out employing the native fluorescence of BSA. A Stern-Volmer constant (KSV) of (2.07±0.12)×10(4) mol(-1) L and a binding constant (Ka) of (8.47±4.39)×10(3) were determined at 310 K. The static quenching caused by the BSA-GA complex formation seems to play a significant role in the overall quenching process. A single binding site on BSA for GA was observed. ΔH=-55.6±0.2 kJ mol(-1) and ΔS=-104.3±0.6 J mol(-1) K(-1) were determined in a set of experiments on the dependence of Ka with the temperature. The binding process is, therefore, spontaneous and enthalpy-driven. Van der Waals forces and hydrogen bonds could also play the major role in the binding mode. The secondary structure changes of BSA in the absence and presence of GA were studied by FTIR and UV-Vis absorption spectroscopy.

  4. Thermodynamic characterization of the interaction between the human Y-box binding protein YB-1 and nucleic acids.

    PubMed

    Tanabe, Yumiko; Nagatoishi, Satoru; Tsumoto, Kouhei

    2015-09-01

    Y-box binding protein 1 (YB-1) binds to both RNA and DNA to control transcription and translation for the regulation of various cellular systems. YB-1 is overexpressed in some cancer cells and is a potential target for treatment of cancer. Herein, we describe isothermal titration calorimetry analyses of the interaction between a number of recombinant YB-1 domains and nucleic acids to identify the RNA and DNA binding sites and their binding mechanisms. These results demonstrated that the C-terminal domain of the protein interacts with single-stranded DNA and RNA by exothermic and endothermic reactions, respectively. The highly conserved cold-shock domain (CSD) also bound to single-stranded RNA and DNA by exothermic and endothermic reactions, respectively. The specific binding manner for RNA is in the CSD, whereas DNA binds with the most affinity to the C-terminal region (amino acids 130-219). We found further that the C-terminal region (amino acids 220-324) regulates the binding stoichiometry of RNA. These quantitative thermodynamic results provide a preliminary indication on the molecular mechanism of binding of the multifunctional protein YB-1 to nucleic acids to regulate its biological function.

  5. The RRM Domain of Human Fused in Sarcoma Protein Reveals a Non-Canonical Nucleic Acid Binding Site

    PubMed Central

    Liu, Xuehui; Niu, Chunyan; Ren, Jintao; Zhang, Jiayu; Xie, Xiaodong; Zhu, Haining; Feng, Wei; Gong, Weimin

    2012-01-01

    Fused in sarcoma (FUS) is involved in many processes of RNA metabolism. FUS and another RNA binding protein, TDP-43, are implicated in amyotrophic lateral sclerosis (ALS). It is significant to characterize the RNA recognition motif (RRM) of FUS as its nucleic acid binding properties are unclear. More importantly, abolishing the RNA binding ability of the RRM domain of TDP43 was reported to suppress the neurotoxicity of TDP-43 in Drosophila. The sequence of FUS-RRM varies significantly from canonical RRMs, but the solution structure of FUS-RRM determined by NMR showed a similar overall folding as other RRMs. We found that FUS-RRM directly bound to RNA and DNA and the binding affinity was in the micromolar range as measured by surface plasmon resonance and NMR titration. The nucleic acid binding pocket in FUS-RRM is significantly distorted since several critical aromatic residues are missing. An exceptionally positively charged loop in FUS-RRM, which is not found in other RRMs, is directly involved in the RNA/DNA binding. Substituting the lysine residues in the unique KK loop impaired the nucleic acid binding and altered FUS subcellular localization. The results provide insights into the nucleic acid binding properties of FUS-RRM and its potential relevance to ALS. PMID:23200923

  6. Effects of chronic celiprolol treatment on brown fat, feeding, and drinking in fa/fa Zucker rats.

    PubMed

    Savontaus, E; Rouru, J; Malminiemi, K; Luukkaa, V; Pesonen, U; Koulu, M; Huupponen, R

    2000-04-01

    Celiprolol is a novel beta-adrenoceptor blocking drug that displays clinically favorable effects on glucose and lipid metabolism. Because some other atypical beta-adrenoceptor blocking drugs have been described to act as agonists on beta(3)-adrenoceptors, we aimed to investigate the effects of celiprolol on brown fat and beta(3)-adrenoceptors. Chronic treatment of obese fa/fa Zucker rats with celiprolol (50 mg/kg/day orally for 20 days) increased GDP binding to brown fat mitochondria by 1.5-fold, whereas beta(3)-adrenoceptor agonist ZD7114 ((S)-4-[2-[(2-hydroxy-3-phenoxypropyl)amino]ethoxy]-N-(2-methoxyet hyl )phenoxyacetamide, 3 mg/kg/day) increased the binding by 3.3-fold. Weight gain was reduced by 19% due to decreased water and food intakes in celiprolol-treated rats. Celiprolol did not activate lipolysis in rat adipocytes in vitro or stimulate human beta(3)-adrenoceptors expressed in Chinese hamster ovary cells as measured with Cytosensor microphysiometer. Therefore, celiprolol does not seem to activate brown fat via beta(3)-adrenoceptors. PMID:10764928

  7. Opuntia ficus indica (nopal) attenuates hepatic steatosis and oxidative stress in obese Zucker (fa/fa) rats.

    PubMed

    Morán-Ramos, Sofía; Avila-Nava, Azalia; Tovar, Armando R; Pedraza-Chaverri, José; López-Romero, Patricia; Torres, Nimbe

    2012-11-01

    Nonalcoholic fatty liver disease (NAFLD) is associated with multiple factors such as obesity, insulin resistance, and oxidative stress. Nopal, a cactus plant widely consumed in the Mexican diet, is considered a functional food because of its antioxidant activity and ability to improve biomarkers of metabolic syndrome. The aim of this study was to assess the effect of nopal consumption on the development of hepatic steatosis and hepatic oxidative stress and on the regulation of genes involved in hepatic lipid metabolism. Obese Zucker (fa/fa) rats were fed a control diet or a diet containing 4% nopal for 7 wk. Rats fed the nopal-containing diet had ∼50% lower hepatic TG than the control group as well as a reduction in hepatomegaly and biomarkers of hepatocyte injury such as alanine and aspartate aminotransferases. Attenuation of hepatic steatosis by nopal consumption was accompanied by a higher serum concentration of adiponectin and a greater abundance of mRNA for genes involved in lipid oxidation and lipid export and production of carnitine palmitoyltransferase-1 and microsomal TG transfer proteins in liver. Hepatic reactive oxygen species and lipid peroxidation biomarkers were significantly lower in rats fed nopal compared with the control rats. Furthermore, rats fed the nopal diet had a lower postprandial serum insulin concentration and a greater liver phosphorylated protein kinase B (pAKT):AKT ratio in the postprandial state. This study suggests that nopal consumption attenuates hepatic steatosis by increasing fatty acid oxidation and VLDL synthesis, decreasing oxidative stress, and improving liver insulin signaling in obese Zucker (fa/fa) rats. PMID:23014486

  8. Opuntia ficus indica (nopal) attenuates hepatic steatosis and oxidative stress in obese Zucker (fa/fa) rats.

    PubMed

    Morán-Ramos, Sofía; Avila-Nava, Azalia; Tovar, Armando R; Pedraza-Chaverri, José; López-Romero, Patricia; Torres, Nimbe

    2012-11-01

    Nonalcoholic fatty liver disease (NAFLD) is associated with multiple factors such as obesity, insulin resistance, and oxidative stress. Nopal, a cactus plant widely consumed in the Mexican diet, is considered a functional food because of its antioxidant activity and ability to improve biomarkers of metabolic syndrome. The aim of this study was to assess the effect of nopal consumption on the development of hepatic steatosis and hepatic oxidative stress and on the regulation of genes involved in hepatic lipid metabolism. Obese Zucker (fa/fa) rats were fed a control diet or a diet containing 4% nopal for 7 wk. Rats fed the nopal-containing diet had ∼50% lower hepatic TG than the control group as well as a reduction in hepatomegaly and biomarkers of hepatocyte injury such as alanine and aspartate aminotransferases. Attenuation of hepatic steatosis by nopal consumption was accompanied by a higher serum concentration of adiponectin and a greater abundance of mRNA for genes involved in lipid oxidation and lipid export and production of carnitine palmitoyltransferase-1 and microsomal TG transfer proteins in liver. Hepatic reactive oxygen species and lipid peroxidation biomarkers were significantly lower in rats fed nopal compared with the control rats. Furthermore, rats fed the nopal diet had a lower postprandial serum insulin concentration and a greater liver phosphorylated protein kinase B (pAKT):AKT ratio in the postprandial state. This study suggests that nopal consumption attenuates hepatic steatosis by increasing fatty acid oxidation and VLDL synthesis, decreasing oxidative stress, and improving liver insulin signaling in obese Zucker (fa/fa) rats.

  9. The binding of folic acid and 5-methyltetrahydrofolate to folate-binding proteins during gastric passage differs in a dynamic in vitro gastrointestinal model.

    PubMed

    Verwei, Miriam; Arkbåge, Karin; Mocking, Hans; Havenaar, Robert; Groten, John

    2004-01-01

    Despite its low natural folate concentration, milk is responsible for 10-15% of the daily folate intake in countries with a high dairy consumption. Milk products can be considered as a potential matrix for folate fortification, e.g., with synthetic folic acid, to enhance the daily intake of folate. In untreated milk, the natural folate, 5-methyltetrahydrofolate (5-CH3-H4folate), is bound to folate-binding proteins (FBP). In this study, the extent of binding to FBP for folic acid and 5-CH3-H4folate was investigated in a dynamic in vitro model simulating human gastric passage. Protein binding of folic acid and 5-CH3-H4folate was characterized using gel-exclusion chromatography. Before gastric passage, folic acid and 5-CH3-H4folate were bound mainly to FBP (76-79%), whereas 7% was free. Folic acid remained bound to FBP to a similar extent after gastric passage. For 5-CH3-H4folate, the FBP-bound fraction gradually decreased from 79 to 5% and the free fraction increased from 7 to 93%. Although folic acid enters the proximal part of the intestine bound to FBP, 5-CH3-H4folate appears to be present mainly as free folate in the duodenal lumen. The stability of FBP was similar in both folate/FBP mixtures, i.e., 70% of the initial FBP content was retained after gastric passage. This study indicated that FBP are partly stable during gastric passage but have different binding characteristics for folic acid and 5-CH3-H4folate in the duodenal lumen. This could result in different bioavailability from folic acid- and 5-CH3-H4folate-fortified milk products.

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

    PubMed

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

    2015-07-21

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

  11. Binding of the substrate UDP-glucuronic acid induces conformational changes in the xanthan gum glucuronosyltransferase.

    PubMed

    Salinas, S R; Petruk, A A; Brukman, N G; Bianco, M I; Jacobs, M; Marti, M A; Ielpi, L

    2016-06-01

    GumK is a membrane-associated glucuronosyltransferase of Xanthomonas campestris that is involved in xanthan gum biosynthesis. GumK belongs to the inverting GT-B superfamily and catalyzes the transfer of a glucuronic acid (GlcA) residue from uridine diphosphate (UDP)-GlcA (UDP-GlcA) to a lipid-PP-trisaccharide embedded in the membrane of the bacteria. The structure of GumK was previously described in its apo- and UDP-bound forms, with no significant conformational differences being observed. Here, we study the behavior of GumK toward its donor substrate UDP-GlcA. Turbidity measurements revealed that the interaction of GumK with UDP-GlcA produces aggregation of protein molecules under specific conditions. Moreover, limited proteolysis assays demonstrated protection of enzymatic digestion when UDP-GlcA is present, and this protection is promoted by substrate binding. Circular dichroism spectroscopy also revealed changes in the GumK tertiary structure after UDP-GlcA addition. According to the obtained emission fluorescence results, we suggest the possibility of exposure of hydrophobic residues upon UDP-GlcA binding. We present in silico-built models of GumK complexed with UDP-GlcA as well as its analogs UDP-glucose and UDP-galacturonic acid. Through molecular dynamics simulations, we also show that a relative movement between the domains appears to be specific and to be triggered by UDP-GlcA. The results presented here strongly suggest that GumK undergoes a conformational change upon donor substrate binding, likely bringing the two Rossmann fold domains closer together and triggering a change in the N-terminal domain, with consequent generation of the acceptor substrate binding site. PMID:27099353

  12. Structural Basis for Activation of Fatty Acid-binding Protein 4

    SciTech Connect

    Gillilan,R.; Ayers, S.; Noy, N.

    2007-01-01

    Fatty acid-binding protein 4 (FABP4) delivers ligands from the cytosol to the nuclear receptor PPAR{gamma} in the nucleus, thereby enhancing the transcriptional activity of the receptor. Notably, FABP4 binds multiple ligands with a similar affinity but its nuclear translocation is activated only by specific compounds. To gain insight into the structural features that underlie the ligand-specificity in activation of the nuclear import of FABP4, we solved the crystal structures of the protein complexed with two compounds that induce its nuclear translocation, and compared these to the apo-protein and to FABP4 structures bound to non-activating ligands. Examination of these structures indicates that activation coincides with closure of a portal loop phenylalanine side-chain, contraction of the binding pocket, a subtle shift in a helical domain containing the nuclear localization signal of the protein, and a resultant change in oligomeric state that exposes the nuclear localization signal to the solution. Comparisons of backbone displacements induced by activating ligands with a measure of mobility derived from translation, libration, screw (TLS) refinement, and with a composite of slowest normal modes of the apo state suggest that the helical motion associated with the activation of the protein is part of the repertoire of the equilibrium motions of the apo-protein, i.e. that ligand binding does not induce the activated configuration but serves to stabilize it. Nuclear import of FABP4 can thus be understood in terms of the pre-existing equilibrium hypothesis of ligand binding.

  13. Binding of Eu(III) to 1,2-hydroxypyridinone-modified peptide nucleic acids.

    PubMed

    de Leon, Arnie R; Olatunde, Abiola O; Morrow, Janet R; Achim, Catalina

    2012-12-01

    Substitution of a nucleobase pair with a pair of 1,2-hydroxypyridinone (1,2-HOPO) ligands in the center of a 10-base-pair peptide nucleic acid (PNA) duplex provides a strong binding site for Eu(III) as evidenced by UV thermal melting curves, UV titrations, and luminescence spectroscopy. Eu(III) excitation spectra and luminescence lifetime data are consistent with Eu(III) bound to both 1,2 HOPO ligands in a PNA-HOPO duplex as the major species present in solution.

  14. Polydiacetylene liposomes functionalized with sialic acid bind and colorimetrically detect influenza virus

    SciTech Connect

    Reichert, A.; Nagy, J.O.; Spevak, W.; Charych, D. )

    1995-01-18

    In this paper we have demonstrated that polymerized liposomes are biomolecular materials that provide a molecular recognition function (sialic acid) and a detection element (polydiacetylene backbone), all within a single supramolecular assembly. The binding event is transduced to a visible color change, readily seen with the naked eye and quantified by absorption spectroscopy. Specificity of the color change was demonstrated by competitive inhibition studies. In addition, nonspecific adsorption, if it occurs. does not appear to affect the color of the liposome solutions. 28 refs., 2 figs.

  15. Functional groups of sialic acids involved in binding to siglecs (sialoadhesins) deduced from interactions with synthetic analogues.

    PubMed

    Kelm, S; Brossmer, R; Isecke, R; Gross, H J; Strenge, K; Schauer, R

    1998-08-01

    The siglecs, formerly called sialoadhesins, are a family of I-type lectins binding to sialic acids on the cell surface. Five members of this family have been identified: sialoadhesin, myelin-associated glycoprotein (MAG), Schwann cell myelin protein (SMP), CD22 and CD33. We have investigated the relevance of substituents at position C-9 and in the N-acetyl group of N-acetylneuraminic acid, using a series of synthetic sialic-acid analogues either on resialylated human erythrocytes or as free alpha-glycosides in hapten inhibition. All five siglecs require the hydroxy group at C-9 for binding, suggesting hydrogen bonding of this substituent with the binding site. Remarkable differences were found among the proteins in their specificity for modifications of the N-acetyl group. Whereas sialoadhesin, MAG and SMP do not tolerate a hydroxy group as in N-glycolylneuraminic acid, they bind to halogenated acetyl residues. In the case of MAG, N-fluoroacetylneuraminic acid is bound about 17-fold better than N-acetylneuraminic acid. In contrast, human and murine CD22 both show good affinity for N-glycolylneuraminic acid, but only human CD22 bound the halogenated compounds. In conclusion, our data indicate that interactions of the hydroxy group at position 9 and the N-acyl substituent contribute significantly to the binding strength. PMID:9738906

  16. Anthramycin binding to deoxyribonucleic acid-mitomycin C complexes. Evidence for drug-induced deoxyribonucleic acid conformational change and cooperativity in mitomycin C binding.

    PubMed

    Kaplan, D J; Hurley, L H

    1981-12-22

    Anthramycin and mitomycin C (MC) are two DNA reactive drugs, which bind covalently to GC pairs producing different effects on DNA: anthramycin stiffening and MC distorsion. This paper describes experiments in which we have used anthramycin as a probe to sense quantitatively the effects on DNA of MC binding. Saturation binding experiments show that both anthramycin and MC partially inhibit the binding of the other drug to DNA (maximum inhibition by MC and anthramycin, 22.4% and 19.7%, respectively) but by a mechanism other than direct site exclusion. This suggests that MC binds in the major groove of DNA, since anthramycin is known to bind in the minor groove. An abrupt reduction in the binding of anthramycin to DNA-MC complexes occurs between MC binding ratios of 0.030 and 0.035, which parallels and probably results from sudden intensification of a MC-induced DNA conformational change occurring between these binding ratios. Dialysis measurements indicate that anthramycin is very possibly binding at sites distant from MC sites and suggest a clustering of closely bound MC chromophores resulting from possible cooperative binding. S1 nuclease digest experiments demonstrate an initial enhancement of nuclease activity in DNA-MC complexes, the magnitude of which correlates well with the reduction of anthramycin binding, relative to the degree of MC binding. The enhanced nuclease activity in these complexes indicates regions of exposed DNA or helix base distortion which is related to or is the result of conformational change. PMID:6798992

  17. GPM Video of In-fa

    NASA Video Gallery

    On Nov. 23, GPM saw In-fa dropping rain at an extreme rate of over 266 mm (10.5 inches) per hour in storms just to the northwest of the typhoon's eye where thunderstorms reached altitudes of over 1...

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

    DOE PAGES

    Kashyap, Sanjay; Woehl, Taylor; Valverde-Tercedor, Carmen; Sánchez-Quesada, Miguel; Jiménez López, Concepción; Prozorov, Tanya

    2014-01-01

    Biological macromolecules are utilized in low-temperature synthetic methods to exert precise control over nanoparticle nucleation and placement. They enable low-temperature formation of a variety of functional nanostructured materials with properties often not achieved via conventional synthetic techniques. Here we report on the in situ visualization of a novel acidic bacterial recombinant protein, MamC, commonly present in the magnetosome membrane of several magnetotactic bacteria, including Magnetococcus marinus , strain MC-1. Our findings provide an insight into the self-assembly of MamC and point to formation of the extended protein surface, which is assumed to play an important role in the formationmore » of biotemplated inorganic nanoparticles. The self-organization of MamC is compared to the behavior of another acidic recombinant iron-binding protein, Mms6.« less

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

    SciTech Connect

    Kashyap, Sanjay; Woehl, Taylor; Valverde-Tercedor, Carmen; Sanchez-Quesada, Miguel; Lopez, Concepcion Jimenez; Prozorov, Tanya

    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.

  20. Analogs of the antituberculous agent pyrazinamide are competitive inhibitors of NADPH binding to M. tuberculosis fatty acid synthase I.

    PubMed

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

    2012-11-01

    Analogs of pyrazinamide (=pyrazine-2-carboxamide; PZA), an essential component of short-course antituberculous chemotherapy, such as 5-chloropyrazinamide (5-Cl-PZA) act as competitive inhibitors of NADPH binding to purified mycobacterial fatty acid synthase I (FAS I) as shown by Saturation Transfer Difference (STD) NMR studies. In addition, pyrazinoic acid esters (POE) and 5-Cl-POE reversibly bind to FAS I with the relatively greater affinity of longer-chain esters for FAS I, clear from the STD amplification factors. The competitive binding of PZA and 5-Cl-PZA clearly illustrates that both agents bind FAS. In contrast to PZA, at low NADPH concentrations 5-Cl-PZA is a cooperative inhibitor of NADPH binding.

  1. Effect of omega-3 fatty acids on the modification of erythrocyte membrane fatty acid content including oleic acid in peritoneal dialysis patients.

    PubMed

    An, W S; Lee, S M; Son, Y K; Kim, S E; Kim, K H; Han, J Y; Bae, H R; Park, Y

    2012-01-01

    Erythrocyte membrane fatty acids (FA), such as oleic acid, are related to acute coronary syndrome. There is no report about the effect of omega-3 FA on oleic acid in peritoneal dialysis (PD) patients. We hypothesized that omega-3 FA can modify erythrocyte membrane FA, including oleic acid, in PD patients. In a double-blind, randomized, placebo-controlled study, 18 patients who were treated with PD for at least 6 months were randomized to treatment for 12 weeks with omega-3 FA or placebo. Erythrocyte membrane FA content was measured by gas chromatography at baseline and after 12 weeks. The erythrocyte membrane content of eicosapentaenoic acid and docosahexaenoic acid was significantly increased and saturated FA and oleic acid were significantly decreased in the omega-3 FA supplementation group after 12 weeks compared to baseline. In conclusion, erythrocyte membrane FA content, including oleic acid, was significantly modified by omega-3 FA supplementation for 12 weeks in PD patients.

  2. Investigations of acetaminophen binding to bovine serum albumin in the presence of fatty acid: Fluorescence and 1H NMR studies

    NASA Astrophysics Data System (ADS)

    Bojko, B.; Sułkowska, A.; Maciążek-Jurczyk, M.; Równicka, J.; Sułkowski, W. W.

    2009-04-01

    The binding of acetaminophen to bovine serum albumin (BSA) was studied by the quenching fluorescence method and the proton nuclear magnetic resonance technique ( 1H NMR). For fluorescence measurements 1-anilino-9-naphthalene sulfonate (ANS) hydrophobic probe was used to verify subdomain IIIA as acetaminophen's likely binding site. Three binding sites of acetaminophen in subdomain IIA of bovine serum albumin were found. Quenching constants calculated by the Stern-Volmer modified method were used to estimate the influence of myristic acid (MYR) on the drug binding to the albumin. The influence of [fatty acid]/[albumin] molar ratios on the affinity of the protein towards acetaminophen was described. Changes of chemical shifts and relaxation times of the drug indicated that the presence of MYR inhibits interaction in the AA-albumin complex. It is suggested that the elevated level of fatty acids does not significantly influence the pharmacokinetics of acetaminophen.

  3. Binding studies of a sialic acid-specific lectin from the horseshoe crab Carcinoscorpius rotunda cauda with various sialoglycoproteins.

    PubMed

    Mohan, S; Thambi Dorai, D; Srimal, S; Bachhawat, B K

    1982-04-01

    Interaction of the sialic acid-specific lectin carcinoscorpin with various sialoglycoproteins was studied by using radioiodinated lectin. The binding of carcinoscorpin was dependent not only on sialic acid content but also on the type of glycosidic linkage and form (branched or linear) of the carbohydrate chains. Carcinoscorpin has different classes of binding sites, and binding follows a phenomenon of positive co-operativity. The effect of Ca2+ concentration on the binding was studied, and the optimal concentration was found to be 0.02 M. Effect of pH, temperature and other bivalent metal ions are also reported. From haemagglutination- and precipitation-inhibition studies, it was concluded that carcinoscorpin has multispecificity towards acidic sugars, and its relation to the biological role of the lectin in the horseshoe crab is discussed.

  4. Two types of fatty acid-binding protein in human kidney. Isolation, characterization and localization.

    PubMed Central

    Maatman, R G; Van Kuppevelt, T H; Veerkamp, J H

    1991-01-01

    Two types of fatty acid-binding protein (FABP) were isolated from human kidney by gel filtration and ion-exchange chromatography. Northern-blot analysis showed the presence of two FABP transcripts in total kidney RNA, hybridizing with cDNA of human liver and muscle FABP respectively. Characterisation based on molecular mass, isoelectric point, fluorescence with dansylaminoundecanoic acid and immunological cross-reactivity showed that one, type B, was fairly similar to human heart FABP. The other, type A, showed, like human liver FABP, a high fluorescence enhancement and a wavelength shift with dansylaminoundecanoic acid as well as the binding of a variety of ligands. Antibodies raised against FABP type A and against liver FABP markedly cross-reacted in e.l.i.s.a., in Western blotting and in indirect immunoperoxidase staining on kidney and liver sections. Differences in amino acid composition and isoelectric points, however, indicate that type A is a new kidney-specific FABP type. The FABP type A is more abundant in kidney than the B type and is predominantly localized in the cortex, especially in the cells of the proximal tubules. The FABP type B is mainly present in the cells of the distal tubules. In conclusion, this study shows the presence of two types of FABP in the kidney. One type seems to be related to heart FABP, while the other type resembles, but is not identical with, liver FABP. Both types have a characteristic cellular distribution along the nephron. Images Fig. 2. Fig. 3. Fig. 6. Fig. 7. Fig. 8. Fig. 9. PMID:1996972

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    PubMed

    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.

  7. Flocculation and Membrane Binding of Outer Membrane Protein F, Porin, at Acidic pH

    NASA Astrophysics Data System (ADS)

    Suzuki, Keiko; Nakae, Taiji; Mitaku, Shigeki

    1998-04-01

    Outer membrane protein F (OmpF), porin, of Escherichia coli is an intrinsic membrane protein made of a β-sheet barrel, the amino acid sequence being as hydrophilic as many soluble proteins in spite of its location in the hydrophobic region of membrane. The binding of porin molecules with a lipid membrane and the flocculation of the protein were studied at various pH, using the combination of centrifugation and intrinsic fluorescence measurements. The binding of porin with the lipid membrane occurred in the pH range below 7, whereas the flocculation of porin in the absence of the membrane was observed only at pH below 5. Porin molecules in the pH range between 5 and 7 were stable as a colloid but spontaneously bound with the lipid membrane soon after the addition of lipid vesicles. The possible mechanism of the structural formation of porin in the outer membrane was discussed based on the pH dependence of the membrane binding ability of this protein.

  8. Structural and biochemical characterization of the lungfish (Lepidosiren paradoxa) liver basic fatty acid binding protein.

    PubMed

    Di Pietro, S M; Santomé, J A

    2001-04-01

    Only one fatty acid-binding protein (FABP) from the liver of the lungfish (Lepidosiren paradoxa) was isolated and characterized. The sequence comparison of lungfish FABP with that of the known members of the liver FABP (L-FABP) and liver basic FABP (Lb-FABP) subfamilies indicates that it is more closely related to chicken, iguana, frog, axolotl, catfish, and shark Lb-FABPs than to mammalian and axolotl L-FABPs. Lungfish liver expression of this single Lb-FABP contrasts with the other fish studied so far which coexpress an Lb-FABP with heart-adipocyte and/or intestinal FABP types. The lungfish liver FABP expression pattern resembles that of tetrapods, which only expresses liver type FABPs. Lungfish Lb-FABP is one of the two FABPs reported to have a disulfide bridge. The molecular modeling of lungfish Lb-FABP predicts that nine of the conserved residues of Lb-FABPs are oriented toward the binding cavity, thus suggesting they are related to the protein binding characteristics.

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

    NASA Astrophysics Data System (ADS)

    Raman, N.; Sobha, S.; Selvaganapathy, M.; Mahalakshmi, R.

    2012-10-01

    Few transition metal complexes of tetradentate N2O2 donor Schiff base ligands containing 2-hydroxybenzylidene-4-aminoantipyrine and amino acids (alanine/valine) abbreviated to KHL1/KHL2 have been synthesized. All the metal complexes have been fully characterized with the help of elemental analyses, molecular weights, molar conductance values, magnetic moments and spectroscopic data. The Schiff bases KHL1/KHL2 are found to act as tetradentate ligands using N2O2 donor set of atoms leading to a square-planar geometry for the complexes around the metal ions. The binding behaviors of the complexes to calf thymus DNA have been investigated by absorption spectra, viscosity measurements and cyclic voltammetry. The DNA binding constants reveal that all these complexes interact with DNA through minor groove binding mode. The studies on mechanism of photocleavage reveal that singlet oxygen (1O2) and superoxide anion radical (O2rad -) may play an important role in the photocleavage. The Schiff bases and their metal complexes have been screened for their in vitro antibacterial activities against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epidermidis, Klebsiella pneumoniae and antifungal activities against Aspergillus niger, Fusarium solani, Culvularia lunata, Rhizoctonia bataicola and Candida albicans by MIC method.

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

    PubMed

    Raman, N; Sobha, S; Selvaganapathy, M; Mahalakshmi, R

    2012-10-01

    Few transition metal complexes of tetradentate N(2)O(2) donor Schiff base ligands containing 2-hydroxybenzylidene-4-aminoantipyrine and amino acids (alanine/valine) abbreviated to KHL(1)/KHL(2) 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 KHL(1)/KHL(2) are found to act as tetradentate ligands using N(2)O(2) 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 ((1)O(2)) and superoxide anion radical (O(2)(-)) 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. PMID:22885083

  11. Adenovirus carrying gene encoding Haliotis discus discus sialic acid binding lectin induces cancer cell apoptosis.

    PubMed

    Yang, Xinyan; Wu, Liqin; Duan, Xuemei; Cui, Lianzhen; Luo, Jingjing; Li, Gongchu

    2014-06-30

    Lectins exist widely in marine bioresources such as bacteria, algae, invertebrate animals and fishes. Some purified marine lectins have been found to elicit cytotoxicity to cancer cells. However, there are few reports describing the cytotoxic effect of marine lectins on cancer cells through virus-mediated gene delivery. We show here that a replication-deficient adenovirus-carrying gene encoding Haliotis discus discus sialic acid binding lectin (Ad.FLAG-HddSBL) suppressed cancer cell proliferation by inducing apoptosis, as compared to the control virus Ad.FLAG. A down-regulated level of anti-apoptosis factor Bcl-2 was suggested to be responsible for the apoptosis induced by Ad.FLAG-HddSBL infection. Further subcellular localization studies revealed that HddSBL distributed in cell membrane, ER, and the nucleus, but not in mitochondria and Golgi apparatus. In contrast, a previously reported mannose-binding lectin Pinellia pedatisecta agglutinin entered the nucleus as well, but did not distribute in inner membrane systems, suggesting differed intracellular sialylation and mannosylation, which may provide different targets for lectin binding. Further cancer-specific controlling of HddSBL expression and animal studies may help to provide insights into a novel way of anti-cancer marine lectin gene therapy. Lectins may provide a reservoir of anti-cancer genes.

  12. Attenuated murine cytomegalovirus binds to N-acetylglucosamine, and shift to virulence may involve recognition of sialic acids.

    PubMed Central

    Ravindranath, R M; Graves, M C

    1990-01-01

    Treatment of cells with lectins specific for N-acetylglucosamine (GlcNAc) blocked infection by mouse cytomegalovirus (MCMV), and GlcNAc pretreatment of the lectin blocked this effect. MCMV failed to infect N-acetylglucosaminidase (GlcNAcase)-treated mouse embryo fibroblasts (MEF). GlcNAc and GlcNAc-containing synthetic oligosaccharides directly inhibited viral infectivity. Ulex lectin inhibition of infection was shown to be due to inhibition of surface adsorption of 35S-labeled virus. Also, GlcNAcase eluted 35S-labeled virus adsorbed to MEF at 4 degrees C and inhibited plaque formation if added after adsorption at this temperature. These findings indicate that GlcNAc binding is involved in attachment rather than in some later step in infection. High-performance thin-layer chromatography overlay of [35S]MCMV indicated that it binds to a GlcNAc-containing asialoglycolipid. Analogous experiments indicated that MCMV made virulent by in vivo salivary gland passage binds to sialic acids in addition to GlcNAc. Treatment of MEF with sialic acid-binding lectins blocked infectivity. Incubation of virus with sialic acids also prevented infection. N-acetylneuraminic acid was 10(3)-fold more potent than N-glycolylneuraminic acid. Sialidase-treated target cells were not efficiently infected by the virus. Thus, MCMV binds to GlcNAc on the cell surface, and the shift to virulence (by in vivo salivary gland passage) correlates with viral recognition of sialic acids. Images PMID:2170680

  13. Lipin 2 binds phosphatidic acid by the electrostatic hydrogen bond switch mechanism independent of phosphorylation.

    PubMed

    Eaton, James M; Takkellapati, Sankeerth; Lawrence, Robert T; McQueeney, Kelley E; Boroda, Salome; Mullins, Garrett R; Sherwood, Samantha G; Finck, Brian N; Villén, Judit; Harris, Thurl E

    2014-06-27

    Lipin 2 is a phosphatidic acid phosphatase (PAP) responsible for the penultimate step of triglyceride synthesis and dephosphorylation of phosphatidic acid (PA) to generate diacylglycerol. The lipin family of PA phosphatases is composed of lipins 1-3, which are members of the conserved haloacid dehalogenase superfamily. Although genetic alteration of LPIN2 in humans is known to cause Majeed syndrome, little is known about the biochemical regulation of its PAP activity. Here, in an attempt to gain a better general understanding of the biochemical nature of lipin 2, we have performed kinetic and phosphorylation analyses. We provide evidence that lipin 2, like lipin 1, binds PA via the electrostatic hydrogen bond switch mechanism but has a lower rate of catalysis. Like lipin 1, lipin 2 is highly phosphorylated, and we identified 15 phosphosites. However, unlike lipin 1, the phosphorylation of lipin 2 is not induced by insulin signaling nor is it sensitive to inhibition of the mammalian target of rapamycin. Importantly, phosphorylation of lipin 2 does not negatively regulate either membrane binding or PAP activity. This suggests that lipin 2 functions as a constitutively active PA phosphatase in stark contrast to the high degree of phosphorylation-mediated regulation of lipin 1. This knowledge of lipin 2 regulation is important for a deeper understanding of how the lipin family functions with respect to lipid synthesis and, more generally, as an example of how the membrane environment around PA can influence its effector proteins. PMID:24811178

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

    PubMed Central

    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

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

    PubMed Central

    Bottasso Arias, Natalia M.; García, Marina; Bondar, Constanza; Guzman, Luciana; Redondo, Agustina; Chopita, Nestor; Córsico, Betina; Chirdo, Fernando G.

    2015-01-01

    Celiac disease (CD) is an immune-mediated enteropathy that develops in genetically susceptible individuals following exposure to dietary gluten. Severe changes at the intestinal mucosa observed in untreated CD patients are linked to changes in the level and in the pattern of expression of different genes. Fully differentiated epithelial cells express two isoforms of fatty acid binding proteins (FABPs): intestinal and liver, IFABP and LFABP, respectively. These proteins bind and transport long chain fatty acids and also have other important biological roles in signaling pathways, particularly those related to PPARγ and inflammatory processes. Herein, we analyze the serum levels of IFABP and characterize the expression of both FABPs at protein and mRNA level in small intestinal mucosa in severe enteropathy and normal tissue. As a result, we observed higher levels of circulating IFABP in untreated CD patients compared with controls and patients on gluten-free diet. In duodenal mucosa a differential FABPs expression pattern was observed with a reduction in mRNA levels compared to controls explained by the epithelium loss in severe enteropathy. In conclusion, we report changes in FABPs' expression pattern in severe enteropathy. Consequently, there might be alterations in lipid metabolism and the inflammatory process in the small intestinal mucosa. PMID:26346822

  16. Structure of shikimate kinase from Mycobacterium tuberculosis reveals the binding of shikimic acid.

    PubMed

    Pereira, José Henrique; de Oliveira, Jaim Simões; Canduri, Fernanda; Dias, Marcio Viniciusbertacine; Palma, Mário Sérgio; Basso, Luiz Augusto; Santos, Diógenes Santiago; de Azevedo, Walter Filgueira

    2004-12-01

    Tuberculosis made a resurgence in the mid-1980s and now kills approximately 3 million people a year. The re-emergence of tuberculosis as a public health threat, the high susceptibility of HIV-infected persons and the proliferation of multi-drug-resistant strains have created a need to develop new drugs. Shikimate kinase and other enzymes in the shikimate pathway are attractive targets for development of non-toxic antimicrobial agents, herbicides and anti-parasitic drugs, because the pathway is essential in these species whereas it is absent from mammals. The crystal structure of shikimate kinase from Mycobacterium tuberculosis (MtSK) complexed with MgADP and shikimic acid (shikimate) has been determined at 2.3 A resolution, clearly revealing the amino-acid residues involved in shikimate binding. This is the first three-dimensional structure of shikimate kinase complexed with shikimate. In MtSK, the Glu61 residue that is strictly conserved in shikimate kinases forms a hydrogen bond and salt bridge with Arg58 and assists in positioning the guanidinium group of Arg58 for shikimate binding. The carboxyl group of shikimate interacts with Arg58, Gly81 and Arg136 and the hydroxyl groups interact with Asp34 and Gly80. The crystal structure of MtSK-MgADP-shikimate will provide crucial information for the elucidation of the mechanism of the shikimate kinase-catalyzed reaction and for the development of a new generation of drugs against tuberculosis.

  17. Hypertension induces tissue-specific gene suppression of a fatty acid binding protein in rat aorta.

    PubMed Central

    Sarzani, R; Claffey, K P; Chobanian, A V; Brecher, P

    1988-01-01

    The effect of hypertension on the expression of a fatty acid binding protein localized in the rat aorta was studied. The presence of rat heart fatty acid binding protein (hFABP) was documented in aortic tissue by using a cDNA probe and polyclonal antibodies. Hypertension was induced in groups of rats by implantation of deoxycorticosterone acetate in conjunction with 1% salt in the drinking water (deoxycorticosterone/salt). By the third week of this treatment a marked reduction (by a factor of 20) in the expression of hFABP mRNA in aorta was found, concomitant with a reduction in immunologically detectable protein, suggesting transcriptional regulation. This effect was tissue specific, since no change in the normal amounts of hFABP mRNA in heart, skeletal muscle, or kidney was found. This reduction in aortic hFABP mRNA was also found in mildly hypertensive uninephrectomized rats given salt but no deoxycorticosterone and in normotensive rats given deoxycorticosterone but no excess salt intake. A marked decrease in aortic hFABP mRNA also was observed in the Goldblatt two kidney-one clip hypertensive model, and administration of angiotensin II for 6 days by osmotic minipump also caused a reduction. These findings suggest that hFABP is under complex regulation in aortic tissue and is suppressed by arterial hypertension. Images PMID:3174661

  18. Molecular cloning of a cDNA encoding a novel fatty acid-binding protein from rat skin.

    PubMed

    Watanabe, R; Fujii, H; Odani, S; Sakakibara, J; Yamamoto, A; Ito, M; Ono, T

    1994-04-15

    A novel skin-type fatty acid-binding protein, termed cutaneous(C)-FABP, has been purified from rat skin and a cDNA clone for this protein has been identified. The purified protein had the ability to bind long chain fatty acids like other rat FABPs. The deduced amino acid sequence of the cDNA clone comprises residues yielding a molecular mass for the polypeptide of 15.1 kDa and exhibits around 50% identity to myelin P2 protein, adipocyte FABP and heart FABP. Our results propose that C-FABP is a new member of the FABP family.

  19. Discovery of arjunolic acid as a novel non-zinc binding carbonic anhydrase II inhibitor.

    PubMed

    Kalyanavenkataraman, Subhalakshmi; Nanjan, Pandurangan; Banerji, Asoke; Nair, Bipin G; Kumar, Geetha B

    2016-06-01

    Elevated levels of carbonic anhydrase II (CA II) have been shown to be associated with cardiac hypertrophy and heart failure. Although arjunolic acid (AA) has a diverse range of therapeutic applications including cardio-protection, there have been no reports on the effect of AA on CA II. The present study describes for the first time, the novel zinc independent inhibition of CA II by AA. The molecular docking studies of AA indicated that the hydroxyl group at C2 of the A-ring, which hydrogen bonds with the catalytic site residues (His64, Asn62 and Asn67), along with the gem-dimethyl group at C20 of the E-ring, greatly influences the inhibitory activity, independent of the catalytic zinc, unlike the inhibition observed with most CA II inhibitors. Among the triterpenoids tested viz. arjunolic acid, arjunic acid, asiatic acid, oleanolic acid and ursolic acid, AA was the most potent in inhibiting CA II in vitro with an IC50 of 9μM. It was interesting to note, that in spite of exhibiting very little differences in their structures, these triterpenoids exhibited vast differences in their inhibitory activities, with IC50 values ranging from 9μM to as high as 333μM. Furthermore, AA also inhibited the cytosolic activity of CA in H9c2 cardiomyocytes, as reflected by the decrease in acidification of the intracellular pH (pHi). The decreased acidification reduced the intracellular calcium levels, which further prevented the mitochondrial membrane depolarization. Thus, these studies provide a better understanding for establishing the novel molecular mechanism involved in CA II inhibition by the non-zinc binding inhibitor AA. PMID:27038848

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

    PubMed

    Katane, Masumi; Matsuda, Satsuki; Saitoh, Yasuaki; Sekine, Masae; Furuchi, Takemitsu; Koyama, Nobuhiro; Nakagome, Izumi; Tomoda, Hiroshi; Hirono, Shuichi; Homma, Hiroshi

    2013-08-20

    d-Amino acid oxidase (DAO) is a degradative enzyme that is stereospecific for d-amino acids, including d-serine and d-alanine, which are believed to be coagonists of the N-methyl-d-aspartate (NMDA) receptor. To identify a new class of DAO inhibitor(s) that can be used to elucidate the molecular details of the active site environment of DAO, manifold biologically active compounds of microbial origin and pre-existing drugs were screened for their ability to inhibit DAO activity, and several compounds were identified as candidates. One of these compounds, acyclovir (ACV), a well-known antiviral drug used for the treatment of herpesvirus infections, was characterized and evaluated as a novel DAO inhibitor in vitro. Analysis showed that ACV acts on DAO as a reversible slow-binding inhibitor, and interestingly, the time required to achieve equilibrium between DAO, ACV, and the DAO/ACV complex was highly dependent on temperature. The binding mechanism of ACV to DAO was investigated in detail by several approaches, including kinetic analysis, structural modeling of DAO complexed with ACV, and site-specific mutagenesis of an active site residue postulated to be involved in the binding of ACV. The results confirm that ACV is a novel, active site-directed inhibitor of DAO that can be a valuable tool for investigating the structure-function relationships of DAO, including the molecular details of the active site environment of DAO. In particular, it appears that ACV can serve as an active site probe to study the structural basis of temperature-induced conformational changes of DAO.

  1. Point mutations in the S protein connect the sialic acid binding activity with the enteropathogenicity of transmissible gastroenteritis coronavirus.

    PubMed Central

    Krempl, C; Schultze, B; Laude, H; Herrler, G

    1997-01-01

    Enteropathogenic transmissible gastroenteritis virus (TGEV), a porcine coronavirus, is able to agglutinate erythrocytes because of sialic acid binding activity. Competitive inhibitors that may mask the sialic acid binding activity can be inactivated by sialidase treatment of virions. Here, we show that TGEV virions with efficient hemagglutinating activity were also obtained when cells were treated with sialidase prior to infection. This method was used to analyze TGEV mutants for hemagglutinating activity. Recently, mutants with strongly reduced enteropathogenicity that have point mutations or a deletion of four amino acids within residues 145 to 155 of the S protein have been described. Here, we show that in addition to their reduced pathogenicity, these mutants also have lost hemagglutinating activity. These results connect sialic acid binding activity with the enteropathogenicity of TGEV. PMID:9060696

  2. The development and amino acid binding ability of nano-materials based on azo derivatives: theory and experiment.

    PubMed

    Shang, Xuefang; Du, Jinge; Yang, Wancai; Liu, Yun; Fu, Zhiyuan; Wei, Xiaofang; Yan, Ruifang; Yao, Ningcong; Guo, Yaping; Zhang, Jinlian; Xu, Xiufang

    2014-05-01

    Two nano-material-containing azo groups have been designed and developed, and the binding ability of nano-materials with various amino acids has been characterized by UV-vis and fluorescence titrations. Results indicated that two nano-materials showed the strongest binding ability for homocysteine among twenty normal kinds of amino acids (alanine, valine, leucine, isoleucine, methionine, aspartic acid, glutamic acid, arginine, glycine, serine, threonine, asparagine, phenylalanine, histidine, tryptophan, proline, lysine, glutamine, tyrosine and homocysteine). The reason for the high sensitivity for homocysteine was that two nano-materials containing an aldehyde group reacted with SH in homocysteine and afforded very stable thiazolidine derivatives. Theoretical investigation further illustrated the possible binding mode in host-guest interaction and the roles of molecular frontier orbitals in molecular interplay. Thus, the two nano-materials can be used as optical sensors for the detection of homocysteine.

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

    PubMed Central

    Gutierrez-Mazariegos, Juliana; Nadendla, Eswar Kumar; Studer, Romain A.; Alvarez, Susana; de Lera, Angel R.; Kuraku, Shigehiro; Bourguet, William; Laudet, Vincent

    2016-01-01

    Whole genome duplications (WGDs) have been classically associated with the origin of evolutionary novelties and the so-called duplication–degeneration–complementation model describes the possible fates of genes after duplication. However, how sequence divergence effectively allows functional changes between gene duplicates is still unclear. In the vertebrate lineage, two rounds of WGDs took place, giving rise to paralogous gene copies observed for many gene families. For the retinoic acid receptors (RARs), for example, which are members of the nuclear hormone receptor (NR) superfamily, a unique ancestral gene has been duplicated resulting in three vertebrate paralogues: RARα, RARβ and RARγ. It has previously been shown that this single ancestral RAR was neofunctionalized to give rise to a larger substrate specificity range in the RARs of extant jawed vertebrates (also called gnathostomes). To understand RAR diversification, the members of the cyclostomes (lamprey and hagfish), jawless vertebrates representing the extant sister group of gnathostomes, provide an intermediate situation and thus allow the characterization of the evolutionary steps that shaped RAR ligand-binding properties following the WGDs. In this study, we assessed the ligand-binding specificity of cyclostome RARs and found that their ligand-binding pockets resemble those of gnathostome RARα and RARβ. In contrast, none of the cyclostome receptors studied showed any RARγ-like specificity. Together, our results suggest that cyclostome RARs cover only a portion of the specificity repertoire of the ancestral gnathostome RARs and indicate that the establishment of ligand-binding specificity was a stepwise event. This iterative process thus provides a rare example for the diversification of receptor–ligand interactions of NRs following WGDs. PMID:27069642

  4. Identification of multiple salicylic acid-binding proteins using two high throughput screens

    PubMed Central

    Manohar, Murli; Tian, Miaoying; Moreau, Magali; Park, Sang-Wook; Choi, Hyong Woo; Fei, Zhangjun; Friso, Giulia; Asif, Muhammed; Manosalva, Patricia; von Dahl, Caroline C.; Shi, Kai; Ma, Shisong; Dinesh-Kumar, Savithramma P.; O'Doherty, Inish; Schroeder, Frank C.; van Wijk, Klass J.; Klessig, Daniel F.

    2014-01-01

    Salicylic acid (SA) is an important hormone involved in many diverse plant processes, including floral induction, stomatal closure, seed germination, adventitious root initiation, and thermogenesis. It also plays critical functions during responses to abiotic and biotic stresses. The role(s) of SA in signaling disease resistance is by far the best studied process, although it is still only partially understood. To obtain insights into how SA carries out its varied functions, particularly in activating disease resistance, two new high throughput screens were developed to identify novel SA-binding proteins (SABPs). The first utilized crosslinking of the photo-reactive SA analog 4-AzidoSA (4AzSA) to proteins in an Arabidopsis leaf extract, followed by immuno-selection with anti-SA antibodies and then mass spectroscopy-based identification. The second utilized photo-affinity crosslinking of 4AzSA to proteins on a protein microarray (PMA) followed by detection with anti-SA antibodies. To determine whether the candidate SABPs (cSABPs) obtained from these screens were true SABPs, recombinantly-produced proteins were generated and tested for SA-inhibitable crosslinking to 4AzSA, which was monitored by immuno-blot analysis, SA-inhibitable binding of the SA derivative 3-aminoethylSA (3AESA), which was detected by a surface plasmon resonance (SPR) assay, or SA-inhibitable binding of [3H]SA, which was detected by size exclusion chromatography. Based on our criteria that true SABPs must exhibit SA-binding activity in at least two of these assays, nine new SABPs are identified here; nine others were previously reported. Approximately 80 cSABPs await further assessment. In addition, the conflicting reports on whether NPR1 is an SABP were addressed by showing that it bound SA in all three of the above assays. PMID:25628632

  5. Nucleic Acid Binding by Mason-Pfizer Monkey Virus CA Promotes Virus Assembly and Genome Packaging

    PubMed Central

    Füzik, Tibor; Píchalová, Růžena; Schur, Florian K. M.; Strohalmová, Karolína; Křížová, Ivana; Hadravová, Romana; Rumlová, Michaela; Briggs, John A. G.

    2016-01-01

    ABSTRACT The Gag polyprotein of retroviruses drives immature virus assembly by forming hexameric protein lattices. The assembly is primarily mediated by protein-protein interactions between capsid (CA) domains and by interactions between nucleocapsid (NC) domains and RNA. Specific interactions between NC and the viral RNA are required for genome packaging. Previously reported cryoelectron microscopy analysis of immature Mason-Pfizer monkey virus (M-PMV) particles suggested that a basic region (residues RKK) in CA may serve as an additional binding site for nucleic acids. Here, we have introduced mutations into the RKK region in both bacterial and proviral M-PMV vectors and have assessed their impact on M-PMV assembly, structure, RNA binding, budding/release, nuclear trafficking, and infectivity using in vitro and in vivo systems. Our data indicate that the RKK region binds and structures nucleic acid that serves to promote virus particle assembly in the cytoplasm. Moreover, the RKK region appears to be important for recruitment of viral genomic RNA into Gag particles, and this function could be linked to changes in nuclear trafficking. Together these observations suggest that in M-PMV, direct interactions between CA and nucleic acid play important functions in the late stages of the viral life cycle. IMPORTANCE Assembly of retrovirus particles is driven by the Gag polyprotein, which can self-assemble to form virus particles and interact with RNA to recruit the viral genome into the particles. Generally, the capsid domains of Gag contribute to essential protein-protein interactions during assembly, while the nucleocapsid domain interacts with RNA. The interactions between the nucleocapsid domain and RNA are important both for identifying the genome and for self-assembly of Gag molecules. Here, we show that a region of basic residues in the capsid protein of the betaretrovirus Mason-Pfizer monkey virus (M-PMV) contributes to interaction of Gag with nucleic acid. This

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

    PubMed

    Huang, Huan; McIntosh, Avery L; Martin, Gregory G; Landrock, Kerstin K; Landrock, Danilo; Gupta, Shipra; Atshaves, Barbara P; Kier, Ann B; Schroeder, Friedhelm

    2014-05-01

    The human liver fatty acid-binding protein (L-FABP) T94A variant, the most common in the FABP family, has been associated with elevated liver triglyceride levels. How this amino acid substitution elicits these effects is not known. This issue was addressed using human recombinant wild-type (WT) and T94A variant L-FABP proteins as well as cultured primary human hepatocytes expressing the respective proteins (genotyped as TT, TC and CC). The T94A substitution did not alter or only slightly altered L-FABP binding affinities for saturated, monounsaturated or polyunsaturated long chain fatty acids, nor did it change the affinity for intermediates of triglyceride synthesis. Nevertheless, the T94A substitution markedly altered the secondary structural response of L-FABP induced by binding long chain fatty acids or intermediates of triglyceride synthesis. Finally, the T94A substitution markedly decreased the levels of induction of peroxisome proliferator-activated receptor α-regulated proteins such as L-FABP, fatty acid transport protein 5 and peroxisome proliferator-activated receptor α itself meditated by the polyunsaturated fatty acids eicosapentaenoic acid and docosahexaenoic acid in cultured primary human hepatocytes. Thus, although the T94A substitution did not alter the affinity of human L-FABP for long chain fatty acids, it significantly altered human L-FABP structure and stability, as well as the conformational and functional response to these ligands.

  7. Biophysical studies of a ruthenium(II) polypyridyl complex binding to DNA and RNA prove that nucleic acid structure has significant effects on binding behaviors.

    PubMed

    Xu, Hong; Liang, Yi; Zhang, Peng; Du, Fen; Zhou, Bing-Rui; Wu, Jun; Liu, Jian-Hong; Liu, Zhi-Gang; Ji, Liang-Nian

    2005-08-01

    The interactions of a metal complex [Ru(phen)(2)PMIP](2+) {Ru=ruthenium, phen=1,10-phenanthroline, PMIP=2-(4-methylphenyl)imidazo[4,5-f]1,10-phenanthroline} with yeast tRNA and calf thymus DNA (CT DNA) have been investigated comparatively by UV-vis spectroscopy, fluorescence spectroscopy, viscosity measurements, isothermal titration calorimetry (ITC), as well as equilibrium dialysis and circular dichroism (CD). Spectroscopic studies together with ITC and viscosity measurements indicate that both binding modes of the Ru(II) polypyridyl complex to yeast tRNA and CT DNA are intercalation and yeast tRNA binding of the complex is stronger than CT DNA binding. ITC experiments show that the interaction of the complex with yeast tRNA is driven by a moderately favorable enthalpy decrease in combination with a moderately favorable entropy increase, while the binding of the complex to CT DNA is driven by a large favorable enthalpy decrease with a less favorable entropy increase. The results from equilibrium dialysis and CD suggest that both interactions are enantioselective and the Delta enantiomer of the complex may bind more favorably to both yeast tRNA and CT DNA than the Lambda enantiomer does, and that the complex is a better candidate for an enantioselective binder to yeast tRNA than to CT DNA. Taken together, these results indicate that the structures of nucleic acids have significant effects on the binding behaviors of metal complexes.

  8. Absorption Spectroscopy Study of Acid-Base and Metal-Binding Properties of Flavanones

    NASA Astrophysics Data System (ADS)

    Shubina, V. S.; Shatalina, Yu. V.

    2013-11-01

    We have used absorption spectroscopy to study the acid-base and metal-binding properties of two structurally similar flavanones: taxifolin and naringenin. We have determined the acid dissociation constants for taxifolin (pKa1 = 7.10 ± 0.05, pKa2 = 8.60 ± 0.09, pKa3 = 8.59 ± 0.19, pKa4 = 11.82 ± 0.36) and naringenin (pKa1 = 7.05 ± 0.05, pKa2 = 8.85 ± 0.09, pKa3 = 12.01 ± 0.38). The appearance of new absorption bands in the visible wavelength region let us determine the stoichiometric composition of the iron (II) complexes of the flavanones. We show that at pH 5, in solution there is a mixture of complexes between taxifolin and iron (II) ions in stoichiometric ratio 2:1 and 1:2, while at pH 7.4 and pH 9, we detect a 1:1 taxifolin:Fe(II) complex. We established that at these pH values, naringenin forms a 2:1 complex with iron (II) ions. We propose structures for the complexes formed. Comprehensive study of the acid-base properties and the metal-binding capability of the two structurally similar flavanones let us determine the structure-properties relation and the conditions under which antioxidant activity of the polyphenols appears, via chelation of variable-valence metal ions.

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

    SciTech Connect

    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.

  10. Two Arginine Residues of Streptococcus gordonii Sialic Acid-Binding Adhesin Hsa Are Essential for Interaction to Host Cell Receptors

    PubMed Central

    Urano-Tashiro, Yumiko; Takahashi, Yukihiro; Oguchi, Riyo; Konishi, Kiyoshi

    2016-01-01

    Hsa is a large, serine-rich protein of Streptococcus gordonii DL1 that mediates binding to α2-3-linked sialic acid termini of glycoproteins, including platelet glycoprotein Ibα, and erythrocyte membrane protein glycophorin A, and band 3. The binding of Hsa to platelet glycoprotein Ibα contributes to the pathogenesis of infective endocarditis. This interaction appears to be mediated by a second non-repetitive region (NR2) of Hsa. However, the molecular details of the interaction between the Hsa NR2 region and these glycoproteins are not well understood. In the present study, we identified the amino acid residues of the Hsa NR2 region that are involved in sialic acid recognition. To identify the sialic acid-binding site of Hsa NR2 region, we prepared various mutants of Hsa NR2 fused with glutathione transferase. Fusion proteins harboring Arg340 to Asn (R340N) or Arg365 to Asn (R365N) substitutions in the NR2 domain exhibited significantly reduced binding to human erythrocytes and platelets. A sugar-binding assay showed that these mutant proteins abolished binding to α2-3-linked sialic acid. Furthermore, we established S. gordonii DL1 derivatives that encoded the corresponding Hsa mutant protein. In whole-cell assays, these mutant strains showed significant reductions in hemagglutination, in platelet aggregation, and in adhesion to human leukocytes. These results indicate that the Arg340 and Arg365 residues of Hsa play an important role in the binding of Hsa to α2-3-linked sialic acid-containing glycoproteins. PMID:27101147

  11. Two Arginine Residues of Streptococcus gordonii Sialic Acid-Binding Adhesin Hsa Are Essential for Interaction to Host Cell Receptors.

    PubMed

    Urano-Tashiro, Yumiko; Takahashi, Yukihiro; Oguchi, Riyo; Konishi, Kiyoshi

    2016-01-01

    Hsa is a large, serine-rich protein of Streptococcus gordonii DL1 that mediates binding to α2-3-linked sialic acid termini of glycoproteins, including platelet glycoprotein Ibα, and erythrocyte membrane protein glycophorin A, and band 3. The binding of Hsa to platelet glycoprotein Ibα contributes to the pathogenesis of infective endocarditis. This interaction appears to be mediated by a second non-repetitive region (NR2) of Hsa. However, the molecular details of the interaction between the Hsa NR2 region and these glycoproteins are not well understood. In the present study, we identified the amino acid residues of the Hsa NR2 region that are involved in sialic acid recognition. To identify the sialic acid-binding site of Hsa NR2 region, we prepared various mutants of Hsa NR2 fused with glutathione transferase. Fusion proteins harboring Arg340 to Asn (R340N) or Arg365 to Asn (R365N) substitutions in the NR2 domain exhibited significantly reduced binding to human erythrocytes and platelets. A sugar-binding assay showed that these mutant proteins abolished binding to α2-3-linked sialic acid. Furthermore, we established S. gordonii DL1 derivatives that encoded the corresponding Hsa mutant protein. In whole-cell assays, these mutant strains showed significant reductions in hemagglutination, in platelet aggregation, and in adhesion to human leukocytes. These results indicate that the Arg340 and Arg365 residues of Hsa play an important role in the binding of Hsa to α2-3-linked sialic acid-containing glycoproteins. PMID:27101147

  12. Differential scanning calorimetry study on the binding of nucleic acids to dimyristoylphosphatidylcholine-sphingosine liposomes.

    PubMed

    Kõiv, A; Mustonen, P; Kinnunen, P K

    1994-03-31

    Binding of DNA and RNA to sphingosine-containing dimyristoylphosphatidylcholine (DMPC) liposomes was characterized by differential scanning calorimetry. The thermal phase behaviour of neat DMPC liposomes was unaffected by the presence of the nucleic acids. However, significant alterations in the melting profiles of the DMPC/sphingosine composite membranes were produced by DNA and RNA, thus revealing their binding to the liposomes. For example, for 79:21 (molar ratio) DMPC/sphingosine liposomes a single endotherm at 29.1 degrees C with an enthalpy of 6.3 kcal/mol lipid was observed. In the presence of DNA at the nucleotide/sphingosine ratio of 0.6 this endotherm separated into three distinct peaks at 28.0, 31.4 and 35.1 degrees C, together with an approximately 22% reduction in the total enthalpy. Further increase in DNA concentration up to 1.5 nucleotides per sphingosine led to complete loss of the original heat absorption peak of the DMPC/sphingosine liposomes, while an endotherm at 34.3 degrees C with delta H of 2.7 kcal/mol developed. By visual inspection, rapid and extensive aggregation of the liposomes due to DNA was evident. Evidence for DNA-induced phase separation was also provided by compression isotherms of sphingosine containing DMPC monolayers recorded over an aqueous buffer both in the presence and absence of DNA. The effects of RNA on the thermal phase behaviour of the composite liposomes were qualitatively similar to those described above for DNA. Notably, the presence of eggPA abolished the nucleic acid induced heat capacity changes for DMPC/sphingosine liposomes probably because of neutralization of the positive charge of sphingosine. The binding of DNA to DMPC/sphingosine liposomes occurred both below and above the lipid phase transition temperature, as shown by fluorescence resonance energy transfer utilizing adriamycin-labelled DNA as a quencher and membrane incorporated pyrene-labelled phospholipid as a donor. However, the apparent binding to

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

    PubMed Central

    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

  14. Coexistence of multiple minor states of fatty acid binding protein and their functional relevance

    PubMed Central

    Yu, Binhan; Yang, Daiwen

    2016-01-01

    Proteins are dynamic over a wide range of timescales, but determining the number of distinct dynamic processes and identifying functionally relevant dynamics are still challenging. Here we present the study on human intestinal fatty acid binding protein (hIFABP) using a novel analysis of 15N relaxation dispersion (RD) and chemical shift saturation transfer (CEST) experiments. Through combined analysis of the two types of experiments, we found that hIFABP exists in a four-state equilibrium in which three minor states interconvert directly with the major state. According to conversion rates from the major “closed” state to minor states, these minor states are irrelevant to the function of fatty acid transport. Based on chemical shifts of the minor states which could not be determined from RD data alone but were extracted from a combined analysis of RD and CEST data, we found that all the minor states are native-like. This conclusion is further supported by hydrogen-deuterium exchange experiments. Direct conversions between the native state and native-like intermediate states may suggest parallel multitrack unfolding/folding pathways of hIFABP. Moreover, hydrogen-deuterium exchange data indicate the existence of another locally unfolded minor state that is relevant to the fatty acid entry process. PMID:27677899

  15. Evaluation of New Diagnostic Biomarkers in Pediatric Sepsis: Matrix Metalloproteinase-9, Tissue Inhibitor of Metalloproteinase-1, Mid-Regional Pro-Atrial Natriuretic Peptide, and Adipocyte Fatty-Acid Binding Protein

    PubMed Central

    Alqahtani, Mashael F.; Smith, Craig M.; Weiss, Scott L.; Dawson, Susan; Ralay Ranaivo, Hantamalala; Wainwright, Mark S.

    2016-01-01

    Elevated plasma concentrations of matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), mid-regional pro-atrial natriuretic peptide (mrProANP), and adipocyte fatty-acid-binding proteins (A-FaBPs) have been investigated as biomarkers for sepsis or detection of acute neurological injuries in adults, but not children. We carried out a single-center, prospective observational study to determine if these measures could serve as biomarkers to identify children with sepsis. A secondary aim was to determine if these biomarkers could identify children with neurologic complications of sepsis. A total of 90 patients ≤ 18 years-old were included in this study. 30 with severe sepsis or septic shock were compared to 30 age-matched febrile and 30 age-matched healthy controls. Serial measurements of each biomarker were obtained, beginning on day 1 of ICU admission. In septic patients, MMP9-/TIMP-1 ratios (Median, IQR, n) were reduced on day 1 (0.024, 0.004–0.174, 13), day 2 (0.020, 0.002–0.109, 10), and day 3 (0.018, 0.003–0.058, 23) compared with febrile (0.705, 0.187–1.778, 22) and healthy (0.7, 0.4–1.2, 29) (p< 0.05) controls. A-FaBP and mrProANP (Median, IQR ng/mL, n) were elevated in septic patients compared to control groups on first 2 days after admission to the PICU (p <0.05). The area under the curve (AUC) for MMP-9/TIMP-1 ratio, mrProANP, and A-FaBP to distinguish septic patients from healthy controls were 0.96, 0.99, and 0.76, respectively. MMP-9/TIMP-1 ratio was inversely and mrProANP was directly related to PIM-2, PELOD, and ICU and hospital LOS (p<0.05). A-FaBP level was associated with PELOD, hospital and ICU length of stay (p<0.05). MMP-9/TIMP-1 ratio associated with poor Glasgow Outcome Score (p<0.05). A-FaBP levels in septic patients with neurological dysfunction (29.3, 17.2–54.6, 7) were significantly increased compared to septic patients without neurological dysfunction (14.6, 13.3–20.6, 11). MMP-9/TIMP-1 ratios

  16. Evaluation of New Diagnostic Biomarkers in Pediatric Sepsis: Matrix Metalloproteinase-9, Tissue Inhibitor of Metalloproteinase-1, Mid-Regional Pro-Atrial Natriuretic Peptide, and Adipocyte Fatty-Acid Binding Protein.

    PubMed

    Alqahtani, Mashael F; Smith, Craig M; Weiss, Scott L; Dawson, Susan; Ralay Ranaivo, Hantamalala; Wainwright, Mark S

    2016-01-01

    Elevated plasma concentrations of matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), mid-regional pro-atrial natriuretic peptide (mrProANP), and adipocyte fatty-acid-binding proteins (A-FaBPs) have been investigated as biomarkers for sepsis or detection of acute neurological injuries in adults, but not children. We carried out a single-center, prospective observational study to determine if these measures could serve as biomarkers to identify children with sepsis. A secondary aim was to determine if these biomarkers could identify children with neurologic complications of sepsis. A total of 90 patients ≤ 18 years-old were included in this study. 30 with severe sepsis or septic shock were compared to 30 age-matched febrile and 30 age-matched healthy controls. Serial measurements of each biomarker were obtained, beginning on day 1 of ICU admission. In septic patients, MMP9-/TIMP-1 ratios (Median, IQR, n) were reduced on day 1 (0.024, 0.004-0.174, 13), day 2 (0.020, 0.002-0.109, 10), and day 3 (0.018, 0.003-0.058, 23) compared with febrile (0.705, 0.187-1.778, 22) and healthy (0.7, 0.4-1.2, 29) (p< 0.05) controls. A-FaBP and mrProANP (Median, IQR ng/mL, n) were elevated in septic patients compared to control groups on first 2 days after admission to the PICU (p <0.05). The area under the curve (AUC) for MMP-9/TIMP-1 ratio, mrProANP, and A-FaBP to distinguish septic patients from healthy controls were 0.96, 0.99, and 0.76, respectively. MMP-9/TIMP-1 ratio was inversely and mrProANP was directly related to PIM-2, PELOD, and ICU and hospital LOS (p<0.05). A-FaBP level was associated with PELOD, hospital and ICU length of stay (p<0.05). MMP-9/TIMP-1 ratio associated with poor Glasgow Outcome Score (p<0.05). A-FaBP levels in septic patients with neurological dysfunction (29.3, 17.2-54.6, 7) were significantly increased compared to septic patients without neurological dysfunction (14.6, 13.3-20.6, 11). MMP-9/TIMP-1 ratios were

  17. Evaluation of New Diagnostic Biomarkers in Pediatric Sepsis: Matrix Metalloproteinase-9, Tissue Inhibitor of Metalloproteinase-1, Mid-Regional Pro-Atrial Natriuretic Peptide, and Adipocyte Fatty-Acid Binding Protein.

    PubMed

    Alqahtani, Mashael F; Smith, Craig M; Weiss, Scott L; Dawson, Susan; Ralay Ranaivo, Hantamalala; Wainwright, Mark S

    2016-01-01

    Elevated plasma concentrations of matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), mid-regional pro-atrial natriuretic peptide (mrProANP), and adipocyte fatty-acid-binding proteins (A-FaBPs) have been investigated as biomarkers for sepsis or detection of acute neurological injuries in adults, but not children. We carried out a single-center, prospective observational study to determine if these measures could serve as biomarkers to identify children with sepsis. A secondary aim was to determine if these biomarkers could identify children with neurologic complications of sepsis. A total of 90 patients ≤ 18 years-old were included in this study. 30 with severe sepsis or septic shock were compared to 30 age-matched febrile and 30 age-matched healthy controls. Serial measurements of each biomarker were obtained, beginning on day 1 of ICU admission. In septic patients, MMP9-/TIMP-1 ratios (Median, IQR, n) were reduced on day 1 (0.024, 0.004-0.174, 13), day 2 (0.020, 0.002-0.109, 10), and day 3 (0.018, 0.003-0.058, 23) compared with febrile (0.705, 0.187-1.778, 22) and healthy (0.7, 0.4-1.2, 29) (p< 0.05) controls. A-FaBP and mrProANP (Median, IQR ng/mL, n) were elevated in septic patients compared to control groups on first 2 days after admission to the PICU (p <0.05). The area under the curve (AUC) for MMP-9/TIMP-1 ratio, mrProANP, and A-FaBP to distinguish septic patients from healthy controls were 0.96, 0.99, and 0.76, respectively. MMP-9/TIMP-1 ratio was inversely and mrProANP was directly related to PIM-2, PELOD, and ICU and hospital LOS (p<0.05). A-FaBP level was associated with PELOD, hospital and ICU length of stay (p<0.05). MMP-9/TIMP-1 ratio associated with poor Glasgow Outcome Score (p<0.05). A-FaBP levels in septic patients with neurological dysfunction (29.3, 17.2-54.6, 7) were significantly increased compared to septic patients without neurological dysfunction (14.6, 13.3-20.6, 11). MMP-9/TIMP-1 ratios were

  18. Layer by layer chitosan/alginate coatings on poly(lactide-co-glycolide) nanoparticles for antifouling protection and Folic acid binding to achieve selective cell targeting.

    PubMed

    Zhou, Jie; Romero, Gabriela; Rojas, Elena; Ma, Lie; Moya, Sergio; Gao, Changyou

    2010-05-15

    Polyelectrolyte multilayers (PEMs) composed of two natural polysaccharides-chitosan (Chi) and alginate (Alg) were deposited by Layer by layer (LbL) assembly on top of biocompatible poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs). Folic acid (FA) or FA grafted poly(ethylene glycol) (PEG-FA) were covalently bounded to the PEMs via carbodiimide chemistry. The assembly of biocompatible PEMs was monitored on planar surfaces by means of the quartz crystal microbalance with dissipation (QCM-D) technique and on top of PLGA NPs by means of ζ-potential measurements. BSA was used as model protein to characterize protein adsorption on PEMs. QCM-D showed protein deposition could not be observed on the Chi/Alg multilayer, for both Chitosan and Alginate as top layers. Finally, cellular uptake experiments were carried out by co-culture of HepG2 cells in presence of NPs. Flow Cytometry and confocal laser scanning microscopy (CLSM) were used to investigate the influence of the surface chemistry of the NPs on uptake. For the HepG2 cell line significantly less uptake of PLGA NPs coated with Chi/Alg than the bare NPs was observed but the uptake increased after attachment of FA molecules.

  19. Binding of basal transcription factor TFIIH to the acidic activation domains of VP16 and p53.

    PubMed Central

    Xiao, H; Pearson, A; Coulombe, B; Truant, R; Zhang, S; Regier, J L; Triezenberg, S J; Reinberg, D; Flores, O; Ingles, C J

    1994-01-01

    Acidic transcriptional activation domains function well in both yeast and mammalian cells, and some have been shown to bind the general transcription factors TFIID and TFIIB. We now show that two acidic transactivators, herpes simplex virus VP16 and human p53, directly interact with the multisubunit human general transcription factor TFIIH and its Saccharomyces cerevisiae counterpart, factor b. The VP16- and p53-binding domains in these factors lie in the p62 subunit of TFIIH and in the homologous subunit, TFB1, of factor b. Point mutations in VP16 that reduce its transactivation activity in both yeast and mammalian cells weaken its binding to both yeast and human TFIIH. This suggests that binding of activation domains to TFIIH is an important aspect of transcriptional activation. Images PMID:7935417

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

    NASA Astrophysics Data System (ADS)

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

    2015-07-01

    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.

  1. A Novel Fatty Acid-Binding Protein-Like Carotenoid-Binding Protein from the Gonad of the New Zealand Sea Urchin Evechinus chloroticus

    PubMed Central

    Pilbrow, Jodi; Sabherwal, Manya; Garama, Daniel; Carne, Alan

    2014-01-01

    A previously uncharacterized protein with a carotenoid-binding function has been isolated and characterized from the gonad of the New Zealand sea urchin Evechinus chloroticus. The main carotenoid bound to the protein was determined by reversed phase-high performance liquid chromatography to be 9′-cis-echinenone and hence this 15 kDa protein has been called an echinenone-binding protein (EBP). Purification of the EBP in quantity from the natural source proved to be challenging. However, analysis of EBP by mass spectrometry combined with information from the Strongylocentrotus purpuratus genome sequence and the recently published E. chloroticus transcriptome database, enabled recombinant expression of wild type EBP and also of a cysteine61 to serine mutant that had improved solubility characteristics. Circular dichroism data and ab initio structure prediction suggests that the EBP adopts a 10-stranded β-barrel fold consistent with that of fatty acid-binding proteins. Therefore, EBP may represent the first report of a fatty acid-binding protein in complex with a carotenoid. PMID:25192378

  2. A novel fatty acid-binding protein-like carotenoid-binding protein from the gonad of the New Zealand sea urchin Evechinus chloroticus.

    PubMed

    Pilbrow, Jodi; Sabherwal, Manya; Garama, Daniel; Carne, Alan

    2014-01-01

    A previously uncharacterized protein with a carotenoid-binding function has been isolated and characterized from the gonad of the New Zealand sea urchin Evechinus chloroticus. The main carotenoid bound to the protein was determined by reversed phase-high performance liquid chromatography to be 9'-cis-echinenone and hence this 15 kDa protein has been called an echinenone-binding protein (EBP). Purification of the EBP in quantity from the natural source proved to be challenging. However, analysis of EBP by mass spectrometry combined with information from the Strongylocentrotus purpuratus genome sequence and the recently published E. chloroticus transcriptome database, enabled recombinant expression of wild type EBP and also of a cysteine61 to serine mutant that had improved solubility characteristics. Circular dichroism data and ab initio structure prediction suggests that the EBP adopts a 10-stranded β-barrel fold consistent with that of fatty acid-binding proteins. Therefore, EBP may represent the first report of a fatty acid-binding protein in complex with a carotenoid.

  3. A novel fatty acid-binding protein-like carotenoid-binding protein from the gonad of the New Zealand sea urchin Evechinus chloroticus.

    PubMed

    Pilbrow, Jodi; Sabherwal, Manya; Garama, Daniel; Carne, Alan

    2014-01-01

    A previously uncharacterized protein with a carotenoid-binding function has been isolated and characterized from the gonad of the New Zealand sea urchin Evechinus chloroticus. The main carotenoid bound to the protein was determined by reversed phase-high performance liquid chromatography to be 9'-cis-echinenone and hence this 15 kDa protein has been called an echinenone-binding protein (EBP). Purification of the EBP in quantity from the natural source proved to be challenging. However, analysis of EBP by mass spectrometry combined with information from the Strongylocentrotus purpuratus genome sequence and the recently published E. chloroticus transcriptome database, enabled recombinant expression of wild type EBP and also of a cysteine61 to serine mutant that had improved solubility characteristics. Circular dichroism data and ab initio structure prediction suggests that the EBP adopts a 10-stranded β-barrel fold consistent with that of fatty acid-binding proteins. Therefore, EBP may represent the first report of a fatty acid-binding protein in complex with a carotenoid. PMID:25192378

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

    PubMed Central

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

    2016-01-01

    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. PMID:26762975

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

    PubMed

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

    2016-04-01

    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. PMID:26762975

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

    PubMed

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

    2016-04-01

    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.

  7. CO2-binding Organic Liquids, an Integrated Acid Gas Capture System

    SciTech Connect

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

    2011-04-01

    Amine systems are effective for CO2 capture, but they are still inefficient because the solvent regeneration energy is largely defined by the amount of water in the process. Most amines form heat-stable salts with SO2 and COS resulting in parasitic solvent loss and degradation. Stripping the CO2-rich solvent is energy intensive it requires temperatures above 100 °C due to the high specific heat and heat of vaporization of water. CO2-capture processes could be much more energy efficient in a water free amine process. In addition, if the capture-material is chemically compatible with other acid gases, less solvent would be lost to heat-stable salts and the process economics would be further improved. One such system that can address these concerns is Binding Organic Liquids (BOLs), a class of switchable ionic liquids.

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

    PubMed

    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. PMID:25966259

  9. Neurologic syndrome associated with homozygous mutation at MAG sialic acid binding site.

    PubMed

    Roda, Ricardo H; FitzGibbon, Edmond J; Boucekkine, Houda; Schindler, Alice B; Blackstone, Craig

    2016-08-01

    The MAG gene encodes myelin-associated glycoprotein (MAG), an abundant protein involved in axon-glial interactions and myelination during nerve regeneration. Several members of a consanguineous family with a clinical syndrome reminiscent of Pelizaeus-Merzbacher disease and demyelinating leukodystrophy on brain MRI were recently found to harbor a homozygous missense p.Ser133Arg MAG mutation. Here, we report two brothers from a nonconsanguineous family afflicted with progressive cognitive impairment, neuropathy, ataxia, nystagmus, and gait disorder. Exome sequencing revealed the homozygous missense mutation p.Arg118His in MAG. This Arg118 residue in immunoglobulin domain 1 is critical for sialic acid binding, providing a compelling mechanistic basis for disease pathogenesis. PMID:27606346

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

    PubMed

    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.

  11. Peptide nucleic acid (PNA) binding-mediated induction of human gamma-globin gene expression.

    PubMed

    Wang, G; Xu, X; Pace, B; Dean, D A; Glazer, P M; Chan, P; Goodman, S R; Shokolenko, I

    1999-07-01

    Peptide nucleic acids (PNAs) can bind to homopurine/homopyrimidine sequences of double-stranded DNA targets in a sequence-specific manner and form [PNA]2/DNA triplexes with single-stranded DNA D-loop structures at the PNA binding sites. These D-loop structures have been found to have a capacity to initiate transcription in vitro. If this strategy can be used to induce transcription of endogenous genes, it may provide a novel approach for gene therapy of many human diseases. Human [beta] globin disorders such as sickle cell anemia and beta-thalassemia are very common genetic diseases that are caused by mutations in the beta-globin gene. When gamma-globin genes are highly expressed in sickle cell patients, the presence of high levels of fetal hemoglobin (HbF, alpha2gamma2) can compensate for the defective beta-globin gene product and such patients have much improved symptoms or are free of disease. However, the gamma-globin genes are developmentally regulated and normally expressed at very low levels (>1%) in adult blood cells. We have investigated the possibility of inducing gamma-globin gene expression with PNAs. Using PNAs designed to bind to the 5' flanking region of the gamma-globin gene, induction of expression of a reporter gene construct was demonstrated both in vitro and in vivo. More importantly, PNA-mediated induction of endogenous gamma-globin gene expression was also demonstrated in K562 human erythroleukemia cells. This result suggests that induction of gamma-globin gene expression with PNAs might provide a new approach for the treatment of sickle cell disease. PNA-induced gene expression strategy also may have implications in gene therapy of other diseases such as genetic diseases, cancer and infectious diseases.

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

    PubMed

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

    2014-04-01

    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.

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

    PubMed Central

    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

  14. Relationship between hyaluronic acid binding assay and outcome in ART: a pilot study.

    PubMed

    Nijs, Martine; Creemers, E; Cox, A; Janssen, M; Vanheusden, E; Van der Elst, J; Ombelet, W

    2010-10-01

    The sperm-hyaluronan binding assay (HBA) is a diagnostic kit for assessing sperm maturity, function and fertility. The aim of this prospective cohort pilot study was to evaluate the relationship between HBA and WHO sperm parameters (motility, concentration and detailed morphology) and possible influence of sperm processing on hyaluronic acid binding. A cohort of 68 patients undergoing a first combo in vitro fertilisation/intracytoplasmic sperm injection treatment after failure of three or more intrauterine insemination cycles were included in the study. Outcome measures studied were fertilisation rate, embryo quality, ongoing pregnancy rate and cumulative pregnancy rate. HBA outcome improved after sperm preparation and culture, but was not correlated to detailed sperm morphology, concentration or motility. HBA did not provide additional information for identifying patients with poor or absent fertilisation, although the latter had more immature sperm cells and cells with cytoplasmic retention present in their semen. HBA outcome in the neat sample was significantly correlated with embryo quality, with miscarriage rates and ongoing pregnancy rates in the fresh cycles, but not with the cumulative ongoing pregnancy rate. No threshold value for HBA and outcome in combo IVF/ICSI treatment could be established. The clinical value for HBA in addition to routine semen analysis for this patient population seems limited.

  15. Steroid binding to Autotaxin links bile salts and lysophosphatidic acid signalling.

    PubMed

    Keune, Willem-Jan; Hausmann, Jens; Bolier, Ruth; Tolenaars, Dagmar; Kremer, Andreas; Heidebrecht, Tatjana; Joosten, Robbie P; Sunkara, Manjula; Morris, Andrew J; Matas-Rico, Elisa; Moolenaar, Wouter H; Oude Elferink, Ronald P; Perrakis, Anastassis

    2016-01-01

    Autotaxin (ATX) generates the lipid mediator lysophosphatidic acid (LPA). ATX-LPA signalling is involved in multiple biological and pathophysiological processes, including vasculogenesis, fibrosis, cholestatic pruritus and tumour progression. ATX has a tripartite active site, combining a hydrophilic groove, a hydrophobic lipid-binding pocket and a tunnel of unclear function. We present crystal structures of rat ATX bound to 7α-hydroxycholesterol and the bile salt tauroursodeoxycholate (TUDCA), showing how the tunnel selectively binds steroids. A structure of ATX simultaneously harbouring TUDCA in the tunnel and LPA in the pocket, together with kinetic analysis, reveals that bile salts act as partial non-competitive inhibitors of ATX, thereby attenuating LPA receptor activation. This unexpected interplay between ATX-LPA signalling and select steroids, notably natural bile salts, provides a molecular basis for the emerging association of ATX with disorders associated with increased circulating levels of bile salts. Furthermore, our findings suggest potential clinical implications in the use of steroid drugs. PMID:27075612

  16. Steroid binding to Autotaxin links bile salts and lysophosphatidic acid signalling

    PubMed Central

    Keune, Willem-Jan; Hausmann, Jens; Bolier, Ruth; Tolenaars, Dagmar; Kremer, Andreas; Heidebrecht, Tatjana; Joosten, Robbie P.; Sunkara, Manjula; Morris, Andrew J.; Matas-Rico, Elisa; Moolenaar, Wouter H.; Oude Elferink, Ronald P.; Perrakis, Anastassis

    2016-01-01

    Autotaxin (ATX) generates the lipid mediator lysophosphatidic acid (LPA). ATX-LPA signalling is involved in multiple biological and pathophysiological processes, including vasculogenesis, fibrosis, cholestatic pruritus and tumour progression. ATX has a tripartite active site, combining a hydrophilic groove, a hydrophobic lipid-binding pocket and a tunnel of unclear function. We present crystal structures of rat ATX bound to 7α-hydroxycholesterol and the bile salt tauroursodeoxycholate (TUDCA), showing how the tunnel selectively binds steroids. A structure of ATX simultaneously harbouring TUDCA in the tunnel and LPA in the pocket, together with kinetic analysis, reveals that bile salts act as partial non-competitive inhibitors of ATX, thereby attenuating LPA receptor activation. This unexpected interplay between ATX-LPA signalling and select steroids, notably natural bile salts, provides a molecular basis for the emerging association of ATX with disorders associated with increased circulating levels of bile salts. Furthermore, our findings suggest potential clinical implications in the use of steroid drugs. PMID:27075612

  17. Novel binding patterns between ganoderic acids and neuraminidase: Insights from docking, molecular dynamics and MM/PBSA studies.

    PubMed

    Yang, Zhiwei; Wu, Fei; Yuan, Xiaohui; Zhang, Lei; Zhang, Shengli

    2016-04-01

    Recently, ganoderic acids (GAs) give rise to the attractive candidates of novel neuraminidase (NA) inhibitors. However, there is still no evident conclusion about their binding patterns. To this end, docking, molecular dynamics and MM/PBSA methods were combined to study the binding profiles of GAs with the N1 protein and familiar H274Y and N294S mutations (A/Vietnam/1203/04 stain). It was found that the binding affinities of ganoderic acid DM and Z (ΔGbind, -16.83 and -10.99 kcal mol(-1)) are comparable to that of current commercial drug oseltamivir (-23.62 kcal mol(-1)). Electrostatic interaction is the main driving force, and should be one important factor to evaluate the binding quality and rational design of NA inhibitors. The 150-loop residues Asp151 and Arg152 played an important role in the binding processes. Further analysis revealed that ganoderic acid DM is a potential source of anti-influenza ingredient, with novel binding pattern and advantage over oseltamivir. It had steric hindrance on the 150 cavity of N1 protein, and exerted activities across the H274Y and N294S mutations. This work also pointed out how to effectively design dual-site NA inhibitors and reinforce their affinities. These findings should prove valuable for the in-depth understanding of interactions between NA and GAs, and warrant the experimental aspects to design novel anti-influenza drugs. PMID:26905206

  18. Solid-State NMR Characterization of Mixed Phosphonic Acid Ligand Binding and Organization on Silica Nanoparticles.

    PubMed

    Davidowski, Stephen K; Holland, Gregory P

    2016-04-01

    As ligand functionalization of nanomaterials becomes more complex, methods to characterize the organization of multiple ligands on surfaces is required. In an effort to further the understanding of ligand-surface interactions, a combination of multinuclear ((1)H, (29)Si, (31)P) and multidimensional solid-state nuclear magnetic resonance (NMR) techniques was utilized to characterize the phosphonic acid functionalization of fumed silica nanoparticles using methylphosphonic acid (MPA) and phenylphosphonic acid (PPA). (1)H → (29)Si cross-polarization (CP)-magic angle spinning (MAS) solid-state NMR was used to selectively detect silicon atoms near hydrogen atoms (primarily surface species); these results indicate that geminal silanols are preferentially depleted during the functionalization with phosphonic acids. (1)H → (31)P CP-MAS solid-state NMR measurements on the functionalized silica nanoparticles show three distinct resonances shifted upfield (lower ppm) and broadened compared to the resonances of the crystalline ligands. Quantitative (31)P MAS solid-state NMR measurements indicate that ligands favor a monodentate binding mode. When fumed silica nanoparticles were functionalized with an equal molar ratio of MPA and PPA, the MPA bound the nanoparticle surface preferentially. Cross-peaks apparent in the 2D (1)H exchange spectroscopy (EXSY) NMR measurements of the multiligand sample at short mixing times indicate that the MPA and PPA are spatially close (≤5 Å) on the surface of the nanostructure. Furthermore, (1)H-(1)H double quantum-single quantum (DQ-SQ) back-to-back (BABA) 2D NMR spectra further confirmed that MPA and PPA are strongly dipolar coupled with observation of DQ intermolecular contacts between the ligands. DQ experimental buildup curves and simulations indicate that the average distance between MPA and PPA is no further than 4.2 ± 0.2 Å. PMID:26914738

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

    PubMed Central

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

    2013-01-01

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

  20. Amphipathic Benzoic Acid Derivativies: Synthesis and Binding in the Hydrophobic Tunnel of the Zinc Deacetylase LpxC

    SciTech Connect

    Shin,H.; Gennadios, H.; Whittington, D.; Christianson, D.

    2007-01-01

    The first committed step in lipid A biosynthesis is catalyzed by uridine diphosphate-(3-O-(R-3-hydroxymyristoyl))-N-acetylglucosamine deacetylase (LpxC), a zinc-dependent deacetylase, and inhibitors of LpxC may be useful in the development of antibacterial agents targeting a broad spectrum of Gram-negative bacteria. Here, we report the design of amphipathic benzoic acid derivatives that bind in the hydrophobic tunnel in the active site of LpxC. The hydrophobic tunnel accounts for the specificity of LpxC toward substrates and substrate analogues bearing a 3-O-myristoyl substituent. Simple benzoic acid derivatives bearing an aliphatic 'tail' bind in the hydrophobic tunnel with micromolar affinity despite the lack of a glucosamine ring like that of the substrate. However, although these benzoic acid derivatives each contain a negatively charged carboxylate 'warhead' intended to coordinate to the active site zinc ion, the 2.25 {angstrom} resolution X-ray crystal structure of LpxC complexed with 3-(heptyloxy)benzoate reveals 'backward' binding in the hydrophobic tunnel, such that the benzoate moiety does not coordinate to zinc. Instead, it binds at the outer end of the hydrophobic tunnel. Interestingly, these ligands bind with affinities comparable to those measured for more complicated substrate analogue inhibitors containing glucosamine ring analogues and hydroxamate 'warheads' that coordinate to the active site zinc ion. We conclude that the intermolecular interactions in the hydrophobic tunnel dominate enzyme affinity in this series of benzoic acid derivatives.

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

    PubMed

    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. PMID:25053052

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

    PubMed

    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.

  3. Acid-base and metal ion binding properties of 2-thiocytidine in aqueous solution.

    PubMed

    Brasuń, Justyna; Matera, Agnieszka; Sochacka, Elzbieta; Swiatek-Kozlowska, Jolanta; Kozlowski, Henryk; Operschall, Bert P; Sigel, Helmut

    2008-06-01

    The thionucleoside 2-thiocytidine (C2S) occurs in nature in transfer RNAs; it receives attention in diverse fields like drug research and nanotechnology. By potentiometric pH titrations we measured the acidity constants of H(C2S)(+) and the stability constants of the M(C2S)(2+) and M(C2S-H)(+) complexes (M(2+) = Zn(2+), Cd(2+)), and we compared these results with those obtained previously for its parent nucleoside, cytidine (Cyd). Replacement of the (C2)=O unit by (C2)=S facilitates the release of the proton from (N3)H(+) in H(C2S)(+) (pK (a) = 3.44) somewhat, compared with H(Cyd)(+) (pK (a) = 4.24). This moderate effect of about 0.8 pK units contrasts with the strong acidification of about 4 pK units of the (C4)NH(2) group in C2S (pK (a) = 12.65) compared with Cyd (pK (a) approximately 16.7); the reason for this result is that the amino-thione tautomer, which dominates for the neutral C2S molecule, is transformed upon deprotonation into the imino-thioate form with the negative charge largely located on the sulfur. In the M(C2S)(2+) complexes the (C2)S group is the primary binding site rather than N3 as is the case in the M(Cyd)(2+) complexes, though owing to chelate formation N3 is to some extent still involved in metal ion binding. Similarly, in the Zn(C2S-H)(+) and Cd(C2S-H)(+) complexes the main metal ion binding site is the (C2)S(-) unit (formation degree above 99.99% compared with that of N3). However, again a large degree of chelate formation with N3 must be surmised for the M(C2S-H)(+) species in accord with previous solid-state studies of related ligands. Upon metal ion binding, the deprotonation of the (C4)NH(2) group (pK (a) = 12.65) is dramatically acidified (pK (a) approximately 3), confirming the very high stability of the M(C2S-H)(+) complexes. To conclude, the hydrogen-bonding and metal ion complex forming capabilities of C2S differ strongly from those of its parent Cyd; this must have consequences for the properties of those RNAs which contain this

  4. Bile acid-binding activity of young persimmon (Diospyros kaki) fruit and its hypolipidemic effect in mice.

    PubMed

    Matsumoto, Kenji; Yokoyama, Shin-ichiro; Gato, Nobuki

    2010-02-01

    The hypolipidemic effects and bile acid-binding properties of young persimmon (Diospyros kaki) fruit were examined. In an animal experiment, male C57BL/6.Cr mice (n = 5) were fed an AIN-76-modified high fat diet supplemented with 2% or 5% (w/w) dried young persimmon fruit (YP) for 10 weeks. The intake of YP significantly enhanced fecal bile acid excretion and lowered the concentration of hepatic lipids and plasma cholesterol. Analysis of gene expression in liver tissue showed that 2% or 5% YP up-regulated the expression of the sterol regulatory element-binding protein-2 gene. In the 5% group, there were increased expressions of the genes for cholesterol 7alpha-hydroxylase and the low-density lipoprotein receptor. Next, the bile acid-binding ability of YP was analysed in vitro using cholic acid (CA). In 100-2000 microM CA solutions, 1% (w/v) YP adsorbed approximately 60% of CA, while dried mature persimmon fruit adsorbed approximately 20% of CA. The positive control, cholestyramine, adsorbed approximately 80% of CA in the 100-2000 microM CA solutions. A crude tannin extract from YP, which contained 54.7% condensed tannins, adsorbed approximately 78% of CA in the 2000 microM CA solutions. These results suggest that the ability of YP to bind bile acid contributes to its hypolipidemic effect in mice. PMID:19585467

  5. Proton and metal ion binding to natural organic polyelectrolytes-II. Preliminary investigation with a peat and a humic acid

    USGS Publications Warehouse

    Marinsky, J.A.; Reddy, M.M.

    1984-01-01

    We summarize here experimental studies of proton and metal ion binding to a peat and a humic acid. Data analysis is based on a unified physico-chemical model for reaction of simple ions with polyelectrolytes employing a modified Henderson-Hasselbalch equation. Peat exhibited an apparent intrinsic acid dissociation constant of 10-4.05, and an apparent intrinsic metal ion binding constant of: 400 for cadmium ion; 600 for zinc ion; 4000 for copper ion; 20000 for lead ion. A humic acid was found to have an apparent intrinsic proton binding constant of 10-2.6. Copper ion binding to this humic acid sample occurred at two types of sites. The first site exhibited reaction characteristics which were independent of solution pH and required the interaction of two ligands on the humic acid matrix to simultaneously complex with each copper ion. The second complex species is assumed to be a simple monodentate copper ion-carboxylate species with a stability constant of 18. ?? 1984.

  6. Proton and aluminum binding properties of organic acids in surface waters of the northeastern U.S.

    PubMed

    Fakhraei, Habibollah; Driscoll, Charles T

    2015-03-01

    A variety of mathematical estimators have been used to quantify the degree of protonation of naturally occurring organic acids. These estimators range from monoprotic, diprotic, and triprotic analog models to the discrete and continuous (Gaussian) distributions of a single proton binding-dissociation. Natural water samples from two long-term monitoring programs in the northeastern U.S. were used to quantify proton- and aluminum-binding properties of naturally occurring organic matter. Water chemistry observations were clustered into 0.05 pH intervals (over 3.75-7.35 pH range) and fit to a triprotic analog model. The model optimization indicates that about 5% of dissolved organic carbon participates in ion binding, and organic acids are composed of both strong and weak acids (i.e., pKa1 = 2.54, pKa2 = 6.19, and pKa3 = 7.52 for Adirondack samples). Binding between organic acids and aluminum can substantially influence the acid behavior of dissolved organic matter and the availability of the toxic form of aluminum (i.e., inorganic monomeric aluminum).

  7. Bile acid-binding activity of young persimmon (Diospyros kaki) fruit and its hypolipidemic effect in mice.

    PubMed

    Matsumoto, Kenji; Yokoyama, Shin-ichiro; Gato, Nobuki

    2010-02-01

    The hypolipidemic effects and bile acid-binding properties of young persimmon (Diospyros kaki) fruit were examined. In an animal experiment, male C57BL/6.Cr mice (n = 5) were fed an AIN-76-modified high fat diet supplemented with 2% or 5% (w/w) dried young persimmon fruit (YP) for 10 weeks. The intake of YP significantly enhanced fecal bile acid excretion and lowered the concentration of hepatic lipids and plasma cholesterol. Analysis of gene expression in liver tissue showed that 2% or 5% YP up-regulated the expression of the sterol regulatory element-binding protein-2 gene. In the 5% group, there were increased expressions of the genes for cholesterol 7alpha-hydroxylase and the low-density lipoprotein receptor. Next, the bile acid-binding ability of YP was analysed in vitro using cholic acid (CA). In 100-2000 microM CA solutions, 1% (w/v) YP adsorbed approximately 60% of CA, while dried mature persimmon fruit adsorbed approximately 20% of CA. The positive control, cholestyramine, adsorbed approximately 80% of CA in the 100-2000 microM CA solutions. A crude tannin extract from YP, which contained 54.7% condensed tannins, adsorbed approximately 78% of CA in the 2000 microM CA solutions. These results suggest that the ability of YP to bind bile acid contributes to its hypolipidemic effect in mice.

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

    PubMed Central

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

    2013-01-01

    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. PMID:23606130

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

    PubMed

    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.

  10. Interaction of aurintricarboxylic acid (ATA) with four nucleic acid binding proteins DNase I, RNase A, reverse transcriptase and Taq polymerase

    NASA Astrophysics Data System (ADS)

    Ghosh, Utpal; Giri, Kalyan; Bhattacharyya, Nitai P.

    2009-12-01

    In the investigation of interaction of aurintricarboxylic acid (ATA) with four biologically important proteins we observed inhibition of enzymatic activity of DNase I, RNase A, M-MLV reverse transcriptase and Taq polymerase by ATA in vitro assay. As the telomerase reverse transcriptase (TERT) is the main catalytic subunit of telomerase holoenzyme, we also monitored effect of ATA on telomerase activity in vivo and observed dose-dependent inhibition of telomerase activity in Chinese hamster V79 cells treated with ATA. Direct association of ATA with DNase I ( Kd = 9.019 μM)), RNase A ( Kd = 2.33 μM) reverse transcriptase ( Kd = 0.255 μM) and Taq polymerase ( Kd = 81.97 μM) was further shown by tryptophan fluorescence quenching studies. Such association altered the three-dimensional conformation of DNase I, RNase A and Taq polymerase as detected by circular dichroism. We propose ATA inhibits enzymatic activity of the four proteins through interfering with DNA or RNA binding to the respective proteins either competitively or allosterically, i.e. by perturbing three-dimensional structure of enzymes.

  11. Mutations of fumarase that distinguish between the active site and a nearby dicarboxylic acid binding site.

    PubMed Central

    Weaver, T.; Lees, M.; Banaszak, L.

    1997-01-01

    Two mutant forms of fumarase C from E. coli have been made using PCR and recombinant DNA. The recombinant form of the protein included a histidine arm on the C-terminal facilitating purification. Based on earlier studies, two different carboxylic acid binding sites, labeled A- and B-, were observed in crystal structures of the wild type and inhibited forms of the enzyme. A histidine at each of the sites was mutated to an asparagine. H188N at the A-site resulted in a large decrease in specific activity, while the H129N mutation at the B-site had essentially no effect. From the results, we conclude that the A-site is indeed the active site, and a dual role for H188 as a potential catalytic base is proposed. Crystal structures of the two mutant proteins produced some unexpected results. Both mutations reduced the affinity for the carboxylic acids at their respective sites. The H129N mutant should be particularly useful in future kinetic studies because it sterically blocks the B-site with the carboxyamide of asparagine assuming the position of the ligand's carboxylate. In the H188N mutation at the active site, the new asparagine side chain still interacts with an active site water that appears to have moved slightly as a result of the mutation. PMID:9098893

  12. Intestinal-fatty acid binding protein and lipid transport in human intestinal epithelial cells

    SciTech Connect

    Montoudis, Alain; Delvin, Edgard; Menard, Daniel

    2006-01-06

    Intestinal-fatty acid binding protein (I-FABP) is a 14-15 kDa cytoplasmic molecule highly expressed in the enterocyte. Although different functions have been proposed for various FABP family members, the specific function of I-FABP in human intestine remains unclear. Here, we studied the role of I-FABP in molecularly modified normal human intestinal epithelial cells (HIEC-6). cDNA transfection resulted in 90-fold I-FABP overexpression compared to cells treated with empty pQCXIP vector. The high-resolution immunogold technique revealed labeling mainly in the cytosol and confirmed the marked phenotype abundance of I-FABP in cDNA transfected cells. I-FABP overexpression was not associated with alterations in cell proliferation and viability. Studies using these transfected cells cultured with [{sup 14}C]oleic acid did not reveal higher efficiency in de novo synthesis or secretion of triglycerides, phospholipids, and cholesteryl esters compared to cells treated with empty pQCXIP vector only. Similarly, the incubation with [{sup 35}S]methionine did not disclose a superiority in the biogenesis of apolipoproteins (apo) A-I, A-IV, B-48, and B-100. Finally, cells transfected with I-FABP did not exhibit an increased production of chylomicrons, VLDL, LDL, and HDL. Our observations establish that I-FABP overexpression in normal HIEC-6 is not related to cell proliferation, lipid esterification, apo synthesis, and lipoprotein assembly, and, therefore, exclude its role in intestinal fat transport.

  13. pH-Activated Targeting Drug Delivery System Based on the Selective Binding of Phenylboronic Acid.

    PubMed

    Zhao, Dan; Xu, Jia-Qi; Yi, Xiao-Qing; Zhang, Quan; Cheng, Si-Xue; Zhuo, Ren-Xi; Li, Feng

    2016-06-15

    Phenylboronic acid (PBA) is a tumor-targeting molecule, but its nonspecific interaction with normal cells or other components containing cis-diol residues undoubtedly limits its potential application in tumor-targeting drug delivery. Herein, we developed fructose-coated mixed micelles via PBA-terminated polyethylene glycol monostearate (PBA-PEG-C18) and Pluronic P123 (PEG20-PPG70-PEG20) to solve this problem, as the stability of borate formed by PBA and fructose was dramatically dependent on pH. The fluorescence spectroscopic results indicated that the borate formed by PBA and fructose decomposed at a decreased pH, and better binding between PBA and sialic acid (SA) was observed at a low pH. These results implied that the fructose groups decorated on the surface of the micelles could be out-competed by SA at a low pH. In vitro uptake and cytotoxicity studies demonstrated that the fructose coating on the mixed micelles improved the endocytosis and enhanced the cytotoxicity of drug-loaded mixed micelles in HepG2 cells but reduced the cytotoxicity in normal cells. These results demonstrate that a simple decorating strategy may facilitate PBA-targeted nanoparticles for tumor-specific drug delivery. PMID:27229625

  14. Development of a Radioiodinated Triazolopyrimidine Probe for Nuclear Medical Imaging of Fatty Acid Binding Protein 4

    PubMed Central

    Onoe, Satoru; Sampei, Sotaro; Kimura, Ikuo; Ono, Masahiro; Saji, Hideo

    2014-01-01

    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. PMID:24732569

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

    PubMed

    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.

  16. Interaction of P-aminobenzoic acid with normal and sickel erythrocyte membrane: photoaffinity labelling of the binding sites

    SciTech Connect

    Premachandra, B.R.

    1986-03-05

    Electron microscopic studies revealed that P-Amino benzoic acid (PABA) could prevent eichinocytosis of red cells in vitro. Equilibrium binding studies with right side out membrane vesicles (ROV) revealed a similar number of binding sites (1.2-1.4 ..mu..mol/mg) and Kd (1.4-1.6 mM) values for both normal and sickle cell membranes. /sup 14/C-Azide analogue of PABA was synthesized as a photoaffinity label to probe its sites of interaction on the erythrocyte membranes. Competitive binding studies of PABA with its azide indicated that both the compounds share common binding sites on the membrane surface since a 20 fold excess of azide inhibited PABA binding in a linear fashion. The azide was covalently incorporated into the membrane components only upon irradiation (52-35% of the label found in the proteins and the rest in lipids). Electrophoretic analysis of photolabelled ROV revealed that the azide interacts chiefly with Band 3 protein. PABA inhibited both high and low affinity calcium (Ca) binding sites situated on either surface of the membrane in a non-competitive manner; however, Ca binding stimulated by Mg-ATP was not affected. Ca transport into inside out vesicles was inhibited by PABA; but it did not affect the calcium ATP-ase activity. The authors studies suggest that the mechanism of action of PABA is mediated by its interaction with Band 3 protein (anion channel), calcium channel and calcium binding sites of erythrocyte membrane.

  17. Photoaffinity Labeling of High Affinity Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP)-Binding Proteins in Sea Urchin Egg*♦

    PubMed Central

    Walseth, Timothy F.; Lin-Moshier, Yaping; Jain, Pooja; Ruas, Margarida; Parrington, John; Galione, Antony; Marchant, Jonathan S.; Slama, James T.

    2012-01-01

    Nicotinic acid adenine dinucleotide phosphate (NAADP) is a messenger that regulates calcium release from intracellular acidic stores. Recent studies have identified two-pore channels (TPCs) as endolysosomal channels that are regulated by NAADP; however, the nature of the NAADP receptor binding site is unknown. To further study NAADP binding sites, we have synthesized and characterized [32P-5-azido]nicotinic acid adenine dinucleotide phosphate ([32P-5N3]NAADP) as a photoaffinity probe. Photolysis of sea urchin egg homogenates preincubated with [32P-5N3]NAADP resulted in specific labeling of 45-, 40-, and 30-kDa proteins, which was prevented by inclusion of nanomolar concentrations of unlabeled NAADP or 5N3-NAADP, but not by micromolar concentrations of structurally related nucleotides such as NAD, nicotinic acid adenine dinucleotide, nicotinamide mononucleotide, nicotinic acid, or nicotinamide. [32P-5N3]NAADP binding was saturable and displayed high affinity (Kd ∼10 nm) in both binding and photolabeling experiments. [32P-5N3]NAADP photolabeling was irreversible in a high K+ buffer, a hallmark feature of NAADP binding in the egg system. The proteins photolabeled by [32P-5N3]NAADP have molecular masses smaller than the sea urchin TPCs, and antibodies to TPCs do not detect any immunoreactivity that comigrates with either the 45-kDa or the 40-kDa photolabeled proteins. Interestingly, antibodies to TPC1 and TPC3 were able to immunoprecipitate a small fraction of the 45- and 40-kDa photolabeled proteins, suggesting that these proteins associate with TPCs. These data suggest that high affinity NAADP binding sites are distinct from TPCs. PMID:22117077

  18. Proton binding to humic acids from organic amendments and amended soils by the NICA-Donnan model.

    PubMed

    Plaza, César; Brunetti, Gennaro; Senesi, Nicola; Polo, Alfredo

    2005-09-01

    The acid-base properties of humic acids (HAs) are known to significantly affect the acid-base buffering capacity of soils, thus having a marked influence on the speciation of cations in the soil solid and liquid phases. Detailed information on the proton binding behavior of humic-like acids (HALs) from organic amendments and humic acids (HAs) from amended soils is, therefore, of intrinsic interest for the evaluation of the agronomic efficacy and environmental impact of soil amendment. In this work, the acid-base properties of HLAs isolated from sewage sludge (SS) and municipal solid waste compost (MSWC), and HAs isolated from soils amended with either SS or MSWC and the corresponding nonamended control soils were investigated by potentiometric titrations at various ionic strengths (0.01, 0.05, 0.1, and 0.3 M) over the pH range from 3.5 to 10.5. The nonideal competitive adsorption (NICA)-Donnan model that describes proton binding by two classes of binding sites with low and high proton affinity, i.e., carboxylic- and phenolic-type groups, was fit to titration data, and a set of fitting parameters was obtained for each HLA and HA sample. The NICA-Donnan model successfully described the shapes of the titration curves, and highlighted substantial differences in site density and proton-binding affinity between the HLAs and HAs examined. With respect to the nonamended control soil HAs, SS-HLA and MSWC-HLA were characterized by smaller carboxylic-type and phenolic-type group contents, larger affinities for proton binding by the carboxylic-type groups, and smaller affinities for proton binding by the phenolic-type groups. Amendment with SS and MSWC determined a number of modifications in soil HAs, including decrease of acidic functional group contents, slight increase of proton affinity of carboxylic-type groups, and slight decrease of the affinities for proton binding by phenolic-type groups. These effects were more evident in the HA fraction from the SS-amended soil than

  19. Excessive Food Intake, Obesity and Inflammation Process in Zucker fa/fa Rat Pancreatic Islets

    PubMed Central

    Chentouf, Myriam; Dubois, Gregor; Jahannaut, Céline; Castex, Françoise; Lajoix, Anne Dominique; Gross, René; Peraldi-Roux, Sylvie

    2011-01-01

    Inappropriate food intake-related obesity and more importantly, visceral adiposity, are major risk factors for the onset of type 2 diabetes. Evidence is emerging that nutriment-induced β-cell dysfunction could be related to indirect induction of a state of low grade inflammation. Our aim was to study whether hyperphagia associated obesity could promote an inflammatory response in pancreatic islets leading to ß-cell dysfunction. In the hyperphagic obese insulin resistant male Zucker rat, we measured the level of circulating pro-inflammatory cytokines and estimated their production as well as the expression of their receptors in pancreatic tissue and β-cells. Our main findings concern intra-islet pro-inflammatory cytokines from fa/fa rats: IL-1β, IL-6 and TNFα expressions were increased; IL-1R1 was also over-expressed with a cellular redistribution also observed for IL-6R. To get insight into the mechanisms involved in phenotypic alterations, abArrays were used to determine the expression profile of proteins implicated in different membrane receptors signaling, apoptosis and cell cycle pathways. Despite JNK overexpression, cell viability was unaffected probably because of decreases in cleaved caspase3 as well as in SMAC/DIABLO and APP, involved in the induction and amplification of apoptosis. Concerning β-cell proliferation, decreases in important cell cycle regulators (Cyclin D1, p35) and increased expression of SMAD4 probably contribute to counteract and restrain hyperplasia in fa/fa rat islets. Finally and probably as a result of IL-1β and IL-1R1 increased expressions with sub-cellular redistribution of the receptor, islets from fa/fa rats were found more sensitive to both stimulating and inhibitory concentrations of the cytokine; this confers some physiopathological relevance to a possible autocrine regulation of β-cell function by IL-1β. These results support the hypothesis that pancreatic islets from prediabetic fa/fa rats undergo an inflammatory

  20. The role of FaBG3 in fruit ripening and B. cinerea fungal infection of strawberry.

    PubMed

    Li, Qian; Ji, Kai; Sun, Yufei; Luo, Hao; Wang, Hongqing; Leng, Ping

    2013-10-01

    In plants, β-glucosidases (BG) have been implicated in developmental and pathogen defense, and are thought to take part in abscisic acid (ABA) synthesis via hydrolysis of ABA glucose ester to release active ABA; however, there is no genetic evidence for the role of BG genes in ripening and biotic/abiotic stress in fruits. To clarify the role of BG genes in fruit, eight Fa/FvBG genes encoding β-glucosidase were isolated using information from the GenBank strawberry nucleotide database. Of the Fa/FvBG genes examined, expression of FaBG3 was the highest, showing peaks at the mature stage, coincident with the changes observed in ABA content. To verify the role of this gene, we suppressed the expression of FaBG3 via inoculation with Agrobacterium tumefaciens containing tobacco rattle virus carrying a FaBG3 fragment (RNAi). The expression of FaBG3 in FaBG3-RNAi-treated fruit was markedly reduced, and the ABA content was lower than that of the control. FaBG3-RNAi-treated fruit did not exhibit full ripening, and were firmer, had lower sugar content, and were pale compared with the control due to down-regulation of ripening-related genes. FaBG3-RNAi-treated fruit with reduced ABA levels were much more resistant to Botrytis cinerea fungus but were more sensitive to dehydration stress than control fruit. These results indicate that FaBG3 may play key roles in fruit ripening, dehydration stress and B. cinerea fungal infection in strawberries via modulation of ABA homeostasis and transcriptional regulation of ripening-related genes.

  1. Calcite growth-rate inhibition by fulvic acid and magnesium ion—Possible influence on biogenic calcite formation

    USGS Publications Warehouse

    Reddy, Michael M.

    2012-01-01

    Increases in ocean surface water dissolved carbon dioxide (CO2) concentrations retard biocalcification by reducing calcite supersaturation (Ωc). Reduced calcification rates may influence growth-rate dependent magnesium ion (Mg) incorporation into biogenic calcite modifying the use of calcifying organisms as paleoclimate proxies. Fulvic acid (FA) at biocalcification sites may further reduce calcification rates. Calcite growth-rate inhibition by FA and Mg, two common constituents of seawater and soil water involved in the formation of biogenic calcite, was measured separately and in combination under identical, highly reproducible experimental conditions. Calcite growth rates (pH=8.5 and Ωc=4.5) are reduced by FA (0.5 mg/L) to 47% and by Mg (10−4 M) to 38%, compared to control experiments containing no added growth-rate inhibitor. Humic acid (HA) is twice as effective a calcite growth-rate inhibitor as FA. Calcite growth rate in the presence of both FA (0.5 mg/L) and Mg (10−4 M) is reduced to 5% of the control rate. Mg inhibits calcite growth rates by substitution for calcium ion at the growth site. In contrast, FA inhibits calcite growth rates by binding multiple carboxylate groups on the calcite surface. FA and Mg together have an increased affinity for the calcite growth sites reducing calcite growth rates.

  2. Uterocalin, a lipocalin provisioning the preattachment equine conceptus: fatty acid and retinol binding properties, and structural characterization.

    PubMed Central

    Suire, S; Stewart, F; Beauchamp, J; Kennedy, M W

    2001-01-01

    The equine conceptus is surrounded by a fibrous capsule that persists until about day 20 of pregnancy, whereupon the capsule is lost, the conceptus attaches to the endometrium and placentation proceeds. Before attachment, the endometrium secretes in abundance a protein of the lipocalin family, uterocalin. The cessation of secretion coincides with the end of the period during which the conceptus is enclosed in its capsule, suggesting that uterocalin is essential for the support of the embryo before direct contact between maternal and foetal tissues is established. Using recombinant protein and fluorescence-based assays, we show that equine uterocalin binds the fluorescent fatty acids 11-(dansylamino)undecanoic acid, dansyl-D,L-alpha-amino-octanoic acid and cis-parinaric acid, and, by competition, oleic, palmitic, arachidonic, docosahexaenoic, gamma-linolenic, cis-eicosapentaenoic and linoleic acids. Uterocalin also binds all-trans-retinol, the binding site for which is coincident or interactive with that for fatty acids. Molecular modelling and intrinsic fluorescence analysis of the wild-type protein and a Trp-->Glu mutant protein indicated that uterocalin has an unusually solvent-exposed Trp side chain projecting from its large helix directly into solvent. This feature is unusual among lipocalins and might relate to binding to, and uptake by, the trophoblast. Uterocalin therefore has the localization and binding activities for the provisioning of the equine conceptus with lipids including those essential for morphogenesis and pattern formation. The possession of a fibrous capsule surrounding the conceptus might be an ancestral condition in mammals; homologues of uterocalin might be essential for early development in marsupials and in eutherians in which there is a prolonged preimplantation period. PMID:11368763

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

    PubMed

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

    2008-06-01

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

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

    PubMed

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

    2008-06-01

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

  5. Competition effects in cation binding to humic acid: Conditional affinity spectra for fixed total metal concentration conditions

    NASA Astrophysics Data System (ADS)

    David, Calin; Mongin, Sandrine; Rey-Castro, Carlos; Galceran, Josep; Companys, Encarnació; Garcés, José Luis; Salvador, José; Puy, Jaume; Cecilia, Joan; Lodeiro, Pablo; Mas, Francesc

    2010-09-01

    Information on the Pb and Cd binding to a purified Aldrich humic acid (HA) is obtained from the influence of different fixed total metal concentrations on the acid-base titrations of this ligand. NICA (Non-Ideal Competitive Adsorption) isotherm has been used for a global quantitative description of the binding, which has then been interpreted by plotting the Conditional Affinity Spectra of the H + binding at fixed total metal concentrations (CAScTM). This new physicochemical tool, here introduced, allows the interpretation of binding results in terms of distributions of proton binding energies. A large increase in the acidity of the phenolic sites as the total metal concentration increases, especially in presence of Pb, is revealed from the shift of the CAScTM towards lower affinities. The variance of the CAScTM distribution, which can be used as a direct measure of the heterogeneity, also shows a significant dependence on the total metal concentration. A discussion of the factors that influence the heterogeneity of the HA under the conditions of each experiment is provided, so that the smoothed pattern exhibited by the titration curves can be justified.

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

    PubMed

    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.

  7. Dominant negative mutant of retinoic acid receptor alpha inhibits retinoic acid-induced P19 cell differentiation by binding to DNA.

    PubMed

    Costa, S L; McBurney, M W

    1996-05-25

    Retinoic acid (RA) is a potent inducer of P19 cell differentiation. RA activity is thought to be mediated by nuclear RA receptors (RARs), transcription factors whose activity is dependent on RA. There are three RARs called alpha, beta, and gamma. We created truncated versions of the three RARs and compared their activities as inhibitors of RA-mediated gene transcription and of P19 cell differentiation. Only mutants of the RAR alpha were inhibitory in these assays. A mutant of RAR alpha carrying a 10-amino-acid insert was able to heterodimerize with RXRbeta or with the normal RAR alpha and the inhibitory activity of this mutant was dependent on an intact DNA binding domain. We conclude that dominant negative mutants of RAR alpha act by heterodimerizing with RXRs or RARs and binding to RA response elements on DNA, thereby preventing binding of the normal receptors to those sites. PMID:8635515

  8. Human Adenovirus 52 Uses Sialic Acid-containing Glycoproteins and the Coxsackie and Adenovirus Receptor for Binding to Target Cells

    PubMed Central

    Lenman, Annasara; Liaci, A. Manuel; Liu, Yan; Årdahl, Carin; Rajan, Anandi; Nilsson, Emma; Bradford, Will; Kaeshammer, Lisa; Jones, Morris S.; Frängsmyr, Lars; Feizi, Ten; Stehle, Thilo; Arnberg, Niklas

    2015-01-01

    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. PMID:25674795

  9. Characterization of ERM transactivation domain binding to the ACID/PTOV domain of the Mediator subunit MED25.

    PubMed

    Landrieu, Isabelle; Verger, Alexis; Baert, Jean-Luc; Rucktooa, Prakash; Cantrelle, François-Xavier; Dewitte, Frédérique; Ferreira, Elisabeth; Lens, Zoé; Villeret, Vincent; Monté, Didier

    2015-08-18

    The N-terminal acidic transactivation domain (TAD) of ERM/ETV5 (ERM38-68), a PEA3 group member of Ets-related transcription factors, directly interacts with the ACID/PTOV domain of the Mediator complex subunit MED25. Molecular details of this interaction were investigated using nuclear magnetic resonance (NMR) spectroscopy. The TAD is disordered in solution but has a propensity to adopt local transient secondary structure. We show that it folds upon binding to MED25 and that the resulting ERM-MED25 complex displays characteristics of a fuzzy complex. Mutational analysis further reveals that two aromatic residues in the ERM TAD (F47 and W57) are involved in the binding to MED25 and participate in the ability of ERM TAD to activate transcription. Mutation of a key residue Q451 in the VP16 H1 binding pocket of MED25 affects the binding of ERM. Furthermore, competition experiments show that ERM and VP16 H1 share a common binding interface on MED25. NMR data confirms the occupancy of this binding pocket by ERM TAD. Based on these experimental data, a structural model of a functional interaction is proposed. This study provides mechanistic insights into the Mediator-transactivator interactions. PMID:26130716

  10. Characterization of ERM transactivation domain binding to the ACID/PTOV domain of the Mediator subunit MED25

    PubMed Central

    Landrieu, Isabelle; Verger, Alexis; Baert, Jean-Luc; Rucktooa, Prakash; Cantrelle, François-Xavier; Dewitte, Frédérique; Ferreira, Elisabeth; Lens, Zoé; Villeret, Vincent; Monté, Didier

    2015-01-01

    The N-terminal acidic transactivation domain (TAD) of ERM/ETV5 (ERM38–68), a PEA3 group member of Ets-related transcription factors, directly interacts with the ACID/PTOV domain of the Mediator complex subunit MED25. Molecular details of this interaction were investigated using nuclear magnetic resonance (NMR) spectroscopy. The TAD is disordered in solution but has a propensity to adopt local transient secondary structure. We show that it folds upon binding to MED25 and that the resulting ERM–MED25 complex displays characteristics of a fuzzy complex. Mutational analysis further reveals that two aromatic residues in the ERM TAD (F47 and W57) are involved in the binding to MED25 and participate in the ability of ERM TAD to activate transcription. Mutation of a key residue Q451 in the VP16 H1 binding pocket of MED25 affects the binding of ERM. Furthermore, competition experiments show that ERM and VP16 H1 share a common binding interface on MED25. NMR data confirms the occupancy of this binding pocket by ERM TAD. Based on these experimental data, a structural model of a functional interaction is proposed. This study provides mechanistic insights into the Mediator–transactivator interactions. PMID:26130716

  11. Identification of functionally important amino acids in the cellulose-binding domain of Trichoderma reesei cellobiohydrolase I.

    PubMed Central

    Linder, M.; Mattinen, M. L.; Kontteli, M.; Lindeberg, G.; Ståhlberg, J.; Drakenberg, T.; Reinikainen, T.; Pettersson, G.; Annila, A.

    1995-01-01

    Cellobiohydrolase I (CBHI) of Trichoderma reesei has two functional domains, a catalytic core domain and a cellulose binding domain (CBD). The structure of the CBD reveals two distinct faces, one of which is flat and the other rough. Several other fungal cellulolytic enzymes have similar two-domain structures, in which the CBDs show a conserved primary structure. Here we have evaluated the contributions of conserved amino acids in CBHI CBD to its binding to cellulose. Binding isotherms were determined for a set of six synthetic analogues in which conserved amino acids were substituted. Two-dimensional NMR spectroscopy was used to assess the structural effects of the substitutions by comparing chemical shifts, coupling constants, and NOEs of the backbone protons between the wild-type CBD and the analogues. In general, the structural effects of the substitutions were minor, although in some cases decreased binding could clearly be ascribed to conformational perturbations. We found that at least two tyrosine residues and a glutamine residue on the flat face were essential for tight binding of the CBD to cellulose. A change on the rough face had only a small effect on the binding and it is unlikely that this face interacts with cellulose directly. PMID:7549870

  12. Production of a Brassica napus low-molecular mass acyl-coenzyme A-binding protein in Arabidopsis alters the acyl-coenzyme A pool and acyl composition of oil in seeds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Low-molecular mass (10 kD) cytosolic acyl-coenzyme A-binding protein (ACBP) has a substantial influence over fatty acid (FA) composition in oilseeds, possibly via an effect on the partitioning of acyl groups between elongation and desaturation pathways. Previously, we demonstrated that the expressio...

  13. 5'-azido-N-1-naphthylphthalamic acid, a photolabile analog of the auxin transport inhibitor, N-1-naphthylphthalamic acid: synthesis and binding properties

    SciTech Connect

    Voet, J.G.; Howley, K.; Shumsky, J.S.

    1987-05-01

    The polar transport of the plant growth regulator, auxin (indole-3-acetic acid, IAAH), is thought to involve the participation of several proteins in the plasma membrane, including a specific, saturable, voltage independent H/sup +//IAA/sup -/ efflux carrier located preferentially at the basal end of each cell. Auxin transport is specifically inhibited by the herbicide, N-1-naphthylphthalamic acid (NPA), which binds specifically to a protein in the plasma membrane, thought to be either the IAA/sup -/ efflux carrier or an allosteric effector protein. They have synthesized and characterized a photolabile analog of NPA, 5'-azido-N-1-naphthylphthalamic acid (Az-NPA). This potential photoaffinity label for the NPA binding protein competes with /sup 3/H-NPA for binding sites on Curcurbita pepo L. (zucchini) stem cell membranes with K/sub j/ = 1.5 x 10/sup -7/ M. The K/sub i/ for NPA under these conditions is 2 x 10/sup -8/M, indicating that the affinity of Az-NPA for the membranes is only 7.5 fold lower than NPA. While the binding of 4.6 x 10/sup -6/ M Az-NPA to NPA binding sites is reversible in the dark, exposure to light results in a 30% loss in /sup 3/H-NPA binding ability. Pretreatment with 10/sup -4/ M NPA protects the membranes against photodestruction of /sup 3/H-NPA binding sites by Az-NPA, supporting the conclusion that Az-NPA destroys these sites by specific covalent attachment.

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

    PubMed

    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.

  15. Chicoric acid binds to two sites and decreases the activity of the YopH bacterial virulence factor

    PubMed Central

    Kuban-Jankowska, Alicja; Sahu, Kamlesh K.; Gorska, Magdalena; Tuszynski, Jack A.; Wozniak, Michal

    2016-01-01

    Chicoric acid (CA) is a phenolic compound present in dietary supplements with a large spectrum of biological properties reported ranging from antioxidant, to antiviral, to immunostimulatory properties. Due to the fact that chicoric acid promotes phagocytic activity and was reported as an allosteric inhibitor of the PTP1B phosphatase, we examined the effect of CA on YopH phosphatase from pathogenic bacteria, which block phagocytic processes of a host cell. We performed computational studies of chicoric acid binding to YopH as well as validation experiments with recombinant enzymes. In addition, we performed similar studies for caffeic and chlorogenic acids to compare the results. Docking experiments demonstrated that, from the tested compounds, only CA binds to both catalytic and secondary binding sites of YopH. Our experimental results showed that CA reduces activity of recombinant YopH phosphatase from Yersinia enterocolitica and human CD45 phosphatase. The inhibition caused by CA was irreversible and did not induce oxidation of catalytic cysteine. We proposed that inactivation of YopH induced by CA is involved with allosteric inhibition by interacting with essential regions responsible for ligand binding. PMID:26735581

  16. Bile acid-binding ability of kaki-tannin from young fruits of persimmon (Diospyros kaki) in vitro and in vivo.

    PubMed

    Matsumoto, Kenji; Kadowaki, Akio; Ozaki, Natsumi; Takenaka, Makiko; Ono, Hiroshi; Yokoyama, Shin-ichiro; Gato, Nobuki

    2011-04-01

    The bile acid-binding ability of a highly polymerized tannin (kaki-tannin) extracted from dried-young fruits of persimmon (Diospyros kaki) was examined. The kaki-tannin was composed mainly of epicatechin, epigallocatechin, epicatechin-3-O-gallate and epigallocatechin-3-O-gallate. Bile acid-binding ability of kaki-tannin was examined against cholic acid, glycocholic acid, taurocholic acid and deoxycholic acid in vitro, and its effect on fecal bile acid excretion in mice was also examined. Although the bile acid-binding ability of kaki-tannin was weaker than that of cholestyramine, kaki-tannin adsorbed all the bile acids tested and significantly promoted fecal bile acid excretion in mice when supplied at 1% (w/w) in the diet. PMID:20922818

  17. Bile acid-binding ability of kaki-tannin from young fruits of persimmon (Diospyros kaki) in vitro and in vivo.

    PubMed

    Matsumoto, Kenji; Kadowaki, Akio; Ozaki, Natsumi; Takenaka, Makiko; Ono, Hiroshi; Yokoyama, Shin-ichiro; Gato, Nobuki

    2011-04-01

    The bile acid-binding ability of a highly polymerized tannin (kaki-tannin) extracted from dried-young fruits of persimmon (Diospyros kaki) was examined. The kaki-tannin was composed mainly of epicatechin, epigallocatechin, epicatechin-3-O-gallate and epigallocatechin-3-O-gallate. Bile acid-binding ability of kaki-tannin was examined against cholic acid, glycocholic acid, taurocholic acid and deoxycholic acid in vitro, and its effect on fecal bile acid excretion in mice was also examined. Although the bile acid-binding ability of kaki-tannin was weaker than that of cholestyramine, kaki-tannin adsorbed all the bile acids tested and significantly promoted fecal bile acid excretion in mice when supplied at 1% (w/w) in the diet.

  18. Rhizobium meliloti mutants that overproduce the R. meliloti acidic Calcofluor-binding exopolysaccharide

    SciTech Connect

    Doherty, D.; Glazebrook, J.; Walker, G.C. ); Leigh, J.A. )

    1988-09-01

    The acidic Calcofluor-binding exopolysaccharide of Rhizobium meliloti Rm1021 plays one or more critical roles in nodule invasion and possible in nodule development. Two loci, exoR and exoS, that effect the regulation of synthesis of this exopolysaccharide were identified by screening for derivatives of strain Rm1021 that formed mucoid colonies that fluoresced extremely brightly under UV light when grown on medium containing Calcofluor. The exopolysaccharide produced in large quantities by the exoR95::Tn5 and exoS96::Tn5 strains was indistinguishable from that produced by the parental strain Rm1021, and its synthesis required the function of at least the exoA, exoB, and exoF genes. Both the exoR and exoS loci were located on the chromosome, and the exo96::Tn5 mutation was 84% linked to the trp-33 mutation by {Phi}M12 transduction. Synthesis of the Calcofluor-binding exopolysaccharide by strain Rm1021 was greatly stimulated by starvation for ammonia. In contrast, the exoR95::Tn5 mutant produced high levels of exopolysaccharide regardless of the presence or absence of ammonia in the medium. The exoS96::Tn5 mutant produced elevated amounts of exopolysaccharide in the presence of ammonia, but higher amounts were observed after starvation for ammonia. The presence of either mutation increased the level of expression of exoF::TnphoA and exoP::TnphoA fusions. Analyses of results obtained when alfalfa seedlings were inoculated with the exoR95::Tn5 strain indicated that the mutant strain could not invade nodules. However, pseudorevertants that retained the original exoR95::Tn5 mutant but acquired unlinked suppressors so that they produced an approximately normal amount of exopolysaccharide were able to invade nodules and fix nitrogen.

  19. RNA-binding protein QKI regulates Glial fibrillary acidic protein expression in human astrocytes.

    PubMed

    Radomska, Katarzyna J; Halvardson, Jonatan; Reinius, Björn; Lindholm Carlström, Eva; Emilsson, Lina; Feuk, Lars; Jazin, Elena

    2013-04-01

    Linkage, association and expression studies previously pointed to the human QKI, KH domain containing, RNA-binding (QKI) as a candidate gene for schizophrenia. Functional studies of the mouse orthologue Qk focused mainly on its role in oligodendrocyte development and myelination, while its function in astroglia remained unexplored. Here, we show that QKI is highly expressed in human primary astrocytes and that its splice forms encode proteins targeting different subcellular localizations. Uncovering the role of QKI in astrocytes is of interest in light of growing evidence implicating astrocyte dysfunction in the pathogenesis of several disorders of the central nervous system. We selectively silenced QKI splice variants in human primary astrocytes and used RNA sequencing to identify differential expression and splice variant composition at the genome-wide level. We found that an mRNA expression of Glial fibrillary acidic protein (GFAP), encoding a major component of astrocyte intermediate filaments, was down-regulated after QKI7 splice variant silencing. Moreover, we identified a potential QKI-binding site within the 3' untranslated region of human GFAP. This sequence was not conserved between mice and humans, raising the possibility that GFAP is a target for QKI in humans but not rodents. Haloperidol treatment of primary astrocytes resulted in coordinated increases in QKI7 and GFAP expression. Taken together, our results provide the first link between QKI and GFAP, two genes with alterations previously observed independently in schizophrenic patients. Our findings for QKI, together with its well-known role in myelination, suggest that QKI is a hub regulator of glia function in humans.

  20. Deciphering the specific high-affinity binding of cucurbit[7]uril to amino acids in water.

    PubMed

    Lee, Jong Wha; Lee, Hyun Hee L; Ko, Young Ho; Kim, Kimoon; Kim, Hugh I

    2015-04-01

    This work presents a systematic study on the host-guest interactions between the macrocyclic host molecule cucurbit[7]uril (CB[7]) and amino acids (AAs) including three basic AAs (Lys, Arg, and His) and three aromatic AAs (Phe, Tyr, and Trp) to elucidate the origin of the high selectivity of CB[7] toward AA residues in proteins. Complex formation between CB[7] and each AA was examined in solution (by isothermal titration calorimetry and NMR) as well as in the gas phase (by ion mobility mass spectrometry and collision-induced dissociation), and the results were further combined with computational investigations. Generally, the aromatic AAs show higher binding affinities than the basic AAs in buffer solutions with various pH values. On the contrary, the gas-phase stabilities of the basic AA complex ions are higher than those of the aromatic AA complex ions, suggesting that the direct ion-dipole interactions between the charged side chains of the basic AAs and the polar carbonyl groups of CB[7] predominate in the absence of water. The ion-dipole interactions are less significant in water, since the original interactions of the guests with water are lost upon complex formation. In contrast, the transfer of the hydrophobic groups from the bulk into the hydrophobic CB[7] cavity suffers less from the desolvation penalty, resulting in higher binding affinities in water. Therefore, initial guest solvation is another key factor which should be considered when designing high-affinity host-guest systems, in addition to the contribution from the release of high-energy water molecules from the CB[7] cavity (J. Am. Chem. Soc. 2012, 134, 15318-15323).

  1. Deciphering the specific high-affinity binding of cucurbit[7]uril to amino acids in water.

    PubMed

    Lee, Jong Wha; Lee, Hyun Hee L; Ko, Young Ho; Kim, Kimoon; Kim, Hugh I

    2015-04-01

    This work presents a systematic study on the host-guest interactions between the macrocyclic host molecule cucurbit[7]uril (CB[7]) and amino acids (AAs) including three basic AAs (Lys, Arg, and His) and three aromatic AAs (Phe, Tyr, and Trp) to elucidate the origin of the high selectivity of CB[7] toward AA residues in proteins. Complex formation between CB[7] and each AA was examined in solution (by isothermal titration calorimetry and NMR) as well as in the gas phase (by ion mobility mass spectrometry and collision-induced dissociation), and the results were further combined with computational investigations. Generally, the aromatic AAs show higher binding affinities than the basic AAs in buffer solutions with various pH values. On the contrary, the gas-phase stabilities of the basic AA complex ions are higher than those of the aromatic AA complex ions, suggesting that the direct ion-dipole interactions between the charged side chains of the basic AAs and the polar carbonyl groups of CB[7] predominate in the absence of water. The ion-dipole interactions are less significant in water, since the original interactions of the guests with water are lost upon complex formation. In contrast, the transfer of the hydrophobic groups from the bulk into the hydrophobic CB[7] cavity suffers less from the desolvation penalty, resulting in higher binding affinities in water. Therefore, initial guest solvation is another key factor which should be considered when designing high-affinity host-guest systems, in addition to the contribution from the release of high-energy water molecules from the CB[7] cavity (J. Am. Chem. Soc. 2012, 134, 15318-15323). PMID:25757499

  2. In vitro mutagenesis study of two critical glutamic acids in the calcium binding loop of the factor IX heavy chain.

    PubMed

    Hamaguchi, N; Stafford, D

    1994-12-01

    We investigated the structural and functional significance of calcium binding in the factor IXa heavy chain by introducing point mutations into the probable calcium binding site (residues 235 and 245). According to factor IXa computer modelling based on trypsin x-ray structure, side chains of two glutamic acid residues, 235 and 245, together with backbone carbonyl groups of residues 237 and 240, bind a calcium ion. Factor IX clotting activity decreased approximately 25 percent on substitution of glutamic acid 235 with lysine. Activity decreased more than 90 percent on substitution of glutamic acid 245 with lysine. Activity also decreased more than 90 percent on substitution of both glutamic acids by lysines. Factor XIa cleavage of factor IXGlu235Lys and factor IXGlu245Lys appeared normal by polyacrylamide gel analysis. (Factor IXGlu235Lys: Factor IX with Lysine substituted for Glutamic acid at residue 235. Factor IXGlu245Lys: Factor IX with Lysine substituted for Glutamic acid at residue 245. Factor IXGlu235&245Lys: Factor IX with Lysine substituted for Glutamic acid at residues 235 and 245.) Activated factor IXGlu235Lys bound the fluorescent active site probe, p-aminobenzamidine, normally, while factor XIa cleaved factor IXGlu245Lys and factor IXGlu235&245Lys failed to bind p-aminobenzamidine. Plasma purified factor IX titrated with terbium showed an increase in luminescence; however, factor IXGlu235Lys and factor IXGlu245Lys had no effect on terbium luminescence. Radioimmunoassays indicate that in calcium's absence, factor IXGlu245Lys adopts a conformation similar to normal factor IX in the presence of calcium. By contrast, factor IXGlu245Lys's conformation in the presence of calcium is similar to that of plasma purified factor IX in the absence of calcium.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7740454

  3. Unraveling the binding mechanism of asiatic acid with human serum albumin and its biological implications.

    PubMed

    Gokara, Mahesh; Malavath, Tirupathi; Kalangi, Suresh Kumar; Reddana, Pallu; Subramanyam, Rajagopal

    2014-01-01

    Asiatic acid (AsA), a naturally occurring pentacyclictriterpenoid found in Centella asiatica, plays a major role in neuroprotection, anticancer, antioxidant, and hepatoprotective activities. Human serum albumin (HSA), a blood plasma protein, participates in the regulation of plasma osmotic pressure and transports endogenous and exogenous substances. The study undertaken to analyze the drug-binding mechanisms of HSA is crucial in understanding the bioavailability of drugs. In this study, we analyzed the cytotoxic activity of AsA on HepG2 (human hepatocellular carcinoma) cell lines and its binding, conformational, docking, molecular simulation studies with HSA under physiological pH 7.2. These studies revealed a clear decrease in the viability of HepG2 cells upon exposure to AsA in a dose-dependent manner with an IC50 of 45 μM. Further studies showed the quenching of intrinsic fluorescence of HSA by AsA with a binding constant of KAsA = 3.86 ± 0.01 × 10(4) M(-1), which corresponds to the free energy of (ΔG) -6.3 kcal M(-1) at 25 °C. Circular dichroism (CD) studies revealed that there is a clear decrease in the α-helical content from 57.50 ± 2.4 to 50% ± 2.3 and an increase in the β-turns from 25 ± 0.65 to 29% ± 0.91 and random coils from 17.5% ± 0.95 to 21% ± 1.2, suggesting partial unfolding of HSA. Autodock studies revealed that the AsA is bound to the subdomain IIA with hydrophobic and hydrophilic interactions. From molecular dynamics, simulation data (RMSD, Rg and RMSF) emphasized the local conformational changes and rigidity of the residues of both HSA and HSA-AsA complexes. PMID:23844909

  4. Functional analysis of peroxisome-proliferator-responsive element motifs in genes of fatty acid-binding proteins

    PubMed Central

    2004-01-01

    Retinoic acids and long-chain fatty acids are lipophilic agonists of nuclear receptors such as RXRs (retinoic X receptors) and PPARs (peroxisome-proliferator-activated receptors) respectively. These agonists are also ligands of intracellular lipid-binding proteins, which include FABPs (fatty acid-binding proteins). We reported previously that L (liver-type)-FABP targets fatty acids to the nucleus of hepatocytes and affects PPARα activation, which binds together with an RXR subtype to a PPRE (peroxisome-proliferator-responsive element). In the present study, we first determined the optimal combination of murine PPAR/RXR subtypes for binding to known murine FABP-PPREs and to those found by computer search and then tested their in vitro functionality. We show that all PPARs bind to L-FABP-PPRE, PPARα, PPARγ1 and PPARγ2 to A (adipocyte-type)-FABP-PPRE. All PPAR/RXR heterodimers transactivate L-FABP-PPRE, best are combinations of PPARα with RXRα or RXRγ. In contrast, PPARα heterodimers do not transactivate A-FABP-PPRE, best combinations are of PPARγ1 with RXRα and RXRγ, and of PPARγ2 with all RXR subtypes. We found that the predicted E (epidermal-type)- and H (heart-type)-FABP-PPREs are not activated by any PPAR/RXR combination without or with the PPAR pan-agonist bezafibrate. In the same way, C2C12 myoblasts transfected with promoter fragments of E-FABP and H-FABP genes containing putative PPREs are also not activated through stimulation of PPARs with bezafibrate applied to the cells. These results demonstrate that only PPREs of L- and A-FABP promoters are functional, and that binding of PPAR/RXR heterodimers to a PPRE in vitro does not necessarily predict transactivation. PMID:15130092

  5. 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.

    PubMed

    Baglo, Yan; Gabrielsen, Mari; Sylte, Ingebrigt; Gederaas, Odrun A

    2013-01-01

    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. PMID:23762315

  6. Synthesis and anticoagulant activity of bioisosteric sulfonic-Acid analogues of the antithrombin-binding pentasaccharide domain of heparin.

    PubMed

    Herczeg, Mihály; Lázár, László; Bereczky, Zsuzsanna; Kövér, Katalin E; Timári, István; Kappelmayer, János; Lipták, András; Antus, Sándor; Borbás, Anikó

    2012-08-20

    Two pentasaccharide sulfonic acids that were related to the antithrombin-binding domain of heparin were prepared, in which two or three primary sulfate esters were replaced by sodium-sulfonatomethyl moieties. The sulfonic-acid groups were formed on a monosaccharide level and the obtained carbohydrate sulfonic-acid esters were found to be excellent donors and acceptors in the glycosylation reactions. Throughout the synthesis, the hydroxy groups to be methylated were masked in the form of acetates and the hydroxy groups to be sulfated were masked with benzyl groups. The disulfonic-acid analogue was prepared in a [2+3] block synthesis by using a trisaccharide disulfonic acid as an acceptor and a glucuronide disaccharide as a donor. For the synthesis of the pentasaccharide trisulfonic acid, a more-efficient approach, which involved elongation of the trisaccharide acceptor with a non-oxidized precursor of the glucuronic acid followed by post-glycosidation oxidation at the tetrasaccharide level and a subsequent [1+4] coupling reaction, was elaborated. In vitro evaluation of the anticoagulant activity of these new sulfonic-acid derivatives revealed that the disulfonate analogue inhibited the blood-coagulation-proteinase factor Xa with outstanding efficacy; however, the introduction of the third sulfonic-acid moiety resulted in a notable decrease in the anti-Xa activity. The difference in the biological activity of the disulfonic- and trisulfonic-acid counterparts could be explained by the different conformation of their L-iduronic-acid residues.

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

    PubMed Central

    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

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

    PubMed Central

    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

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

    PubMed

    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.

  10. Temporal profile of intestinal tissue expression of intestinal fatty acid-binding protein in a rat model of necrotizing enterocolitis

    PubMed Central

    Simões, Ana Leda Bertoncini; Figueira, Rebeca Lopes; Gonçalves, Frances Lilian Lanhellas; Mitidiero, Luís Felipe Tsuyoshi; Silva, Orlando Castro e; Peiró, José Luis; Sbragia, Lourenço

    2016-01-01

    OBJECTIVES: Necrotizing enterocolitis is a severe multifactorial intestinal disorder that primarily affects preterm newborns, causing 20-40% mortality and morbidity. Intestinal fatty acid-binding protein has been reported to be a biomarker for the detection of intestinal injuries. Our aim was to assess intestinal tissue injury and the molecular expression of intestinal fatty acid-binding protein over time in a necrotizing enterocolitis model. METHODS: A total of 144 Newborn rats were divided into two groups: 1) Control, which received breastfeeding (n=72) and 2) Necrotizing Enterocolitis, which received formula feeding and underwent hypoxia and hypothermia (n=72). A total of six time points of ischemia (2 times a day for 3 days; 12 pups for each time point) were examined. Samples were collected for analysis of body weight, morphological and histological characteristics, intestinal weight, intestinal weight/body weight ratio, injury grade, and intestinal fatty acid-binding protein levels. RESULTS: Body and intestinal weights were lower in the Necrotizing Enterocolitis group than in the Control group (p<0.005 and p<0.0005, respectively). The intestinal weight/body weight ratio was higher in the Necrotizing Enterocolitis group than in the Control group (p<0.005) only at the sixth ischemia time point. The Necrotizing Enterocolitis group displayed higher expression of intestinal fatty acid-binding protein (p<0.0005) and showed greater tissue damage than the Control group. CONCLUSION: Intestinal fatty acid-binding protein was an efficient marker of ischemic injury to the intestine and a good correlation was demonstrated between the time of ischemic injury and the grade of intestinal injury. PMID:27464299

  11. Development of novel ferulic acid derivatives as potent histone deacetylase inhibitors.

    PubMed

    Wang, Fang; Lu, Wen; Zhang, Tao; Dong, Jinyun; Gao, Hongping; Li, Pengfei; Wang, Sicen; Zhang, Jie

    2013-11-15

    Histone deacetylase inhibitors (HDACIs) offer a promising strategy for cancer therapy. The discovery of potent ferulic acid-based HDACIs with hydroxamic acid or 2-aminobenzamide group as zinc binding group was reported. The halogeno-acetanilide was introduced as novel surface recognition moiety (SRM). The majority of title compounds displayed potent HDAC inhibitory activity. In particular, FA6 and FA16 exhibited significant enzymatic inhibitory activities, with IC50 values of 3.94 and 2.82 μM, respectively. Furthermore, these compounds showed moderate antiproliferative activity against a panel of human cancer cells. FA17 displayed promising profile as an antitumor candidate. The results indicated that these ferulic acid derivatives could serve as promising lead compounds for further optimization. PMID:24095016

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

    SciTech Connect

    Lummis, S.C.R.; Johnston, G.A.R. ); Nicoletti, G. ); Holan, G. )

    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.

  13. 15N NMR investigation of the covalent binding of reduced TNT amines to soil humic acid, model compounds, and lignocellulose

    USGS Publications Warehouse

    Thorn, K.A.; Kennedy, K.R.

    2002-01-01

    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.

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

    PubMed Central

    Thanos, Panayotis K.; Clavin, Brendan H.; Hamilton, John; O’Rourke, Joseph R.; Maher, Thomas; Koumas, Christopher; Miao, Erick; Lankop, Jessenia; Elhage, Aya; Haj-Dahmane, Samir; Deutsch, Dale; Kaczocha, Martin

    2016-01-01

    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, has 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. 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 (SN) given its recently reported anti-inflammatory and analgesic properties. Male C57BL mice were split into four treatment groups and conditioned with 5.0, 20.0, 40.0 mg/kg SBFI26, or vehicle during a conditioned place preference (CPP) paradigm. Following CPP, mice underwent a battery of behavioral tests [open field, novel object recognition (NOR), social interaction (SI), and SN] paired with acute SBFI26 administration. Results showed that SBFI26 did not produce CPP 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. PMID:27092087

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

    PubMed

    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.

  16. Identification by affinity chromatography of the eukaryotic ribosomal proteins that bind to 5.8 S ribosomal ribonucleic acid.

    PubMed

    Ulbrich, N; Lin, A; Wool, I G

    1979-09-10

    The proteins that bind to rat liver 5.8 S ribosomal ribonucleic acid were identified by affinity chromatography. The nucleic acid was oxidized with periodate and coupled by its 3'-terminus to Sepharose 4B through and adipic acid dihydrazide spacer. The ribosomal proteins that associate with the immobilized 5.8 S rRNA were identified by polyacrylamide gel electrophoresiss: they were L19, L8, and L6 from the 60 S subunit; and S13 and S9 from the small subparticle. Small amounts of L14, L17', L18, L27/L27', and L35', and of S11, S15, S23/S24, and S26 also were bound to the affinity column, but whether they associate directly and specifically with 5.8 S rRNA is not known. Escherichia coli ribosomal proteins did not bind to the rat liver 5.8 S rRNA affinity column. PMID:468846

  17. Structure-function studies of human deoxyhypusine synthase: identification of amino acid residues critical for the binding of spermidine and NAD.

    PubMed

    Lee, C H; Um, P Y; Park, M H

    2001-05-01

    Deoxyhypusine synthase catalyses the first step in the biosynthesis of hypusine [N(epsilon)-(4-amino-2-hydroxybutyl)lysine]. The crystal structure of human deoxyhypusine synthase in complex with NAD revealed four NAD-binding sites per enzyme tetramer, and led to a prediction of the spermidine-binding pocket. We have replaced each of the seven amino acid residues at the predicted spermidine-binding site, and eleven residues that contact NAD, on an individual basis with alanine. Of the amino acid residues at the spermidine site, substitution of Asp-243, Trp-327, His-288, Asp-316 or Glu-323 with alanine caused an almost complete loss of spermidine binding and enzyme activity; only the mutation Tyr-305-->Ala showed partial binding and activity. His-288-->Ala was also deficient in terms of binding NAD. NAD binding was significantly reduced in all of the NAD-site mutant enzymes, except for Glu-137-->Ala, which showed a normal binding of NAD, but was totally lacking in spermidine binding. Of the NAD-site mutant enzymes, Asp-342-->Ala, Asp-313-->Ala and Asp-238-->Ala displayed the lowest binding of NAD. These enzymes and His-288Ala also showed a reduced binding of spermidine, presumably because spermidine binding is dependent on NAD. These findings permit the positive identification of amino acid residues critical for binding of spermidine and NAD, and provide a new insight into the complex molecular interactions involved in the deoxyhypusine synthase reaction. PMID:11311149

  18. A surface-associated retinol- and fatty acid-binding protein (Gp-FAR-1) from the potato cyst nematode Globodera pallida: lipid binding activities, structural analysis and expression pattern.

    PubMed Central

    Prior, A; Jones, J T; Blok, V C; Beauchamp, J; McDermott, L; Cooper, A; Kennedy, M W

    2001-01-01

    Parasitic nematodes produce at least two structurally novel classes of small helix-rich retinol- and fatty-acid-binding proteins that have no counterparts in their plant or animal hosts and thus represent potential targets for new nematicides. Here we describe a protein (Gp-FAR-1) from the plant-parasitic nematode Globodera pallida, which is a member of the nematode-specific fatty-acid- and retinol-binding (FAR) family of proteins but localizes to the surface of this species, placing it in a strategic position for interaction with the host. Recombinant Gp-FAR-1 was found to bind retinol, cis-parinaric acid and the fluorophore-tagged lipids 11-(dansylamino)undecanoic acid and dansyl-D,L-alpha-amino-octanoic acid. The fluorescence emission characteristics of the dansylated analogues indicated that the entire ligand enters the binding cavity. Fluorescence competition experiments showed that Gp-FAR-1 binds fatty acids in the range C(11) to C(24), with optimal binding at C(15). Intrinsic fluorescence analysis of a mutant protein into which a tryptophan residue had been inserted supported computer-based predictions of the position of this residue at the protein's interior and possibly also at the binding site. Of direct relevance to plant defence systems was the observation that Gp-FAR-1 binds two lipids (linolenic and linoleic acids) that are precursors of plant defence compounds and the jasmonic acid signalling pathway. Moreover, Gp-FAR-1 was found to inhibit the lipoxygenase-mediated modification of these substrates in vitro. Thus not only does Gp-FAR-1 function as a broad-spectrum retinol- and fatty-acid-binding protein, the results are consistent with the idea that Gp-FAR-1 is involved in the evasion of primary host plant defence systems. PMID:11368765

  19. Fatty acid binding protein 7 regulates phagocytosis and cytokine production in Kupffer cells during liver injury.

    PubMed

    Miyazaki, Hirofumi; Sawada, Tomoo; Kiyohira, Miwa; Yu, Zhiqian; Nakamura, Keiji; Yasumoto, Yuki; Kagawa, Yoshiteru; Ebrahimi, Majid; Islam, Ariful; Sharifi, Kazem; Kawamura, Saki; Kodama, Takanori; Yamamoto, Yui; Adachi, Yasuhiro; Tokuda, Nobuko; Terai, Shuji; Sakaida, Isao; Ishikawa, Toshizo; Owada, Yuji

    2014-09-01

    Kupffer cells (KCs) are involved in the progression of liver diseases such as hepatitis and liver cancer. Several members of the fatty acid binding proteins (FABPs) are expressed by tissue macrophages, and FABP7 is localized only in KCs. To clarify the role of FABP7 in the regulation of KC function, we evaluated pathological changes of Fabp7 knockout mice during carbon tetrachloride-induced liver injury. During liver injury in Fabp7 knockout mice, serum liver enzymes were increased, cytokine expression (tumor necrosis factor-α, monocyte chemoattractant protein-1, and transforming growth factor-β) was decreased in the liver, and the number of KCs in the liver necrotic area was significantly decreased. Interestingly, in the FABP7-deficient KCs, phagocytosis of apoptotic cells was impaired, and expression of the scavenger receptor CD36 was markedly decreased. In chronic liver injury, Fabp7 knockout mice showed less fibrogenic response to carbon tetrachloride compared with wild-type mice. Taken together, FABP7 is involved in the liver injury process through its regulation of KC phagocytic activity and cytokine production. Such modulation of KC function by FABP7 may provide a novel therapeutic approach to the treatment of liver diseases.

  20. Plasma Fatty Acid Binding Protein 4 and Risk of Sudden Cardiac Death in Older Adults

    PubMed Central

    Djoussé, Luc; Maziarz, Marlena; Biggs, Mary L.; Ix, Joachim H.; Zieman, Susan J.; Kizer, Jorge R.; Lemaitre, Rozenn N.; Mozaffarian, Dariush; Tracy, Russell P.; Mukamal, Kenneth J.; Siscovick, David S.; Sotoodehnia, Nona

    2013-01-01

    Although fatty acid binding protein 4 (FABP4) may increase risk of diabetes and exert negative cardiac inotropy, it is unknown whether plasma concentrations of FABP4 are associated with incidence of sudden cardiac death (SCD). We prospectively analyzed data on 4,560 participants of the Cardiovascular Health Study. FABP4 was measured at baseline using ELISA, and SCD events were adjudicated through review of medical records. We used Cox proportional hazards to estimate effect measures. During a median followup of 11.8 years, 146 SCD cases occurred. In a multivariable model adjusting for demographic, lifestyle, and metabolic factors, relative risk of SCD associated with each higher standard deviation (SD) of plasma FABP4 was 1.15 (95% CI: 0.95–1.38), P = 0.15. In a secondary analysis stratified by prevalent diabetes status, FABP4 was associated with higher risk of SCD in nondiabetic participants, (RR per SD higher FABP4: 1.33 (95% CI: 1.07–1.65), P = 0.009) but not in diabetic participants (RR per SD higher FABP4: 0.88 (95% CI: 0.62–1.27), P = 0.50), P for diabetes-FABP4 interaction 0.049. In summary, a single measure of plasma FABP4 obtained later in life was not associated with the risk of SCD in older adults overall. Confirmation of our post-hoc results in nondiabetic people in other studies is warranted. PMID:24455402

  1. Nonstationary disposition of valproic acid during prolonged intravenous infusion: contributions of unbound clearance and protein binding.

    PubMed

    Arens, T L; Pollack, G M

    2001-09-01

    Circadian variations in disposition have been observed for a variety of agents, including anticonvulsants. Valproic acid (VPA), an anticonvulsant used to control generalized and partial seizures, has exhibited diurnal oscillations in steady-state concentrations during long-term administration to humans and non-human primates. The present study was conducted to assess potential diurnal changes in the disposition of VPA during prolonged i.v. infusion in rats. Animals, maintained on a strict 12-h per day light cycle, were equipped with venous cannulae and an arterial microdialysis probe. VPA was administered as a 50-mg/kg loading dose followed by a 42 mg/kg/h infusion for 70 h. Blood and microdialysate samples were obtained at timed intervals after establishment of steady-state throughout two complete light/dark cycles; and total (serum) and unbound (microdialysate) VPA was determined by gas chromatography. Modest oscillations (6-7 h period) in total and unbound VPA were observed; clearance and binding parameters were not different between light and dark periods. However, unbound clearance increased, and unbound fraction decreased, with time over the course of the infusion. These results suggest that time-dependent changes in VPA disposition occur in rats, although oscillations in steady-state concentrations do not appear to be diurnal in nature. PMID:11754040

  2. Association of androgen with gender difference in serum adipocyte fatty acid binding protein levels

    PubMed Central

    Hu, Xiang; Ma, Xiaojing; Pan, Xiaoping; Luo, Yuqi; Xu, Yiting; Xiong, Qin; Bao, Yuqian; Jia, Weiping

    2016-01-01

    Clinical investigations have indicated women have higher levels of adipocyte fatty acid binding protein (A-FABP) than men. The present study aimed to identify factors related to gender difference in serum A-FABP levels. A total of 507 participants (194 men, 132 premenopausal women, and 181 postmenopausal women) were enrolled in the present study. Serum A-FABP levels increased in the order from men to premenopausal women to postmenopausal women in both body mass index categories (<25.0 and ≥25.0 kg/m2; all P < 0.05). Multiple stepwise regression analyses showed that after adjustment for factors related to serum A-FABP levels, the trunk fat mass was an independent and positive factor of serum A-FABP levels. For men, total testosterone was associated independently and inversely with serum A-FABP levels. For pre- and postmenopausal women, bioavailable testosterone and total testosterone were independent and positive factors associated with serum A-FABP levels, respectively. The present study demonstrated that the androgen was correlated with the serum A-FABP levels negatively in men, but positively in women. With these effects on the fat content, especially trunk fat, androgen might contribute to the gender difference in serum A-FABP levels. PMID:27270834

  3. Exploring and Expanding the Fatty-Acid-Binding Protein Superfamily in Fasciola Species.

    PubMed

    Morphew, Russell M; Wilkinson, Toby J; Mackintosh, Neil; Jahndel, Veronika; Paterson, Steve; McVeigh, Paul; Abbas Abidi, Syed M; Saifullah, Khalid; Raman, Muthusamy; Ravikumar, Gopalakrishnan; LaCourse, James; Maule, Aaron; Brophy, Peter M

    2016-09-01

    The liver flukes Fasciola hepatica and F. gigantica infect livestock worldwide and threaten food security with climate change and problematic control measures spreading disease. Fascioliasis is also a foodborne disease with up to 17 million humans infected. In the absence of vaccines, treatment depends on triclabendazole (TCBZ), and overuse has led to widespread resistance, compromising future TCBZ control. Reductionist biology from many laboratories has predicted new therapeutic targets. To this end, the fatty-acid-binding protein (FABP) superfamily has proposed multifunctional roles, including functions intersecting vaccine and drug therapy, such as immune modulation and anthelmintic sequestration. Research is hindered by a lack of understanding of the full FABP superfamily complement. Although discovery studies predicted FABPs as promising vaccine candidates, it is unclear if uncharacterized FABPs are more relevant for vaccine formulations. We have coupled genome, transcriptome, and EST data mining with proteomics and phylogenetics to reveal a liver fluke FABP superfamily of seven clades: previously identified clades I-III and newly identified clades IV-VII. All new clade FABPs were analyzed using bioinformatics and cloned from both liver flukes. The extended FABP data set will provide new study tools to research the role of FABPs in parasite biology and as therapy targets. PMID:27495901

  4. Astrocyte fatty acid binding protein-7 is a marker for neurogenic niches in the rat hippocampus.

    PubMed

    Young, John K; Heinbockel, Thomas; Gondré-Lewis, Marjorie C

    2013-12-01

    Recent research has determined that newborn neurons in the dentate gyrus of the hippocampus of the macaque are frequently adjacent to astrocytes immunoreactive for fatty acid binding protein-7 (FABP7). To investigate if a similar relationship between FABP7-positive (FABP7+) astrocytes and proliferating cells exists in the rodent brain, sections of brains from juvenile rats were stained by immunohistochemistry to demonstrate newborn cells (antibody to Ki67 protein) and FABP7+ astrocytes. In rat brains, FABP7+ astrocytes were particularly abundant in the dentate gyrus of the hippocampus and were frequently close to dividing cells immunoreactive for Ki67 protein. FABP7+ astrocytes were also present in the olfactory bulbs, arcuate nucleus of the hypothalamus, and in the dorsal medulla subjacent to the area postrema, sites where more modest numbers of newborn neurons can also be found. These data suggest that regional accumulations of FABP7+ astrocytes may represent reservoirs of cells having the potential for neurogenesis. Because FABP7+ astrocytes are particularly abundant in the hippocampus, and since the gene for FABP7 has been linked to Alzheimer's disease, age-related changes in FABP7+ astrocytes (mitochondrial degeneration) may be relevant to age-associated disorders of the hippocampus.

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

    PubMed

    Marcos, Enrique; Crehuet, Ramon; Bahar, Ivet

    2011-09-01

    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.

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

    PubMed

    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.

  7. Serologic Intestinal-Fatty Acid Binding Protein in Necrotizing Enterocolitis Diagnosis: A Meta-Analysis

    PubMed Central

    Cheng, Shupeng; Yu, Jialin; Zhou, Min; Tu, Yan; Lu, Qi

    2015-01-01

    Background. Previous studies showed that intestinal-fatty acid binding protein (I-FABP) may be a valid and promising serologic biomarker for early diagnosis of necrotizing enterocolitis (NEC). Objective. To investigate the early diagnostic value of serologic I-FABP in NEC for the premature neonates. Methods. All major databases were searched from January 1, 1990, to May 1, 2015. We used Meta-Disc 1.4 and Revman5.0 software to calculate the diagnostic accuracy. Results. Seven studies with 444 subjects were identified. The pooled sensitivity of I-FABP was 0.67 for NEC I, 0.74 for NEC II, and 0.83 for NEC III, and the pooled specificity was 0.84, respectively, which showed a moderate diagnostic accuracy. The area under curve (AUC) for each stage was 0.75 (Q⁎ = 0.69), 0.82 (Q⁎ = 0.76), and 0.91 (Q⁎ = 0.84). The diagnostic threshold analysis showed no significant difference in threshold effect. The metaregression showed that the cut-off value has the largest effect on heterogeneity. The funnel plots indicated the existence of publication bias. Conclusion. I-FABP is a valid serologic biomarker for early diagnosis in NEC for the premature neonates with a moderate accuracy. PMID:26798632

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

    PubMed

    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.

  9. Unsaturated fatty acids bind Myc-Max transcription factor and inhibit Myc-Max-DNA complex formation.

    PubMed

    Chung, Sunah; Park, Seyeon; Yang, Chul Hak

    2002-12-15

    Oncoprotein Myc, hetero-dimerized with Max through a b/HLH/Zip region, is a transcription factor that governs important cellular processes such as cell cycle entry, proliferation and differentiation. We found that linoleic acid, isolated from Pollen Typhae, and other unsaturated fatty acids have strong inhibitory effects on the binding of Myc-Max heterodimer to an E-box DNA site (CA(C/T)GTG). The interaction of a fatty acid with a protein dimer, not with DNA, is assumed to block the entire Myc-Max-DNA complex formation. Unsaturated fatty acids also showed cytotoxicity against a SNU16 human stomach cancer cell line and conjugated linoleic acid suppressed mRNA expression of several myc-target genes; ornithine decarboxylase, p53, cdc25a in the SNU16 cells.

  10. Characterization of fatty acid binding and transfer from Δ98Δ, a functional all-β abridged form of IFABP.

    PubMed

    Sawicki, Luciana Rodriguez; Guerbi, María Ximena; Falomir Lockhart, Lisandro Jorge; Curto, Lucrecia María; Delfino, José María; Córsico, Betina; Franchini, Gisela Raquel

    2014-12-01

    Intestinal fatty acid binding protein (IFABP) is an intracellular lipid binding protein whose specific functions within the cell are still uncertain. An abbreviated version of IFABP encompassing residues 29-126, dubbed Δ98Δ is a stable product of limited proteolysis with clostripain of holo-IFABP. Cumulative evidence shows that Δ98Δ adopts a stable, monomeric and functional fold, with compact core and loose periphery. In agreement with previous results, this abridged variant indicates that the helical domain is-not necessary to preserve the general topology of IFABP's β-barrel and that the helix-turn-helix motif is a fundamental element of the portal region involved in ligand binding and protein-membrane interactions. Results presented here suggest that Δ98Δ binds fatty acids with affinities lower than IFABP but higher than those shown by previous helix-less variants, shows a 'diffusional' fatty acid transfer mechanism and it interacts with artificial membranes. This work highlights the importance of the β-barrel of IFABP for its specific functions. PMID:25311169

  11. The human intestinal fatty acid binding protein (hFABP2) gene is regulated by HNF-4{alpha}

    SciTech Connect

    Klapper, Maja . E-mail: klapper@molnut.uni-kiel.de; Boehme, Mike; Nitz, Inke; Doering, Frank

    2007-04-27

    The cytosolic human intestinal fatty acid binding protein (hFABP2) is proposed to be involved in intestinal absorption of long-chain fatty acids. The aim of this study was to investigate the regulation of hFABP2 by the endodermal hepatocyte nuclear factor 4{alpha} (HNF-4{alpha}), involved in regulation of genes of fatty acid metabolism and differentiation. Electromobility shift assays demonstrated that HNF-4{alpha} binds at position -324 to -336 within the hFABP2 promoter. Mutation of this HNF-4 binding site abolished the luciferase reporter activity of hFABP2 in postconfluent Caco-2 cells. In HeLa cells, this mutation reduced the activation of the hFABP2 promoter by HNF-4{alpha} by about 50%. Thus, binding element at position -336/-324 essentially determines the transcriptional activity of promoter and may be important in control of hFABP2 expression by dietary lipids and differentiation. Studying genotype interactions of hFABP2 and HNF-4{alpha}, that are both candidate genes for diabetes type 2, may be a powerful approach.

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

    SciTech Connect

    Bielmann, Regula; Habann, Matthias; Eugster, Marcel R.; Lurz, Rudi; Calendar, Richard; Klumpp, Jochen; Loessner, Martin J.

    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.

  13. A receptor-binding protein of Campylobacter jejuni bacteriophage NCTC 12673 recognizes flagellin glycosylated with acetamidino-modified pseudaminic acid.

    PubMed

    Javed, Muhammad Afzal; van Alphen, Lieke B; Sacher, Jessica; Ding, Wen; Kelly, John; Nargang, Cheryl; Smith, David F; Cummings, Richard D; Szymanski, Christine M

    2015-01-01

    Bacteriophage receptor-binding proteins (RBPs) confer host specificity. We previously identified a putative RBP (Gp047) from the campylobacter lytic phage NCTC 12673 and demonstrated that Gp047 has a broader host range than its parent phage. While NCTC 12673 recognizes the capsular polysaccharide (CPS) of a limited number of Campylobacter jejuni isolates, Gp047 binds to a majority of C. jejuni and related Campylobacter coli strains. In this study, we demonstrate that Gp047 also binds to acapsular mutants, suggesting that unlike the parent phage, CPS is not the receptor for Gp047. Affinity chromatography and far-western analyses of C. jejuni lysates using Gp047 followed by mass spectrometry indicated that Gp047 binds to the major flagellin protein, FlaA. Because C. jejuni flagellin is extensively glycosylated, we investigated this binding specificity further and demonstrate that Gp047 only recognizes flagellin decorated with acetamidino-modified pseudaminic acid. This binding activity is localized to the C-terminal quarter of the protein and both wild-type and coccoid forms of C. jejuni are recognized. In addition, Gp047 treatment agglutinates vegetative cells and reduces their motility. Because Gp047 is highly conserved among all campylobacter phages sequenced to date, it is likely that this protein plays an important role in the phage life cycle.

  14. Protein binding of fentanyl and its metabolite nor-fentanyl in human plasma, albumin and α-1 acid glycoprotein.

    PubMed

    Bista, Sudeep Raj; Haywood, Alison; Hardy, Janet; Lobb, Michael; Tapuni, Angela; Norris, Ross

    2015-03-01

    1.Fentanyl is a highly lipophilic opioid commonly used to treat cancer pain. Plasma protein binding (PPB) of fentanyl in human plasma is reported as 80-85%, however it is unclear whether fentanyl binds primarily to albumin (ALB) or α-1 acid glycoprotein (AAG) and no studies have been conducted on the metabolite, nor-fentanyl. Fentanyl is also known to bind to plasticware and ultrafiltration (UF) devices which impacts adversely on binding experiments. 2.PPB of fentanyl and nor-fentanyl to ALB and AAG in isotonic phosphate buffer solution and seeded human plasma was quantified. PPB was also performed in plasma samples obtained from cancer patients receiving transdermal fentanyl. The adsorption of fentanyl and nor-fentanyl to UF devices and plasticware commonly used in PPB studies was also assessed. 3.Fentanyl was shown to bind primarily to ALB as opposed to AAG, with nor-fentanyl exhibiting negligible binding to plasma proteins. Total PPB of fentanyl was 86-89% in seeded human plasma. PPB in 56 cancer patient samples was 95.1 ± 3.52% for fentanyl and 32.4 ± 21.9% for nor-fentanyl. 4.UF was shown to be a reliable and convenient method for PPB studies, thereby removing the need for complex testing for adsorption of the drug to plasticware during UF.

  15. Key Amino Acid Residues of Ankyrin-Sensitive Phosphatidylethanolamine/Phosphatidylcholine-Lipid Binding Site of βI-Spectrin

    PubMed Central

    Wolny, Marcin; Grzybek, Michał; Bok, Ewa; Chorzalska, Anna; Lenoir, Marc; Czogalla, Aleksander; Adamczyk, Klaudia; Kolondra, Adam; Diakowski, Witold; Overduin, Michael; Sikorski, Aleksander F.

    2011-01-01

    It was shown previously that an ankyrin-sensitive, phosphatidylethanolamine/phosphatidylcholine (PE/PC) binding site maps to the N-terminal part of the ankyrin-binding domain of β-spectrin (ankBDn). Here we have identified the amino acid residues within this domain which are responsible for recognizing monolayers and bilayers composed of PE/PC mixtures. In vitro binding studies revealed that a quadruple mutant with substituted hydrophobic residues W1771, L1775, M1778 and W1779 not only failed to effectively bind PE/PC, but its residual PE/PC-binding activity was insensitive to inhibition with ankyrin. Structure prediction and analysis, supported by in vitro experiments, suggests that “opening” of the coiled-coil structure underlies the mechanism of this interaction. Experiments on red blood cells and HeLa cells supported the conclusions derived from the model and in vitro lipid-protein interaction results, and showed the potential physiological role of this binding. We postulate that direct interactions between spectrin ankBDn and PE-rich domains play an important role in stabilizing the structure of the spectrin-based membrane skeleton. PMID:21738695

  16. A receptor-binding protein of Campylobacter jejuni bacteriophage NCTC 12673 recognizes flagellin glycosylated with acetamidino-modified pseudaminic acid.

    PubMed

    Javed, Muhammad Afzal; van Alphen, Lieke B; Sacher, Jessica; Ding, Wen; Kelly, John; Nargang, Cheryl; Smith, David F; Cummings, Richard D; Szymanski, Christine M

    2015-01-01

    Bacteriophage receptor-binding proteins (RBPs) confer host specificity. We previously identified a putative RBP (Gp047) from the campylobacter lytic phage NCTC 12673 and demonstrated that Gp047 has a broader host range than its parent phage. While NCTC 12673 recognizes the capsular polysaccharide (CPS) of a limited number of Campylobacter jejuni isolates, Gp047 binds to a majority of C. jejuni and related Campylobacter coli strains. In this study, we demonstrate that Gp047 also binds to acapsular mutants, suggesting that unlike the parent phage, CPS is not the receptor for Gp047. Affinity chromatography and far-western analyses of C. jejuni lysates using Gp047 followed by mass spectrometry indicated that Gp047 binds to the major flagellin protein, FlaA. Because C. jejuni flagellin is extensively glycosylated, we investigated this binding specificity further and demonstrate that Gp047 only recognizes flagellin decorated with acetamidino-modified pseudaminic acid. This binding activity is localized to the C-terminal quarter of the protein and both wild-type and coccoid forms of C. jejuni are recognized. In addition, Gp047 treatment agglutinates vegetative cells and reduces their motility. Because Gp047 is highly conserved among all campylobacter phages sequenced to date, it is likely that this protein plays an important role in the phage life cycle. PMID:25354466

  17. Binding site multiplicity with fatty acid ligands: implications for the regulation of PKR kinase autophosphorylation with palmitate.

    PubMed

    Fang, Liang; Cho, Hyun Ju; Chan, Christina; Feig, Michael

    2014-10-01

    Saturated long chain-free fatty acids (FFAs), especially palmitate, have been implicated in apoptosis by inhibiting the activity of PKR (double-stranded RNA-dependent protein kinase). We recently found evidence that palmitate interacts directly with the kinase domain of PKR, subsequently inhibiting the autophosphorylation of PKR. To investigate the interactions of palmitate with PKR and its effects on PKR autophosphorylation, we performed extensive unbiased MD simulations combined with biochemical and biophysical experiments. The simulations predict multiple putative binding sites of palmitate on both the phosphorylated and unphosphorylated PKR with similar binding affinities. Ligand-protein interactions involving a large variety of different binding modes challenge the conventional view of highly specific, single binding sites. Key interactions of palmitate involve the αC-helix of PKR, especially near residue R307. Experimental mutation of R307 was found to affect palmitate binding and reduce its inhibitory effect. Based on this study a new allosteric mechanism is proposed where palmitate binding to the αC-helix prevents the inactive-to-active transition of PKR and subsequently reduces its ability to autophosphorylate.

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

    NASA Technical Reports Server (NTRS)

    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.

  19. Erythrocyte-binding antigen 175 mediates invasion in Plasmodium falciparum utilizing sialic acid-dependent and -independent pathways

    PubMed Central

    Duraisingh, Manoj T.; Maier, Alexander G.; Triglia, Tony; Cowman, Alan F.

    2003-01-01

    The Plasmodium falciparum erythrocyte-binding antigen 175 (EBA-175) is a ligand for merozoite invasion into human erythrocytes that binds to glycophorin A in a sialic acid-dependent manner. P. falciparum strain W2mef depends on sialic acid for invasion of erythrocytes, whereas 3D7 is sialic acid-independent. We generated parasites that lack expression or express truncated forms of EBA-175 in W2mef and 3D7. Lack of EBA-175 expression in W2mef parasites was associated with a switch to sialic acid-independent invasion. 3D7 parasites lacking expression of EBA-175 showed no alteration in their ability to utilize sialic acid-independent pathways. Strikingly, both W2mef and 3D7 parasites lacking EBA-175 expression invaded chymotrypsin-treated erythrocytes inefficiently compared with the parental lines. This loss of function suggests that the EBA-175/glycophorin A ligand–receptor interaction is the major chymotrypsin-resistant invasion pathway. Parasite lines with truncated EBA-175 had invasion phenotypes equivalent to parasites lacking expression of EBA-175. The EBA-175 ligand is functional in erythrocyte invasion by merozoites that utilize either sialic acid-dependent or -independent invasion pathways. This finding suggests a model where a minimal affinity supplied by multiple ligand–receptor interactions is required for successful invasion and has implications for EBA-175 as a malaria vaccine candidate. PMID:12672957

  20. A Photocytes-Associated Fatty Acid-Binding Protein from the Light Organ of Adult Taiwanese Firefly, Luciola cerata

    PubMed Central

    Goh, King-Siang; Li, Chia-Wei

    2011-01-01

    Background Intracellular fatty acid-binding proteins (FABPs) are considered to be an important energy source supplier in lipid metabolism; however, they have never been reported in any bioluminescent tissue before. In this study, we determined the structural and functional characteristics of a novel FABP (lcFABP) from the light organ of adult Taiwanese firefly, Luciola cerata, and showed anatomical association of lcFABP with photocytes. Principal Findings Our results demonstrated the primary structure of lcFABP deduced from the cDNA clone of light organ shares structural homologies with other insect and human FABPs. In vitro binding assay indicated the recombinant lcFABP binds saturated long chain fatty acids (C14-C18) more strongly than other fatty acids and firefly luciferin. In addition, tissue distribution screening assay using a rabbit antiserum specifically against the N-terminal sequence of lcFABP confirmed the light organ-specific expression of lcFABP. In the light organ, the lcFABP constituted about 15% of total soluble proteins, and was detected in both cytosol and nucleus of photocytes. Conclusions The specific localization of abundant lcFABP in the light organ suggests that sustained bioluminescent flashes in the light organ might be a high energy demanding process. In photocytes, lcFABP might play a key role in providing long chain fatty acids to peroxisomes for the luciferase-catalyzed long chain acyl-CoA synthetic reaction. PMID:22242133

  1. Binding interaction of a gamma-aminobutyric acid derivative with serum albumin: an insight by fluorescence and molecular modeling analysis.

    PubMed

    Pal, Uttam; Pramanik, Sumit Kumar; Bhattacharya, Baisali; Banerji, Biswadip; C Maiti, Nakul

    2016-01-01

    gamma-Aminobutyric acid (GABA) is a naturally occurring inhibitory neurotransmitter and some of its derivatives showed potential to act as neuroprotective agents. With the aim of developing potential leads for anti-Alzheimer's drugs, in this study we synthesized a novel GABA derivative, methyl 4-(4-((2-(tert-butoxy)-2-oxoethyl)(4-methoxyphenyl)amino)benzamido)butanoate by a unique method of Buchwald-Hartwig cross coupling synthesis; with some modification the yield was significant (97 %) and spectroscopic analysis confirmed that the compound was highly pure (98.8 % by HPLC). The druglikeness properties such as logP, logS, and polar surface area were 3.87, -4.86 and 94.17 Å(2) respectively and it satisfied the Lipinski's rule of five. We examined the binding behavior of the molecule to human serum albumin (HSA) and bovine serum albumin (BSA) which are known as universal drug carrier proteins. The molecule binds to the proteins with low micromolar efficiency and the calculated binding constants were 3.85 and 2.75 micromolar for BSA and HSA, respectively. Temperature dependent study using van't Hoff equation established that the binding was thermodynamically favorable and the changes in the Gibb's free energy, ΔG for the binding process was negative. However, the binding of the molecule to HSA was enthalpy driven and the change of enthalpy (ΔH) was -10.63 kJ/mol, whereas, the binding to BSA was entropy driven and the change in entropy ΔS was 222 J/mol. The molecular docking analysis showed that the binding sites of the molecule lie in the groove between domain I and domain III of BSA, whereas it is within the domain I in case of HSA, which also supported the different thermodynamic nature of binding with HSA and BSA. Molecular dynamics analysis suggested that the binding was stable with time and provided further details of the binding interaction. Molecular dynamics study also highlighted the effect of this ligand binding on the serum albumin structure. PMID

  2. A single amino acid substitution (R441A) in the receptor-binding domain of SARS coronavirus spike protein disrupts the antigenic structure and binding activity

    SciTech Connect

    He Yuxian . E-mail: yhe@nybloodcenter.org; Li Jingjing; Jiang Shibo

    2006-05-26

    The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) has two major functions: interacting with the receptor to mediate virus entry and inducing protective immunity. Coincidently, the receptor-binding domain (RBD, residues 318-510) of SAR-CoV S protein is a major antigenic site to induce neutralizing antibodies. Here, we used RBD-Fc, a fusion protein containing the RBD and human IgG1 Fc, as a model in the studies and found that a single amino acid substitution in the RBD (R441A) could abolish the immunogenicity of RBD to induce neutralizing antibodies in immunized mice and rabbits. With a panel of anti-RBD mAbs as probes, we observed that R441A substitution was able to disrupt the majority of neutralizing epitopes in the RBD, suggesting that this residue is critical for the antigenic structure responsible for inducing protective immune responses. We also demonstrated that the RBD-Fc bearing R441A mutation could not bind to soluble and cell-associated angiotensin-converting enzyme 2 (ACE2), the functional receptor for SARS-CoV and failed to block S protein-mediated pseudovirus entry, indicating that this point mutation also disrupted the receptor-binding motif (RBM) in the RBD. Taken together, these data provide direct evidence to show that a single amino acid residue at key position in the RBD can determine the major function of SARS-CoV S protein and imply for designing SARS vaccines and therapeutics.

  3. Thermodynamics of metal cation binding by a solid soil derived humic acid. 2. Binding of Mn(II), Co(NH3)6aq3+ and Hg(II).

    PubMed

    Ghabbour, Elham A; Shaker, Medhat; El-Toukhy, Ahmed; Abid, Ismail M; Davies, Geoffrey

    2006-07-01

    Metal binding is an important function of humic acids (HAs) in soils, sediments and waters. At pH 2.0, Mn(II) and Co(NH3)6aq3+ bind tightly in one step labeled A to a solid humic acid NHA isolated from a New Hampshire soil. Two consecutive steps are observed for Hg(II) binding. All the binding isotherms fit the Langmuir model in the temperature range 10.0-50.0 degrees C. Stoichiometric site capacities indicate predominant binding by charge-neutralizing HA carboxylate groups for Mn(II) and the second step A of Hg(II) binding. The binding affinity order in step A is Co(NH3)(6)3+>Hg(II)>Mn(II). Metal binding enthalpy and entropy changes fit the linear correlation found previously for binding of other metal cations by solid HAs. Free energy buffering from cooperative enthalpy and entropy changes and lower enthalpies for metal-HA interactions in solution suggest that desolvation of the cations and HA binding sites as well as HA conformational changes to allow for inner-sphere complexation predominate metal binding by hydrated solid HAs.

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

    PubMed

    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.

  5. SEROTONIN BINDING TO NERVE-ENDING PARTICLES OF THE RAT BRAIN AND ITS INHIBITION BY LYSERGIC ACID DIETHYLAMIDE.

    PubMed

    MARCHBANKS, R M; ROSENBLATT, F; O'BRIEN, R D

    1964-05-29

    The binding of serotonin to nerve-ending particles and other preparations from rat brain has been examined. By investigating the amount bound as a function of serotonin concentration from 10(-7)M to 1O(-2)M, it was possible to identify three major components having K(assoc) (association constant) values of 2 x 10(6), 5 x 10(4), and 5 x 10(2). The component having the highest binding constant was not present in liver and appeared to be confined to the cortex and midbrain regions. This component is inhibited by d-lysergic acid diethylamide at low concentrations. Solubilization of this binding component has been achieved.

  6. Rapid, Structure-Based Exploration of Pipecolic Acid Amides as Novel Selective Antagonists of the FK506-Binding Protein 51.

    PubMed

    Gaali, Steffen; Feng, Xixi; Hähle, Andreas; Sippel, Claudia; Bracher, Andreas; Hausch, Felix

    2016-03-24

    The FK506-binding protein 51 (FKBP51) is a key regulator of stress hormone receptors and an established risk factor for stress-related disorders. Drug development for FKBP51 has been impaired by the structurally similar but functionally opposing homologue FKBP52. High selectivity between FKBP51 and FKBP52 can be achieved by ligands that stabilize a recently discovered FKBP51-favoring conformation. However, drug-like parameters for these ligands remained unfavorable. In the present study, we replaced the potentially labile pipecolic ester group of previous FKBP51 ligands by various low molecular weight amides. This resulted in the first series of pipecolic acid amides, which had much lower molecular weights without affecting FKBP51 selectivity. We discovered a geminally substituted cyclopentyl amide as a preferred FKBP51-binding motif and elucidated its binding mode to provide a new lead structure for future drug optimization. PMID:26954324

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

    USGS Publications Warehouse

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

    1999-01-01

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

  8. Divalent cations enhance fluoride binding to Streptococcus mutans and Streptococcus sanguinis cells and subsequently inhibit bacterial acid production.

    PubMed

    Domon-Tawaraya, H; Nakajo, K; Washio, J; Ashizawa, T; Ichino, T; Sugawara, H; Fukumoto, S; Takahashi, N

    2013-01-01

    One preventive effect of topical fluoride application is derived from the fact that fluoride can inhibit bacterial acid production. Furthermore, divalent cations such as Ca(2+) and Mg(2+) increase the binding of fluoride to bacterial cells. These findings suggest that exposure of oral bacteria to fluoride in the presence of divalent cations increases fluoride binding to bacterial cells and subsequently enhances fluoride-induced inhibition of bacterial acid production. This study investigated the effects of fluoride exposure (0-20,000 ppm F) in the presence of Ca(2+) or Mg(2+) prior to glucose challenge on pH fall ability by bacterial sugar fermentation, as well as fluoride binding to bacterial cells by exposure to fluoride, and fluoride release from bacterial cells during bacterial sugar fermentation, using caries-related bacteria, Streptococcus mutans and Streptococcus sanguinis. The pH fall by both streptococci was inhibited by exposure to over 250 ppm F in the presence of Ca(2+) (p < 0.01), whereas in the presence of Mg(2+), the pH fall by S. mutans and S. sanguinis was inhibited after exposure to over 250 and 950 ppm F, respectively (p < 0.05). The amounts of fluoride binding to and released from streptococcal cells increased with the concentration of fluoride the cells were exposed to in the presence of Mg(2+), but were high enough even after 250 ppm F exposure in the presence of Ca(2+). The enhanced inhibition of acid production in the presence of divalent cations is probably due to the improved efficiency of fluoride binding to bacterial cells being improved via these divalent cations.

  9. Pressurized water extraction of β-glucan enriched fractions with bile acids-binding capacities obtained from edible mushrooms.

    PubMed

    Palanisamy, Marimuthu; Aldars-García, Laila; Gil-Ramírez, Alicia; Ruiz-Rodríguez, Alejandro; Marín, Francisco R; Reglero, Guillermo; Soler-Rivas, Cristina

    2014-01-01

    A pressurized water extraction (PWE) method was developed in order to extract β-glucans with bile acids-binding capacities from cultivated mushrooms (Agaricus bisporus, Lentinula edodes, and Pleurotus ostreatus) to be used as supplements to design novel foods with hypocholesterolemic properties. Extraction yields were higher in individual than sequential extractions being the optimal extraction parameters: 200°C, 5 cycles of 5 min each at 10.3 MPa. The crude polysaccharide (PSC) fractions, isolated from the PWE extracts contained mainly β-glucans (including chitooligosaccharides deriving from chitin hydrolysis), α-glucans, and other PSCs (hetero-/proteo-glucans) depending on the extraction temperature and mushroom strain considered. The observed bile acids-binding capacities of some extracts were similar to a β-glucan enriched fraction obtained from cereals. PMID:24399760

  10. Nuclear magnetic resonance and molecular modeling study on mycophenolic acid: implications for binding to inosine monophosphate dehydrogenase.

    PubMed

    Makara, G M; Keserû, G M; Kajtár-Peredy, M; Anderson, W K

    1996-03-15

    The conformation of the sodium salt of mycophenolic acid (MPA), a potent inhibitor of inosine monophosphate dehydrogenase (IMPD), derived from 1D DIFNOE and 2D ROESY experiments in water and molecular dynamics (MD) is described. The hexenoic acid side chain conformation consistent with the NMR data was similar to that seen in the X-ray structure of MPA. The solution conformation was applied in a molecular modeling study in order to explore the potential features of enzyme binding. Our results, based on striking similarities in molecular volume and electrostatic isopotential between MPA and cofactor NAD+, lead to the suggestion that MPA is capable of binding to the nicotinamide site of IMPD and mimicking the NAD+ inverse regulation of the enzyme. In addition, our proposed model is in good agreement with the observed high affinity of the dinucleotide analogues thiazole- and selenazole-4-carboxamide adenine dinucleotide to IMPD.

  11. Cell cytotoxicity and serum albumin binding capacity of the morin-Cu(ii) complex and its effect on deoxyribonucleic acid.

    PubMed

    Roy, Atanu Singha; Samanta, Sintu Kumar; Ghosh, Pooja; Tripathy, Debi Ranjan; Ghosh, Sudip Kumar; Dasgupta, Swagata

    2016-08-16

    The dietary components, flavonoids, are important for their anti-oxidant properties and the ability to act as metal ion chelators. The characterization of the morin-Cu(ii) complex is executed using elemental analysis, FTIR and mass spectroscopy. DNA cleaving and cell cytotoxicity properties followed by serum albumin binding have been investigated in this report. The morin-Cu(ii) complex was found to cleave plasmid pBR322 DNA via an oxidative pathway as revealed by agarose gel based assay performed in the presence of some scavengers and reactive oxygen species. The breaking of the deoxyribose ring of calf thymus DNA (ct-DNA) was also confirmed by the formation of thiobarbituric acid reacting species (TBARS) between thiobarbituric acid and malonaldehyde. The morin-Cu(ii) complex is able to inhibit the growth of human HeLa cells. Fluorescence studies revealed that the morin-Cu(ii) complex can quench the intrinsic fluorescence of serum albumins (SAs) via a static quenching method. The binding constants were found to be in the order of 10(5) M(-1) and observed to increase with temperature. Both ΔH° and ΔS° are positive for the binding of the morin-Cu(ii) complex with serum albumins which indicated the presence of hydrophobic forces. Site-selectivity studies reveal that the morin-Cu(ii) complex binds to both site 1 (subdomain IIA) and site 2 (subdomain IIIA) of human serum albumin (HSA) and bovine serum albumin (BSA). Circular dichroism (CD) studies showed the structural perturbation of SAs during binding with the morin-Cu(ii) complex. The results from binding studies confirmed that after complexation with the Cu(ii) ion, morin alters its mode of interaction with SAs which could have differential implications on its other biological and pharmaceutical properties.

  12. Cell cytotoxicity and serum albumin binding capacity of the morin-Cu(ii) complex and its effect on deoxyribonucleic acid.

    PubMed

    Roy, Atanu Singha; Samanta, Sintu Kumar; Ghosh, Pooja; Tripathy, Debi Ranjan; Ghosh, Sudip Kumar; Dasgupta, Swagata

    2016-08-16

    The dietary components, flavonoids, are important for their anti-oxidant properties and the ability to act as metal ion chelators. The characterization of the morin-Cu(ii) complex is executed using elemental analysis, FTIR and mass spectroscopy. DNA cleaving and cell cytotoxicity properties followed by serum albumin binding have been investigated in this report. The morin-Cu(ii) complex was found to cleave plasmid pBR322 DNA via an oxidative pathway as revealed by agarose gel based assay performed in the presence of some scavengers and reactive oxygen species. The breaking of the deoxyribose ring of calf thymus DNA (ct-DNA) was also confirmed by the formation of thiobarbituric acid reacting species (TBARS) between thiobarbituric acid and malonaldehyde. The morin-Cu(ii) complex is able to inhibit the growth of human HeLa cells. Fluorescence studies revealed that the morin-Cu(ii) complex can quench the intrinsic fluorescence of serum albumins (SAs) via a static quenching method. The binding constants were found to be in the order of 10(5) M(-1) and observed to increase with temperature. Both ΔH° and ΔS° are positive for the binding of the morin-Cu(ii) complex with serum albumins which indicated the presence of hydrophobic forces. Site-selectivity studies reveal that the morin-Cu(ii) complex binds to both site 1 (subdomain IIA) and site 2 (subdomain IIIA) of human serum albumin (HSA) and bovine serum albumin (BSA). Circular dichroism (CD) studies showed the structural perturbation of SAs during binding with the morin-Cu(ii) complex. The results from binding studies confirmed that after complexation with the Cu(ii) ion, morin alters its mode of interaction with SAs which could have differential implications on its other biological and pharmaceutical properties. PMID:27345944

  13. A possible role for acid phosphatase with thiamin-binding activity encoded by PHO3 in yeast.

    PubMed

    Nosaka, K; Kaneko, Y; Nishimura, H; Iwashima, A

    1989-07-01

    Periplasmic soluble thiamin-binding protein in Saccharomyces cerevisiae (Iwashima, A. et al. (1979) Biochim. Biophys. Acta 577, 217-220) was demonstrated to be encoded by PHO3 gene that codes for thiamin repressible acid phosphatase (Schweingruber, M.E. et al. (1986) J. Biol. Chem. 261, 15877-15882) by genetic analysis. The pho3 mutant cells of S. cerevisiae in contrast to the parent cells have markedly reduced activity of the uptake of [14C]thiamin phosphates, suggesting that thiamin repressible acid phosphatase plays a role in the hydrolysis of thiamin phosphates in the periplasmic space prior to the uptake of their thiamin moieties by S. cerevisiae.

  14. Identification of IgE-binding peptide and critical amino acids of Jatropha curcas allergen involved in allergenic response.

    PubMed

    Crespo, Livia Maia; de Oliveira, Natalia Deus; Damatta, Renato Augusto; do Nascimento, Viviane Veiga; Soares, Thais Pacheco; Machado, Olga Lima Tavares

    2016-01-01

    Increasing energy demand has spurred interest in the use of biofuels. Jatropha curcas (physic nut), an inedible oilseed, is a potential source of bioenergy. The seeds, however, contain allergens such as Jat c 1, a 2S albumin that can induce hypersensitivity reactions in humans and result in allergic diseases. Recent advances in identifying and characterizing plant allergens and, in particular, their immunoglobulin E (IgE)-binding epitopes have produced a wealth of information. We identified IgE-binding regions and the critical amino acids involved in the degranulation of mast cells and the release of histamine, preliminary steps for the prevention and treatment of this allergy. Four IgE-binding regions were identified in the sequence of Jat c 1. We identified and demonstrated the fundamental role of two glutamic acid residues in IgE binding. The sequence LEKQLEEGEVGS produces a random loop on the most exposed part of Jat c 1. This region is important to the stimulation of the allergic response. The possibility of using this information to produce vaccines and other pharmacological agents for allergy treatment is discussed. PMID:27119058

  15. Structure of ginseng major latex-like protein 151 and its proposed lysophosphatidic acid-binding mechanism.

    PubMed

    Choi, Sun Hye; Hong, Myoung Ki; Kim, Hyeon Joong; Ryoo, Nayeon; Rhim, Hyewhon; Nah, Seung Yeol; Kang, Lin Woo

    2015-05-01

    Lysophosphatidic acid (LPA) is a phospholipid growth factor with myriad effects on biological systems. LPA is usually present bound to animal plasma proteins such as albumin or gelsolin. When LPA complexes with plasma proteins, it binds to its cognate receptors with higher affinity than when it is free. Recently, gintonin from ginseng was found to bind to LPA and to activate mammalian LPA receptors. Gintonin contains two components: ginseng major latex-like protein 151 (GLP) and ginseng ribonuclease-like storage protein. Here, the crystal structure of GLP is reported, which belongs to the plant Bet v 1 superfamily, and a model is proposed for how GLP binds LPA. Amino-acid residues of GLP recognizing LPA were identified using site-directed mutagenesis and isothermal titration calorimetry. The resulting GLP mutants were used to study the activation of LPA receptor-dependent signalling pathways. In contrast to wild-type GLP, the H147A mutant did not bind LPA, elicit intracellular Ca(2+) transients in neuronal cells or activate Ca(2+)-dependent Cl(-) channels in Xenopus oocytes. Based on these results, a mechanism by which GLP recognizes LPA and its requirement to activate G protein-coupled LPA receptors to elicit diverse biological responses were proposed. PMID:25945569

  16. Adaptive Evolution of Eel Fluorescent Proteins from Fatty Acid Binding Proteins Produces Bright Fluorescence in the Marine Environment

    PubMed Central

    Gruber, David F.; Gaffney, Jean P.; Mehr, Shaadi; DeSalle, Rob; Sparks, John S.; Platisa, Jelena; Pieribone, Vincent A.

    2015-01-01

    We report the identification and characterization of two new members of a family of bilirubin-inducible fluorescent proteins (FPs) from marine chlopsid eels and demonstrate a key region of the sequence that serves as an evolutionary switch from non-fluorescent to fluorescent fatty acid-binding proteins (FABPs). Using transcriptomic analysis of two species of brightly fluorescent Kaupichthys eels (Kaupichthys hyoproroides and Kaupichthys n. sp.), two new FPs were identified, cloned and characterized (Chlopsid FP I and Chlopsid FP II). We then performed phylogenetic analysis on 210 FABPs, spanning 16 vertebrate orders, and including 163 vertebrate taxa. We show that the fluorescent FPs diverged as a protein family and are the sister group to brain FABPs. Our results indicate that the evolution of this family involved at least three gene duplication events. We show that fluorescent FABPs possess a unique, conserved tripeptide Gly-Pro-Pro sequence motif, which is not found in non-fluorescent fatty acid binding proteins. This motif arose from a duplication event of the FABP brain isoforms and was under strong purifying selection, leading to the classification of this new FP family. Residues adjacent to the motif are under strong positive selection, suggesting a further refinement of the eel protein’s fluorescent properties. We present a phylogenetic reconstruction of this emerging FP family and describe additional fluorescent FABP members from groups of distantly related eels. The elucidation of this class of fish FPs with diverse properties provides new templates for the development of protein-based fluorescent tools. The evolutionary adaptation from fatty acid-binding proteins to fluorescent fatty acid-binding proteins raises intrigue as to the functional role of bright green fluorescence in this cryptic genus of reclusive eels that inhabit a blue, nearly monochromatic, marine environment. PMID:26561348

  17. Discovery of FDA-approved drugs as inhibitors of fatty acid binding protein 4 using molecular docking screening.

    PubMed

    Wang, Yan; Law, Wai-Kit; Hu, Jian-Shu; Lin, Huang-Quan; Ip, Tsz-Ming; Wan, David Chi-Cheong

    2014-11-24

    We first identified fluorescein, ketazolam, antrafenine, darifenacin, fosaprepitant, paliperidone, risperidone, pimozide, trovafloxacin, and levofloxacin as inhibitors of fatty acid binding protein 4 using molecular docking s