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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. 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.

  3. Blood compatibility of a ferulic acid (FA)-eluting PHBHHx system for biodegradable magnesium stent application.

    PubMed

    Zhang, Erlin; Shen, Feng

    2015-01-01

    Magnesium stent has shown potential application as a new biodegradable stent. However, the fast degradation of magnesium stent limited its clinic application. Recently, a biodegradable and drug-eluting coating system was designed to prevent magnesium from fast degradation by adding ferulic acid (FA) in poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) by a physical method. In vitro study has demonstrated that the FA-eluting system exhibited strong promotion to the endothelialization, which might be a choice for the stent application. In this paper, the hemolysis rate, the plasma recalcification time (PRT), the plasma prothrombin time (PT) and the kinetic clotting time of the FA-eluting films were investigated and the platelet adhesion was observed in order to assess the blood compatibility of the FA-eluting PHBHHx films in comparison with PHBHHx film. The results have shown that the addition of FA had no influence on the hemolysis, but prolonged PRT, PT and the clotting time and reduced the platelet adhesion and activation, displaying that the FA-eluting PHBHHx exhibited better blood compatibility than PHBHHx. In addition, the effect of alkali treatment on the blood compatibility of FA-eluting PHBHHx was also studied. It was indicated that alkali treatment had no effect on the hemolysis and the coagulation time, but enhanced slightly the platelet adhesion. All these demonstrated that FA-eluting PHBHHx film had good blood compatibility and might be a candidate surface coating for the biodegradable magnesium stent.

  4. Composition and copper binding properties of aquatic fulvic acids in eutrophic Taihu Lake, China.

    PubMed

    Li, Weiwei; Zhang, Fenfen; Ye, Qi; Wu, Dan; Wang, Liying; Yu, Yihua; Deng, Bing; Du, Jinzhou

    2017-04-01

    Fulvic acid (FA) plays a significant role in biogenic-elemental cycling in aquatic ecosystems which is highly dependent on their organic composition. In this study, the aquatic FA contents and binding properties during bloom and non-bloom periods in Taihu Lake were investigated by two-dimensional correlation spectroscopy Fourier transform infrared spectroscopy (2D-COS-FTIR), nuclear magnetic resonance (NMR) and elemental analysis. Compared with non-bloom FA, bloom FA was of lower nitrogen content and higher C/N ratio. It contained more carboxylic and aliphatic groups while less amide groups. 2D-COS-FTIR spectra evidenced the carboxyl groups in bloom FA had the fastest response to Cu(II) binding. Also, polysaccharide in bloom FA was more susceptive to Cu(II) concentrations than that in non-bloom FA. While comparing with bloom FA, the N-rich organic compounds in non-bloom FA exhibited faster binding sequence with Cu(II). A comprehensive scheme about the interaction process of FA-Cu(II) showed that both nitrogenous and oxygenic groups in FAs were active in binding to Cu(II). The alteration in binding behaviors of organic groups in FAs to Cu(II) may have been driven by algal products and microbial community variety in Taihu Lake. Our results here have the potential to contribute significantly to future studies of dissolved organic matter dynamic biogeochemistry processes and trace metal cycling processes in eutrophic lakes.

  5. 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

  6. Glucomannan- and glucomannan plus spirulina-enriched pork affect liver fatty acid profile, LDL receptor expression and antioxidant status in Zucker fa/fa rats fed atherogenic diets

    PubMed Central

    González-Torres, Laura; Matos, Cátia; Vázquez-Velasco, Miguel; Santos-López, Jorge A.; Sánchez-Martínez, Iria; García–Fernández, Camino; Bastida, Sara; Benedí, Juana; Sánchez-Muniz, Francisco J.

    2017-01-01

    ABSTRACT We evaluated the effects of glucomannan or glucomannan plus spirulina-restructured pork (RP) on liver fatty acid profile, desaturase/elongase enzyme activities and oxidative status of Zucker fa/fa rats for seven weeks. Control (C), glucomannan (G) and glucomannan/spirulina (GS)-RP; HC (cholesterol-enriched control), HG and HGS (cholesterol-enriched glucomannan and glucomannan/spirulina-RP) experimental diets were tested. Increased metabolic syndrome markers were found in C, G and GS rats. Cholesterol feeding increased liver size, fat, and cholesterol and reduced antioxidant enzyme levels and expressions. Cholesterolemia was lower in HG and HGS than in HC. GS vs. G showed higher stearic but lower oleic levels. SFA and PUFA decreased while MUFA increased by cholesterol feeding. The arachidonic/linoleic and docosahexaenoic/alpha-linolenic ratios were lower in HC, HG, and HGS vs. C, G, and GS, respectively, suggesting a delta-6-elongase-desaturase system inhibition. Moreover, cholesterol feeding, mainly in HGS, decreased low-density-lipoprotein receptor expression and the delta-5-desaturase activity and increased the delta-9-desaturase activity. In conclusion, the liver production of highly unsaturated fatty acids was limited to decrease their oxidation in presence of hypercholesterolaemia. Glucomannan or glucomannan/spirulina-RP has added new attributes to their functional properties in meat, partially arresting the negative effects induced by high-fat-high-cholesterol feeding on the liver fatty acid and antioxidant statuses. PMID:28325998

  7. Americium binding to humic acid.

    PubMed

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

    2001-09-01

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

  8. Vanadium-fulvic acid chemistry: conformational and binding studies by electron spin probe techniques

    NASA Astrophysics Data System (ADS)

    Templeton, G. Daniel, III; Chasteen, N. Dennis

    1980-05-01

    Two fractions of soil fulvic acid (FA) were separated by gel filtration chromatography. An observed increase in volume of the heavier fraction (FA I) with increasing pH was attributed to aggregation, intramolecular negative charge repulsions and the rupture of hydrogen bonds, which control molecular conformation. Optical absorption properties and elemental analyses of both fractions were determined. The stability constants and stoichiometries of FA complexes with vanadyl, VO 2+, at pH 5.0 and ionic strength of 0.04 M were measured by electron paramagnetic resonance (EPR) spectroscopy. EPR spectra of model VO 2+ complexes with phthalic and salicylic acids, which are the probable functional groups present in FA, are identical to those of the VO 2+-FA complexes. Aggregation of FA I occurs in the presence of VO 2+ to form a complex that can be approximated as '(VO) 2(FA I) 6'. The average site distance between vanadyl ions in this complex is shown to be greater than 1.2 nm. EPR parameters for FA I suggest binding by carboxylate groups. These parameters are compared with those of other vanadyl complexes with fulvic and humic acids reported by others. Reduction of VO 3- to VO 2+ by these materials is discussed.

  9. Synergistic effect of chemo-photothermal for breast cancer therapy using folic acid (FA) modified zinc oxide nanosheet.

    PubMed

    Vimala, Karuppaiya; Shanthi, Krishnamurthy; Sundarraj, Shenbagamoorthy; Kannan, Soundarapandian

    2017-02-15

    Modern therapies for malignant breast cancer in clinics are not efficacious and often result in deprived patient compliance owing to squat therapeutic effectiveness and strong systemic side effects. In order to overcome this, we combined chemo-photothermal targeted therapy of breast cancer within one novel multifunctional drug delivery system. Folic Acid-functionalized polyethylene glycol coated Zinc Oxide nanosheet (FA-PEG-ZnO NS), was successfully synthesized, characterized and introduced to the drug delivery field for the first time. A doxorubicin (DOX)-loaded FA-PEG-ZnO NS based system (DOX-FA-PEG-ZnO NS) showed stimulative effect of heat, pH responsive and sustained drug release properties. Cytotoxicity experiments confirmed that combined therapy mediated the maximum rate of death in breast cancer cells compared to that of single chemotherapy or photothermal therapy. In vivo toxicity evaluation showed that the DOX-FA-PEG-ZnO NS contains minimum systemic toxicity in the mice model system. The findings of the present study provided an ideal drug delivery system for breast cancer therapy due to the advanced chemo-photothermal synergistic targeted therapy and good drug release properties of DOX-FA-PEG-ZnO NS, which could effectively avoid frequent and invasive dosing and improve patient compliance. Thus, functionalized-ZnO NS could be used as a novel nanomaterial for selective chemo-photothermal therapy.

  10. Induction of the fatty acid 2-hydroxylase (FA2H) gene by Δ(9)-tetrahydrocannabinol in human breast cancer cells.

    PubMed

    Takeda, Shuso; Harada, Mari; Su, Shengzhong; Okajima, Shunsuke; Miyoshi, Hiroko; Yoshida, Kazutaka; Nishimura, Hajime; Okamoto, Yoshiko; Amamoto, Toshiaki; Watanabe, Kazuhito; Omiecinski, Curtis J; Aramaki, Hironori

    2013-01-01

    To investigate gene(s) being regulated by ∆(9)-tetrahydrocannabinol (∆(9)-THC), we performed DNA microarray analysis of human breast cancer MDA-MB-231 cells, which are poorly differentiated breast cancer cells, treated with ∆(9)-THC for 48 hr at an IC50 concentration of approximately 25 µM. Among the highly up-regulated genes (> 10-fold) observed, fatty acid 2-hydroxylase (FA2H) was significantly induced (17.8-fold). Although the physiological role of FA2H has not yet been fully understood, FA2H has been shown to modulate cell differentiation. The results of Oil Red O staining after ∆(9)-THC exposure showed the distribution of lipid droplets (a sign of the differentiated phenotype) in cells. Taken together, the results obtained here indicate that FA2H is a novel ∆(9)-THC-regulated gene, and that ∆(9)-THC induces differentiation signal(s) in poorly differentiated MDA-MB-231 cells.

  11. 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

  12. Crystal Structure of the Receptor-Binding Domain of Botulinum Neurotoxin Type HA, Also Known as Type FA or H

    PubMed Central

    Yao, Guorui; Lam, Kwok-ho; Perry, Kay; Weisemann, Jasmin; Rummel, Andreas; Jin, Rongsheng

    2017-01-01

    Botulinum neurotoxins (BoNTs), which have been exploited as cosmetics and muscle-disorder treatment medicines for decades, are well known for their extreme neurotoxicity to humans. They pose a potential bioterrorism threat because they cause botulism, a flaccid muscular paralysis-associated disease that requires immediate antitoxin treatment and intensive care over a long period of time. In addition to the existing seven established BoNT serotypes (BoNT/A–G), a new mosaic toxin type termed BoNT/HA (aka type FA or H) was reported recently. Sequence analyses indicate that the receptor-binding domain (HC) of BoNT/HA is ~84% identical to that of BoNT/A1. However, BoNT/HA responds differently to some potent BoNT/A-neutralizing antibodies (e.g., CR2) that target the HC. Therefore, it raises a serious concern as to whether BoNT/HA poses a new threat to our biosecurity. In this study, we report the first high-resolution crystal structure of BoNT/HA-HC at 1.8 Å. Sequence and structure analyses reveal that BoNT/HA and BoNT/A1 are different regarding their binding to cell-surface receptors including both polysialoganglioside (PSG) and synaptic vesicle glycoprotein 2 (SV2). Furthermore, the new structure also provides explanations for the ~540-fold decreased affinity of antibody CR2 towards BoNT/HA compared to BoNT/A1. Taken together, these new findings advance our understanding of the structure and function of this newly identified toxin at the molecular level, and pave the way for the future development of more effective countermeasures. PMID:28282873

  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. 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

    2014-12-04

    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.

  15. 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).

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

    PubMed

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

    2017-03-21

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

  17. [Enzymatic conversion of cephalosporin C to glutaryl-7-aminocephalosporanic acid using whole cells of the yeast Trigonopsis variabilis FA10].

    PubMed

    Chen, J; Zhu, T B; Zhang, Y F; Yang, Y L; Jiao, R S

    2001-03-01

    A process for the production of glutaryl-7-aminocephalosporanic acid (GL-7ACA) from cephalosporin C(CPC) using permeabilized cells of yeast Trigonopsis variabilis FA10 containing D-amino acid oxidase (DAO) is described. It was found that the bioconversion of CPC to GL-7ACA was interfered by the catalase activity presented in the cells that hydrolyzed the hydrogen peroxide and resulted in the accumulation of alpha-keto-adipyl-7-ACA (AKA-7ACA) and decrease of GL-7ACA yield. the methods to overcome this problem including the addition of extra H2O2 and use of catalase inhibitor, NaN3, were developed and the rate of GL-7ACA from CPC were 73% and 70.1%, respectively. Another alternative method was to incubate the permeabilized FA10 cells at pH10.5-11.0 for 30 minutes at 20 degrees C which served to selectively inactivate the catalase. In the bioconversion of CPC to GL-7ACA using pH10.5-treated cells without catalase activity, the high reaction yield of GL-7ACA(84%) was achieved.

  18. Structural analysis of binding functionality of folic acid-PEG dendrimers against folate receptor.

    PubMed

    Sampogna-Mireles, Diana; Araya-Durán, Ingrid D; Márquez-Miranda, Valeria; Valencia-Gallegos, Jesús A; González-Nilo, Fernando D

    2017-03-01

    Dendrimers functionalized with folic acid (FA) are drug delivery systems that can selectively target cancer cells with folate receptors (FR-α) overexpression. Incorporation of polyethylene glycol (PEG) can enhance dendrimers solubility and pharmacokinetics, but ligand-receptor binding must not be affected. In this work we characterized, at atomic level, the binding functionality of conventional site-specific dendrimers conjugated with FA with PEG 750 or PEG 3350 as a linker. After Molecular Dynamics simulation, we observed that both PEG's did not interfere over ligand-receptor binding functionality. Although binding kinetics could be notably affected, the folate fragment from both dendrimers remained exposed to the solvent before approaching selectively to FR-α. PEG 3350 provided better solubility and protection from enzymatic degradation to the dendrimer than PEG 750. Also, FA-PEG3350 dendrimer showed a slightly better interaction with FR-α than FA-PEG750 dendrimer. Therefore, theoretical evidence supports that both dendrimers are suitable as drug delivery systems for cancer therapies.

  19. Fatty acid binding sites of human and bovine albumins: Differences observed by spin probe ESR

    NASA Astrophysics Data System (ADS)

    Muravsky, Vladimir; Gurachevskaya, Tatjana; Berezenko, Stephen; Schnurr, Kerstin; Gurachevsky, Andrey

    2009-09-01

    Bovine and human serum albumins and recombinant human albumin, all non-covalently complexed with 5- and 16-doxyl stearic acids, were investigated by ESR spectroscopy in solution over a range of pH values (5.5-8.0) and temperatures (25-50 °C), with respect to the allocation and mobility of fatty acid (FA) molecules bound to the proteins and conformation of the binding sites. In all proteins bound FA undergo a permanent intra-albumin migration between the binding sites and inter-domain residence. Nature identity of the recombinant human albumin to its serum-derived analog was observed. However, the binding sites of bovine albumin appeared shorter in length and wider in diameter than those of human albumin. Presumably, less tightly folded domains in bovine albumin allow better penetration of water molecules in the interior of the globule that resulted in higher activation energy of FA dissociation from the binding site. Thus, the sensitive technique based on ESR non-covalent spin labeling allowed quantitative analysis and reliable comparison of the fine features of binding proteins.

  20. 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

  1. 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.

  2. Dissolution Profile of Mefenamic Acid Solid Dosage Forms in Two Compendial and Biorelevant (FaSSIF) Media.

    PubMed

    Nurhikmah, Wilda; Sumirtapura, Yeyet Cahyati; Pamudji, Jessie Sofia

    2016-01-01

    Mefenamic acid is a non-steroidal anti-inflammatory drug (NSAID) that is widely used for the treatment of mild-to-moderate pain. Mefenamic acid belongs to the Biopharmaceutical Classification System (BCS) class II drug which has lower water solubility but high permeability. There are two different compendial methods available for dissolution tests of mefenamic acid solid dosage forms, i.e. methods of United States Pharmacopeia 37 (USP) and Pharmacopoeia of the People's Republic of China 2010 (PPRC). Indonesian Pharmacopeia V ed. (FI) adopted the USP method. On the other hand, many researches focused on the use of a 'biorelevant' medium to develop the dissolution test method. The aim of this research was to study the dissolution profile of mefenamic acid from its solid dosage forms (caplet and capsule) available in the Indonesian market with three different dissolution medium: USP, PPRC, and biorelevant fasted simulated small intestinal fluid (FaSSIF) media. The tested products consisted of the innovator's product (available only in caplet dosage form, FN caplet) and generic products (available as caplet and capsule). The dissolution test of the drug products in all dissolution media was performed in 900 mL of medium using apparatus II (paddle) at a temperature of 37°C and rotation speed of 75 rpm, except for the capsule product and for USP medium, both of which tests were done using apparatus I (basket) with rotation speed of 100 rpm. The solubility test of mefenamic acid was carried out in all media at temperature of 37°C. The result obtained from the solubility test showed that the the highest solubility of mefenamic acid was obtained in USP medium (approximately 2 mg/mL), followed by PPRC medium (about 0.5 mg/mL), and FaSSIF medium (approximately 0.06 mg/ml). In the dissolution test, percentage of drug dissolved in in the USP and PPRC media after 45 min for all products reached more than 75%, except for the PN caplet in USP medium which reached only about 44

  3. 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.

  4. Shared midgut binding sites for Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac and Cry1Fa proteins from Bacillus thuringiensis in two important corn pests, Ostrinia nubilalis and Spodoptera frugiperda.

    PubMed

    Hernández-Rodríguez, Carmen Sara; Hernández-Martínez, Patricia; Van Rie, Jeroen; Escriche, Baltasar; Ferré, Juan

    2013-01-01

    First generation of insect-protected transgenic corn (Bt-corn) was based on the expression of Cry1Ab or Cry1Fa proteins. Currently, the trend is the combination of two or more genes expressing proteins that bind to different targets. In addition to broadening the spectrum of action, this strategy helps to delay the evolution of resistance in exposed insect populations. One of such examples is the combination of Cry1A.105 with Cry1Fa and Cry2Ab to control O. nubilalis and S. frugiperda. Cry1A.105 is a chimeric protein with domains I and II and the C-terminal half of the protein from Cry1Ac, and domain III almost identical to Cry1Fa. The aim of the present study was to determine whether the chimeric Cry1A.105 has shared binding sites either with Cry1A proteins, with Cry1Fa, or with both, in O. nubilalis and in S. frugiperda. Brush-border membrane vesicles (BBMV) from last instar larval midguts were used in competition binding assays with (125)I-labeled Cry1A.105, Cry1Ab, and Cry1Fa, and unlabeled Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac, Cry1Fa, Cry2Ab and Cry2Ae. The results showed that Cry1A.105, Cry1Ab, Cry1Ac and Cry1Fa competed with high affinity for the same binding sites in both insect species. However, Cry2Ab and Cry2Ae did not compete for the binding sites of Cry1 proteins. Therefore, according to our results, the development of cross-resistance among Cry1Ab/Ac, Cry1A.105, and Cry1Fa proteins is possible in these two insect species if the alteration of shared binding sites occurs. Conversely, cross-resistance between these proteins and Cry2A proteins is very unlikely in such case.

  5. 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.

  6. FCA does not bind abscisic acid.

    PubMed

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

    2008-12-11

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

  7. 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.

  8. Urinary Excretion of Liver Type Fatty Acid Binding Protein Accurately Reflects the Degree of Tubulointerstitial Damage

    PubMed Central

    Yokoyama, Takeshi; Kamijo-Ikemori, Atsuko; Sugaya, Takeshi; Hoshino, Seiko; Yasuda, Takashi; Kimura, Kenjiro

    2009-01-01

    To investigate the relationship between liver-type fatty acid-binding protein (L-FABP), a biomarker of chronic kidney disease, in the kidney and the degree of tubulointerstitial damage, folic acid (FA)-induced nephropathy was studied in a mouse model system. As renal L-FABP is not expressed in wild-type mice, human L-FABP (hL-FABP) transgenic mice were used in this study. hL-FABP is expressed in the renal proximal tubules of the transgenic mice that were injected intraperitoneally with FA in NaHCO3 (the FA group) or only NaHCO3 (the control group) and oral saline solution daily during the experimental period. The FA group developed severe tubulointerstitial damage with the infiltration of macrophages and the deposition of type I collagen on days 3 and 7 and recovered to the control level on day 14. The gene and protein expression levels of hL-FABP in the kidney were significantly enhanced on days 3 and 7. Urinary hL-FABP in the FA group was elevated on days 3 and 7 and decreased to the control level on day 14. The protein expression levels of hL-FABP in both the kidney and urine significantly correlated with the degree of tubulointerstitial damage, the infiltration of macrophages, and the deposition of type I collagen. In conclusion, renal expression and urinary excretion of hL-FABP significantly reflected the severity of tubulointerstitial damage in FA-induced nephropathy. PMID:19435794

  9. Kinetics of ligand binding to nucleic acids.

    PubMed

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

    2006-02-01

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

  10. Ferulic acid (FA) abrogates γ-radiation induced oxidative stress and DNA damage by up-regulating nuclear translocation of Nrf2 and activation of NHEJ pathway.

    PubMed

    Das, Ujjal; Manna, Krishnendu; Khan, Amitava; Sinha, Mahuya; Biswas, Sushobhan; Sengupta, Aaveri; Chakraborty, Anindita; Dey, Sanjit

    2017-01-01

    The present study was aimed to evaluate the radioprotective effect of ferulic acid (FA), a naturally occurring plant flavonoid in terms of DNA damage and damage related alterations of repair pathways by gamma radiation. FA was administered at a dose of 50 mg/kg body weight for five consecutive days prior to exposing the swiss albino mice to a single dose of 10 Gy gamma radiation. Ionising radiation induces oxidative damage manifested by decreased expression of Cu, Zn-SOD (SOD stands for super oxide dismutase), Mn-SOD and catalase. Gamma radiation promulgated reactive oxygen species (ROS) mediated DNA damage and modified repair pathways. ROS enhanced nuclear translocation of p53, activated ATM (ataxia telangiectasia-mutated protein), increased expression of GADD45a (growth arrest and DNA-damage-inducible protein) gene and inactivated Non homologous end joining (NHEJ) repair pathway. The comet formation in irradiated mice peripheral blood mononuclear cells (PBMC) reiterated the DNA damage in IR exposed groups. FA pretreatment significantly prevented the comet formation and regulated the nuclear translocation of p53, inhibited ATM activation and expression of GADD45a gene. FA promoted the nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and activated NHEJ repair pathway to overcome ROS mediated oxidative stress and DNA damage. Therefore, the current study stated that FA can challenge the oxidative stress by (i) inducing nuclear translocation of Nrf2, (ii) scavenging ROS, and (iii) activating NHEJ DNA repair process.

  11. 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.

  12. Cloning and expression of Tsaiya duck liver fatty acid binding protein.

    PubMed

    Ko, Y H; Cheng, C H; Shen, T F; Ding, S T

    2004-11-01

    Liver basic fatty acid (FA)-binding protein (Lb-FABP) cDNA was cloned from the livers of laying Tsaiya ducks and used to generate probes for quantification of the Lb-FABP mRNA in Tsaiya ducks. The full-length Lb-FABP cDNA of the Tsaiya duck was highly homologous with that of the mallard (99%), chicken (88%), and iguana (73%). The amino acid sequence was also highly homologous to Lb-FABP found in birds and reptiles, indicating a similar function of the Tsaiya duck Lb-FABP to those species. The calculated molecular weight for the cloned duck Lb-FABP was 14,043g/mol. The Lb-FABP was highly expressed in the liver of laying Tsaiya ducks and not detectable in heart, ovary, intestine, or adipose tissues. The expression of Tsaiya duck Lb-FABP in the skeletal muscle was also detected, and the sequence was confirmed. The greater expression of the hepatic Lb-FABP in the egg-laying Tsaiya ducks than the prelaying ducks paralleled the higher FA use by the laying ducks. These results suggest that hepatic Lb-FABP may be needed for egg production when FA metabolism is high for the ducks. Feeding laying Tsaiya ducks with diets enriched with 2% docosahexaenoic acid (DHA) oil for 2 wk significantly increased hepatic DHA content compared with in ducks fed a 2% butter basal diet. There was no effect of dietary DHA enrichment on the expression of Lb-FABP in the liver of Tsaiya ducks. The results suggest that even though the Lb-FABP may be involved in hepatic FA metabolism, the effect of individual FA on liver Lb-FABP in laying Tsaiya ducks needs to be further studied.

  13. Shared Midgut Binding Sites for Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac and Cry1Fa Proteins from Bacillus thuringiensis in Two Important Corn Pests, Ostrinia nubilalis and Spodoptera frugiperda

    PubMed Central

    Hernández-Rodríguez, Carmen Sara; Hernández-Martínez, Patricia; Van Rie, Jeroen; Escriche, Baltasar; Ferré, Juan

    2013-01-01

    First generation of insect-protected transgenic corn (Bt-corn) was based on the expression of Cry1Ab or Cry1Fa proteins. Currently, the trend is the combination of two or more genes expressing proteins that bind to different targets. In addition to broadening the spectrum of action, this strategy helps to delay the evolution of resistance in exposed insect populations. One of such examples is the combination of Cry1A.105 with Cry1Fa and Cry2Ab to control O. nubilalis and S. frugiperda. Cry1A.105 is a chimeric protein with domains I and II and the C-terminal half of the protein from Cry1Ac, and domain III almost identical to Cry1Fa. The aim of the present study was to determine whether the chimeric Cry1A.105 has shared binding sites either with Cry1A proteins, with Cry1Fa, or with both, in O. nubilalis and in S. frugiperda. Brush-border membrane vesicles (BBMV) from last instar larval midguts were used in competition binding assays with 125I-labeled Cry1A.105, Cry1Ab, and Cry1Fa, and unlabeled Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac, Cry1Fa, Cry2Ab and Cry2Ae. The results showed that Cry1A.105, Cry1Ab, Cry1Ac and Cry1Fa competed with high affinity for the same binding sites in both insect species. However, Cry2Ab and Cry2Ae did not compete for the binding sites of Cry1 proteins. Therefore, according to our results, the development of cross-resistance among Cry1Ab/Ac, Cry1A.105, and Cry1Fa proteins is possible in these two insect species if the alteration of shared binding sites occurs. Conversely, cross-resistance between these proteins and Cry2A proteins is very unlikely in such case. PMID:23861865

  14. Prediction of fatty acid-binding residues on protein surfaces with three-dimensional probability distributions of interacting atoms.

    PubMed

    Mahalingam, Rajasekaran; Peng, Hung-Pin; Yang, An-Suei

    2014-08-01

    Protein-fatty acid interaction is vital for many cellular processes and understanding this interaction is important for functional annotation as well as drug discovery. In this work, we present a method for predicting the fatty acid (FA)-binding residues by using three-dimensional probability density distributions of interacting atoms of FAs on protein surfaces which are derived from the known protein-FA complex structures. A machine learning algorithm was established to learn the characteristic patterns of the probability density maps specific to the FA-binding sites. The predictor was trained with five-fold cross validation on a non-redundant training set and then evaluated with an independent test set as well as on holo-apo pair's dataset. The results showed good accuracy in predicting the FA-binding residues. Further, the predictor developed in this study is implemented as an online server which is freely accessible at the following website, http://ismblab.genomics.sinica.edu.tw/.

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

    PubMed Central

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

    2016-01-01

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

  16. Three Dimensional Structure Prediction of Fatty Acid Binding Site on Human Transmembrane Receptor CD36.

    PubMed

    Tarhda, Zineb; Semlali, Oussama; Kettani, Anas; Moussa, Ahmed; Abumrad, Nada A; Ibrahimi, Azeddine

    2013-01-01

    CD36 is an integral membrane protein which is thought to have a hairpin-like structure with alpha-helices at the C and N terminals projecting through the membrane as well as a larger extracellular loop. This receptor interacts with a number of ligands including oxidized low density lipoprotein and long chain fatty acids (LCFAs). It is also implicated in lipid metabolism and heart diseases. It is therefore important to determine the 3D structure of the CD36 site involved in lipid binding. In this study, we predict the 3D structure of the fatty acid (FA) binding site [127-279 aa] of the CD36 receptor based on homology modeling with X-ray structure of Human Muscle Fatty Acid Binding Protein (PDB code: 1HMT). Qualitative and quantitative analysis of the resulting model suggests that this model was reliable and stable, taking in consideration over 97.8% of the residues in the most favored regions as well as the significant overall quality factor. Protein analysis, which relied on the secondary structure prediction of the target sequence and the comparison of 1HMT and CD36 [127-279 aa] secondary structures, led to the determination of the amino acid sequence consensus. These results also led to the identification of the functional sites on CD36 and revealed the presence of residues which may play a major role during ligand-protein interactions.

  17. Three Dimensional Structure Prediction of Fatty Acid Binding Site on Human Transmembrane Receptor CD36

    PubMed Central

    Tarhda, Zineb; Semlali, Oussama; Kettani, Anas; Moussa, Ahmed; Abumrad, Nada A.; Ibrahimi, Azeddine

    2013-01-01

    CD36 is an integral membrane protein which is thought to have a hairpin-like structure with alpha-helices at the C and N terminals projecting through the membrane as well as a larger extracellular loop. This receptor interacts with a number of ligands including oxidized low density lipoprotein and long chain fatty acids (LCFAs). It is also implicated in lipid metabolism and heart diseases. It is therefore important to determine the 3D structure of the CD36 site involved in lipid binding. In this study, we predict the 3D structure of the fatty acid (FA) binding site [127–279 aa] of the CD36 receptor based on homology modeling with X-ray structure of Human Muscle Fatty Acid Binding Protein (PDB code: 1HMT). Qualitative and quantitative analysis of the resulting model suggests that this model was reliable and stable, taking in consideration over 97.8% of the residues in the most favored regions as well as the significant overall quality factor. Protein analysis, which relied on the secondary structure prediction of the target sequence and the comparison of 1HMT and CD36 [127–279 aa] secondary structures, led to the determination of the amino acid sequence consensus. These results also led to the identification of the functional sites on CD36 and revealed the presence of residues which may play a major role during ligand-protein interactions. PMID:24348024

  18. 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.

  19. Cellular Retinoic Acid Binding Protein and Breast Cancer

    DTIC Science & Technology

    2006-05-01

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

  20. Evaluation of Carbohydrate-Derived Fulvic Acid (CHD-FA) as a Topical Broad-Spectrum Antimicrobial for Drug-Resistant Wound Infections

    DTIC Science & Technology

    2014-10-01

    molecular mechanisms during wound healing with CHD-FA. Figure 5. Wound images of rats infected with A. baumannii (ATCC BAA-747) and treated with CHD-FA...host gene expression profiling to better assess the cellular and molecular mechanisms during wound healing with CHD-FA. 13 Figure 6. Wound...expression profiling will be performed to better assess the cellular and molecular mechanisms during wound healing with CHD-FA. 20 Figure 5

  1. Paralogous proteins comprising the 150 kDa hydrophobic-ligand-binding-protein complex of the Taenia solium metacestode have evolved non-overlapped binding affinities toward fatty acid analogs.

    PubMed

    Kim, Seon-Hee; Bae, Young-An; Yang, Yichao; Hong, Sung-Tae; Kong, Yoon

    2011-09-01

    We previously identified a hydrophobic-ligand-binding protein (HLBP) of the Taenia solium metacestode (TsM), which might be involved in the uptake of fatty acids (FAs) from host environments. The TsM 150kDa HLBP was a hetero-oligomeric complex composed of multiple 7kDa (RS1) and 10kDa (CyDA, b1 and m13h) subunits, and displayed a wide spectrum of binding affinities toward various FA analogs. In this study, we analysed biochemical properties and phylogenetic relationships of the individual subunits. Despite the low sequence identity (average 26.5%), these subunit proteins conserved an α-helix-rich structural domain and the first introns inserted in each of the respective chromosomal genes were found to be orthologous to one another, suggesting their common evolutionary origin. The recombinant RS1 protein bound strongly to all of the FA analogs examined including 11-[(5-dimethylaminonaphthalene-1-sulfonyl)amino]undecanoic acid (DAUDA), but not to 16-(9-anthroyloxy)palmitic acid (16-AP). The interactive binding between RS1 and FA analogs was specifically interfered with by the addition of non-fluorescent FA molecules or antibodies specific to the 150kDa protein. Conversely, the 10kDa members reacted only with the palmitic acid-derived 16-AP, whose interactive force was strengthened by the presence of other FA molecules. The use of mutagenic RS1 proteins demonstrated that a structural/electrostatic integrity around the second α-helix, rather than the conventional Trp residue, was the major factor governing the hydrophobic interaction. The 7 and 10kDa proteins exhibited distinctive immunoreactive patterns against sera from neurocysticercosis patients. These collective data suggest that the paralogous protein family have gained diverse functions during their evolution, to ensure the maintenance of metabolic homeostasis and survival of TsMs in hostile host environments.

  2. 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.

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

    PubMed

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

    2006-01-01

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

  4. Nucleic acid-binding specificity of human FUS protein

    PubMed Central

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

    2015-01-01

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

  5. 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.

  6. 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.

  7. 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.

  8. A critical role of fatty acid binding protein 4 and 5 (FABP4/5) in the systemic response to fasting.

    PubMed

    Syamsunarno, Mas Rizky A A; Iso, Tatsuya; Hanaoka, Hirofumi; Yamaguchi, Aiko; Obokata, Masaru; Koitabashi, Norimichi; Goto, Kosaku; Hishiki, Takako; Nagahata, Yoshiko; Matsui, Hiroki; Sano, Motoaki; Kobayashi, Masaki; Kikuchi, Osamu; Sasaki, Tsutomu; Maeda, Kazuhisa; Murakami, Masami; Kitamura, Tadahiro; Suematsu, Makoto; Tsushima, Yoshito; Endo, Keigo; Hotamisligil, Gökhan S; Kurabayashi, Masahiko

    2013-01-01

    During prolonged fasting, fatty acid (FA) released from adipose tissue is a major energy source for peripheral tissues, including the heart, skeletal muscle and liver. We recently showed that FA binding protein 4 (FABP4) and FABP5, which are abundantly expressed in adipocytes and macrophages, are prominently expressed in capillary endothelial cells in the heart and skeletal muscle. In addition, mice deficient for both FABP4 and FABP5 (FABP4/5 DKO mice) exhibited defective uptake of FA with compensatory up-regulation of glucose consumption in these tissues during fasting. Here we showed that deletion of FABP4/5 resulted in a marked perturbation of metabolism in response to prolonged fasting, including hyperketotic hypoglycemia and hepatic steatosis. Blood glucose levels were reduced, whereas the levels of non-esterified FA (NEFA) and ketone bodies were markedly increased during fasting. In addition, the uptake of the (125)I-BMIPP FA analogue in the DKO livers was markedly increased after fasting. Consistent with an increased influx of NEFA into the liver, DKO mice showed marked hepatic steatosis after a 48-hr fast. Although gluconeogenesis was observed shortly after fasting, the substrates for gluconeogenesis were reduced during prolonged fasting, resulting in insufficient gluconeogenesis and enhanced hypoglycemia. These metabolic responses to prolonged fasting in DKO mice were readily reversed by re-feeding. Taken together, these data strongly suggested that a maladaptive response to fasting in DKO mice occurred as a result of an increased influx of NEFA into the liver and pronounced hypoglycemia. Together with our previous study, the metabolic consequence found in the present study is likely to be attributed to an impairment of FA uptake in the heart and skeletal muscle. Thus, our data provided evidence that peripheral uptake of FA via capillary endothelial FABP4/5 is crucial for systemic metabolism and may establish FABP4/5 as potentially novel targets for the

  9. 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

  10. Decreased plasma arachidonic acid binding capacity in neonates.

    PubMed

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

    1987-01-01

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

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

    PubMed

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

    2012-02-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2014-06-01

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

  14. Water-mediated recognition of simple alkyl chains by heart-type fatty-acid-binding protein.

    PubMed

    Matsuoka, Shigeru; Sugiyama, Shigeru; Matsuoka, Daisuke; Hirose, Mika; Lethu, Sébastien; Ano, Hikaru; Hara, Toshiaki; Ichihara, Osamu; Kimura, S Roy; Murakami, Satoshi; Ishida, Hanako; Mizohata, Eiichi; Inoue, Tsuyoshi; Murata, Michio

    2015-01-26

    Long-chain fatty acids (FAs) with low water solubility require fatty-acid-binding proteins (FABPs) to transport them from cytoplasm to the mitochondria for energy production. However, the precise mechanism by which these proteins recognize the various lengths of simple alkyl chains of FAs with similar high affinity remains unknown. To address this question, we employed a newly developed calorimetric method for comprehensively evaluating the affinity of FAs, sub-Angstrom X-ray crystallography to accurately determine their 3D structure, and energy calculations of the coexisting water molecules using the computer program WaterMap. Our results clearly showed that the heart-type FABP (FABP3) preferentially incorporates a U-shaped FA of C10-C18 using a lipid-compatible water cluster, and excludes longer FAs using a chain-length-limiting water cluster. These mechanisms could help us gain a general understanding of how proteins recognize diverse lipids with different chain lengths.

  15. 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.

  16. Modeling lanthanide series binding sites on humic acid.

    PubMed

    Pourret, Olivier; Martinez, Raul E

    2009-02-01

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

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. 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.

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

    PubMed

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

    2013-01-01

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

  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.

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

    SciTech Connect

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

    1991-08-01

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

  8. Direct Comparison of Mice Null for Liver or Intestinal Fatty Acid-binding Proteins Reveals Highly Divergent Phenotypic Responses to High Fat Feeding*

    PubMed Central

    Gajda, Angela M.; Zhou, Yin Xiu; Agellon, Luis B.; Fried, Susan K.; Kodukula, Sarala; Fortson, Walter; Patel, Khamoshi; Storch, Judith

    2013-01-01

    The enterocyte expresses two fatty acid-binding proteins (FABP), intestinal FABP (IFABP; FABP2) and liver FABP (LFABP; FABP1). LFABP is also expressed in liver. Despite ligand transport and binding differences, it has remained uncertain whether these intestinally coexpressed proteins, which both bind long chain fatty acids (FA), are functionally distinct. Here, we directly compared IFABP−/− and LFABP−/− mice fed high fat diets containing long chain saturated or unsaturated fatty acids, reasoning that providing an abundance of dietary lipid would reveal unique functional properties. The results showed that mucosal lipid metabolism was indeed differentially modified, with significant decreases in FA incorporation into triacylglycerol (TG) relative to phospholipid (PL) in IFABP−/− mice, whereas LFABP−/− mice had reduced monoacylglycerol incorporation in TG relative to PL, as well as reduced FA oxidation. Interestingly, striking differences were found in whole body energy homeostasis; LFABP−/− mice fed high fat diets became obese relative to WT, whereas IFABP−/− mice displayed an opposite, lean phenotype. Fuel utilization followed adiposity, with LFABP−/− mice preferentially utilizing lipids, and IFABP−/− mice preferentially metabolizing carbohydrate for energy production. Changes in body weight and fat may arise, in part, from altered food intake; mucosal levels of the endocannabinoids 2-arachidonoylglycerol and arachidonoylethanolamine were elevated in LFABP−/−, perhaps contributing to increased energy intake. This direct comparison provides evidence that LFABP and IFABP have distinct roles in intestinal lipid metabolism; differential intracellular functions in intestine and in liver, for LFABP−/− mice, result in divergent downstream effects at the systemic level. PMID:23990461

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

    PubMed

    Miao, Zhichao; Westhof, Eric

    2015-06-23

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

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

    PubMed Central

    Tonsgard, J H; Meredith, S C

    1991-01-01

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

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

    PubMed

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

    1995-07-14

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

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

    NASA Astrophysics Data System (ADS)

    Ritchie, Jason D.; Perdue, E. Michael

    2003-01-01

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

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

    PubMed

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

    2009-12-18

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

  16. 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

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

    PubMed

    Bertucci, C; Nanni, B; Salvadori, P

    1999-01-01

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

  18. 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.

  19. 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

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

    PubMed

    Hanus, Martin; Zhorov, Eugene

    2006-12-01

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

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

    SciTech Connect

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

    1981-11-16

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

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. 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

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

    PubMed

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

    2001-09-01

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

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

    PubMed Central

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

    1984-01-01

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

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

    PubMed

    Bourassa, P; Tajmir-Riahi, H A

    2012-01-12

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

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

    PubMed

    Albani, J R

    2006-11-06

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

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

    PubMed

    Ponnuraj, Karthe; Saravanan, Konda Mani

    2017-04-01

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

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

    PubMed

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

    2011-08-15

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-11-01

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

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

    PubMed Central

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

    2016-01-01

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

  15. 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

  16. 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.

  17. 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.

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

    PubMed

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

    1991-09-01

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

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

    SciTech Connect

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

    1998-08-15

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

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

    SciTech Connect

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

    1987-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Rhea, James R.; Young, Thomas C.

    1987-10-01

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

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

    PubMed

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

    2006-07-01

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

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

    PubMed

    Shao, Qing; Hall, Carol K

    2016-08-09

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

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

    PubMed

    Hughes, Austin L; Piontkivska, Helen

    2011-12-15

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

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    1999-02-02

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

  8. 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

  9. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2017-01-01

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

  11. 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.

  12. 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

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

    PubMed

    Avanzati, B; Catalá, A

    1983-07-01

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

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

    PubMed

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

    2015-12-07

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

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

    PubMed

    Hu, Guohua; Yu, Wenjian

    2013-06-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed

    Wei, Qing; La, David; Kihara, Daisuke

    2017-01-01

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

  18. 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

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

    PubMed

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

    2011-03-01

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

  20. 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

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

    PubMed

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

    2008-03-15

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

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

    PubMed Central

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

    1994-01-01

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

  3. 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.

  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. 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.

  6. 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

  7. 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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    2012-01-01

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

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

    DOEpatents

    Heldebrant, David J.; Yonker, Clement R.

    2010-09-21

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

  12. 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.

  13. 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.

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

    PubMed Central

    Mandal, C; Basu, S; Mandal, C

    1989-01-01

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

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

    PubMed

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

    2007-07-01

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

  17. Computing Optimal Binding of Two Nucleic Acid Chains

    NASA Astrophysics Data System (ADS)

    Hodas, Nathan O.; Aalberts, Daniel P.

    2004-03-01

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

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

    PubMed

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

    2009-10-01

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

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

    PubMed

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

    2014-11-11

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

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

    USGS Publications Warehouse

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

    1995-01-01

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

  1. 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.

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed

    Yoshida; Sokhakian; Dubin

    1998-09-15

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

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

    SciTech Connect

    Terasaki, T.; Pardridge, W.M.

    1988-05-17

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

  5. 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.

  6. 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.

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

    PubMed

    Chowrira, B M; Burke, J M

    1991-09-03

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

  8. 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

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

    PubMed Central

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

    2010-01-01

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

  10. 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.

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

    PubMed

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

    2014-02-05

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

  12. 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.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-11-23

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

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

    PubMed

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

    2015-11-12

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

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

    NASA Astrophysics Data System (ADS)

    Tsuji, Motonori; Shudo, Koichi; Kagechika, Hiroyuki

    2015-10-01

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

  17. Structure-Function of CD36 and Importance of Fatty Acid Signal Transduction in Fat Metabolism

    PubMed Central

    Pepino, Marta Yanina; Kuda, Ondrej; Samovski, Dmitri; Abumrad, Nada A

    2015-01-01

    CD36 is a scavenger receptor that functions in high affinity tissue uptake of long chain fatty acids (FA) and contributes under excessive fat supply to lipid accumulation and metabolic dysfunction. This review describes recent evidence regarding the CD36 FA binding site and a potential mechanism for FA transfer. It also presents the view that CD36 and FA signaling coordinate fat utilization based on newly identified CD36 actions that involve oral fat perception, intestinal fat absorption, secretion of the peptides cholecystokinin and secretin, regulation of hepatic lipoprotein output, activation of beta oxidation by muscle and regulation of the production of the FA derived bioactive eicosanoids. Thus abnormalities of fat metabolism and the associated pathology might involve dysfunction of CD36-mediated signal transduction in addition to the changes of FA uptake. PMID:24850384

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

    PubMed

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

    1995-09-01

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

  19. 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.

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

    PubMed

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

    2014-03-01

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

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

    PubMed

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

    1998-10-01

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

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

    PubMed

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

    2015-07-23

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

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

    PubMed

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

    2016-03-01

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

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

    PubMed Central

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

    1993-01-01

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

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

    PubMed Central

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

    1993-01-01

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

  6. Human Serum Albumin and HER2-Binding Affibody Fusion Proteins for Targeted Delivery of Fatty Acid-Modified Molecules and Therapy.

    PubMed

    Dong, Daoyuan; Xia, Guanjun; Li, Zhijun; Li, Zhiyu

    2016-10-03

    Human epidermal growth factor receptor 2 (HER2) is a well-studied therapeutic target as well as a biomarker of breast cancer. HER2-targeting affibody (ZHER2:342) is a novel small scaffold protein with an extreme high affinity against HER2 screened by phage display. However, the small molecular weight of ZHER2:342 has limited its pharmaceutical application. Human serum albumin (HSA) and ZHER2:342 fusion protein may not only extend the serum half-life of ZHER2:342 but also preserve the biological function of HSA to bind and transport fatty acids, which can be used to deliver fatty acid-modified therapeutics to HER2-positive cancer cells. Two HSA and ZHER2:342 fusion proteins, one with a single ZHER2:342 domain fused to the C terminus of HSA (rHSA-ZHER2) and another with two tandem copies of ZHER2:342 fused to the C terminus of HSA (rHSA-(ZHER2)2), have been constructed, expressed, and purified. Both fusion proteins possessed the HER2 and fatty acid (FA) binding abilities demonstrated by in vitro assays. Interestingly, rHSA-(ZHER2)2, not rHSA-ZHER2, was able to inhibit the proliferation of SK-BR-3 cells at a relatively low concentration, and the increase of HER2 and ERK1/2 phosphorylation followed by rHSA-(ZHER2)2 treatment has been observed. HSA fusion proteins are easy and economical to express, purify, and formulate. As expected, HSA fusion proteins and fusion protein-bound fatty acid-modified FITC could be efficiently taken up by cells. These results proved the feasibility of using HSA fusion proteins as therapeutic agents as well as carriers for targeted drug delivery.

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

    PubMed Central

    Ockner, Robert K.; Manning, Joan A.

    1974-01-01

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

  8. Fatty Acid Flippase Activity Of UCP2 Is Essential For Its Proton Transport In Mitochondria

    PubMed Central

    Berardi, Marcelo J.; Chou, James J.

    2014-01-01

    SUMMARY Modulation of cellular energy expenditure is fundamental to normal and pathological cell growth and differentiation. Mitochondria stores energy as a proton gradient across their inner membrane. Uncoupling proteins (UCPs) can dissipate the gradient to produce heat or regulate metabolite fluxes. UCP-mediated proton currents require fatty acids (FA) and are blocked by nucleotides, but the molecular basis of these processes is unknown. We find, by nuclear magnetic resonance and functional mutagenesis, that UCP2 can bind FA laterally thru its peripheral site, and this intramembrane molecular recognition is essential for UCP2-catalyzed FA flipping across the membrane, which in turn is essential for proton translocation. The antagonist GDP binds inside the UCP2 cavity and perturbs its conformation, which can displace FA from the peripheral site as a mean of inhibiting proton currents. Our data provide a biophysical perspective of the intricate interplay of UCPs, FA, and nucleotides in determining proton fluxes in mitochondria. PMID:25127353

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

    PubMed

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

    2013-10-15

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

  10. 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.

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

    PubMed

    Zhao, Haiyan; Gabbaï, François P

    2010-11-01

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    1987-06-01

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

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

    PubMed Central

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

    2012-01-01

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

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

    SciTech Connect

    Mailman, D.; Rose, C. )

    1990-01-01

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

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

    PubMed

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

    2002-07-01

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

  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. 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...

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

    NASA Astrophysics Data System (ADS)

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

    2015-02-01

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

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

    PubMed

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

    2017-02-16

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    DTIC Science & Technology

    2005-08-01

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

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

    PubMed

    Deutscher, Murray P

    2009-01-01

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

  6. 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}).

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    Trainer, Vera L; Bill, Brian D

    2004-08-10

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

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

    PubMed

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

    2015-03-01

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

  10. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2014-04-01

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

  12. 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.

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

    PubMed

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

    2014-01-01

    Two fatty acid binding proteins, MvFABPa and MvFABPb were identified in the parasite Mesocestoides vogae (Platyhelmithes, Cestoda). Fatty acid binding proteins are small intracellular proteins whose members exhibit great diversity. Proteins of this family have been identified in many organisms, of which Platyhelminthes are among the most primitive. These proteins have particular relevance in flatworms since de novo synthesis of fatty acids is absent. Fatty acids should be captured from the media needing an efficient transport system to uptake and distribute these molecules. While HLBPs could be involved in the shuttle of fatty acids to the surrounding host tissues and convey them into the parasite, FABPs could be responsible for the intracellular trafficking. In an effort to understand the role of MvFABPs in fatty acid transport of M. vogae larvae, we analysed the intracellular localization of both MvFABPs and the co-localization with in vivo uptake of fatty acid analogue BODIPY FL C16. Immunohistochemical studies on larvae sections using specific antibodies, showed a diffuse cytoplasmic distribution of each protein with some expression in nuclei and mitochondria. MvFABPs distribution was confirmed by mass spectrometry identification from 2D-electrophoresis of larvae subcellular fractions. This work is the first report showing intracellular distribution of MvFABPs as well as the co-localization of these proteins with the BODIPY FL C16 incorporated from the media. Our results suggest that fatty acid binding proteins could target fatty acids to cellular compartments including nuclei. In this sense, M. vogae FABPs could participate in several cellular processes fulfilling most of the functions attributed to vertebrate's counterparts.

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

    PubMed Central

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

    2014-01-01

    Two fatty acid binding proteins, MvFABPa and MvFABPb were identified in the parasite Mesocestoides vogae (Platyhelmithes, Cestoda). Fatty acid binding proteins are small intracellular proteins whose members exhibit great diversity. Proteins of this family have been identified in many organisms, of which Platyhelminthes are among the most primitive. These proteins have particular relevance in flatworms since de novo synthesis of fatty acids is absent. Fatty acids should be captured from the media needing an efficient transport system to uptake and distribute these molecules. While HLBPs could be involved in the shuttle of fatty acids to the surrounding host tissues and convey them into the parasite, FABPs could be responsible for the intracellular trafficking. In an effort to understand the role of MvFABPs in fatty acid transport of M. vogae larvae, we analysed the intracellular localization of both MvFABPs and the co-localization with in vivo uptake of fatty acid analogue BODIPY FL C16. Immunohistochemical studies on larvae sections using specific antibodies, showed a diffuse cytoplasmic distribution of each protein with some expression in nuclei and mitochondria. MvFABPs distribution was confirmed by mass spectrometry identification from 2D-electrophoresis of larvae subcellular fractions. This work is the first report showing intracellular distribution of MvFABPs as well as the co-localization of these proteins with the BODIPY FL C16 incorporated from the media. Our results suggest that fatty acid binding proteins could target fatty acids to cellular compartments including nuclei. In this sense, M. vogae FABPs could participate in several cellular processes fulfilling most of the functions attributed to vertebrate’s counterparts. PMID:25347286

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

    DOE PAGES

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

    2015-09-18

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

  16. 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.

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

    PubMed

    Torabi, Forough; Binduraihem, Adel; Miller, David

    2017-03-01

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

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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

  3. A 25-Amino Acid Sequence of the Arabidopsis TGD2 Protein Is Sufficient for Specific Binding of Phosphatidic Acid*

    PubMed Central

    Lu, Binbin; Benning, Christoph

    2009-01-01

    Genetic analysis suggests that the TGD2 protein of Arabidopsis is required for the biosynthesis of endoplasmic reticulum derived thylakoid lipids. TGD2 is proposed to be the substrate-binding protein of a presumed lipid transporter consisting of the TGD1 (permease) and TGD3 (ATPase) proteins. The TGD1, -2, and -3 proteins are localized in the inner chloroplast envelope membrane. TGD2 appears to be anchored with an N-terminal membrane-spanning domain into the inner envelope membrane, whereas the C-terminal domain faces the intermembrane space. It was previously shown that the C-terminal domain of TGD2 binds phosphatidic acid (PtdOH). To investigate the PtdOH binding site of TGD2 in detail, the C-terminal domain of the TGD2 sequence lacking the transit peptide and transmembrane sequences was fused to the C terminus of the Discosoma sp. red fluorescent protein (DR). This greatly improved the solubility of the resulting DR-TGD2C fusion protein following production in Escherichia coli. The DR-TGD2C protein bound PtdOH with high specificity, as demonstrated by membrane lipid-protein overlay and liposome association assays. Internal deletion and truncation mutagenesis identified a previously undescribed minimal 25-amino acid fragment in the C-terminal domain of TGD2 that is sufficient for PtdOH binding. Binding characteristics of this 25-mer were distinctly different from those of TGD2C, suggesting that additional sequences of TGD2 providing the proper context for this 25-mer are needed for wild type-like PtdOH binding. PMID:19416982

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

    PubMed

    Makarska-Bialokoz, Magdalena

    2017-01-31

    The binding interaction between human hemoglobin and uric acid has been studied for the first time, by UV-vis absorption and steady-state, synchronous and three-dimensional fluorescence techniques. Characteristic effects observed for human hemoglobin intrinsic fluorescence during interaction with uric acid at neutral pH point at the formation of stacking non-covalent and non-fluorescent complexes. All the calculated parameters, the binding, fluorescence quenching and bimolecular quenching rate constants, as well as Förster resonance energy transfer parameters confirm the existence of static quenching. The results of synchronous fluorescence measurements indicate that the fluorescence quenching of human hemoglobin originates both from Trp and Tyr residues and that the addition of uric acid could significantly hinder the physiological functions of human hemoglobin.

  5. 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.

  6. 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...

  7. 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.

  8. 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

  9. 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/.

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

    PubMed Central

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

    2015-01-01

    Over four decades have passed since liver fatty acid binding protein (FABP)1 was first isolated. There are few protein families for which most of the complete tertiary structures, binding properties, and tissue occurrences are described in such detail and yet new functions are being uncovered for this protein. FABP1 is known to be critical for fatty acid uptake and intracellular transport and also has an important role in regulating lipid metabolism and cellular signaling pathways. FABP1 is an important endogenous cytoprotectant, minimizing hepatocyte oxidative damage and interfering with ischemia-reperfusion and other hepatic injuries. The protein may be targeted for metabolic activation through the cross-talk among many transcriptional factors and their activating ligands. Deficiency or malfunction of FABP1 has been reported in several diseases. FABP1 also influences cell proliferation during liver regeneration and may be considered as a prognostic factor for hepatic surgery. FABP1 binds and modulates the action of many molecules such as fatty acids, heme, and other metalloporphyrins. The ability to bind heme is another cytoprotective property and one that deserves closer investigation. The role of FABP1 in substrate availability and in protection from oxidative stress suggests that FABP1 plays a pivotal role during intracellular bacterial/viral infections by reducing inflammation and the adverse effects of starvation (energy deficiency). PMID:26443794

  11. Improved binding of acidic bone matrix proteins to cationized filters during solid phase assays.

    PubMed

    Farach-Carson, M C; Wright, G C; Butler, W T

    1992-01-01

    A number of commercially available matrix filter supports have been designed for the immobilization of proteins following either electrotransfer from sodium dodecyl sulfate (SDS) polyacrylamide gels or direct application during dot blotting assays. These matrices differ with respect to chemical composition, charge, pore size, and degree of hydrophobicity. It follows that the properties of the protein(s) of interest will greatly influence the degree to which they interact with and ultimately bind to various filters. Acidic bone proteins contain diverse post-translational modifications that influence their interactions with solid phase matrices such as those used in immunoblotting (Western or dot blotting) or ion binding (overlay) procedures. This communication describes the results of a study comparing binding of various mixtures of non-collagenous acidic bone matrix phosphoproteins as well as purified osteopontin and osteocalcin to various filters including nitrocellulose and cationized paper or nylon. Based on our findings, we recommend the use of cationized filters for solid phase assays requiring the binding of these acidic macromolecules to background supports.

  12. 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...

  13. Genetic effects of sterol regulatory element binding proteins and fatty acid-binding protein4 on the fatty acid composition of Korean cattle (Hanwoo)

    PubMed Central

    Oh, Dong-Yep; Lee, Jea-Young; Jang, Ji-Eun; Lee, Seung-Uk

    2017-01-01

    Objective This study identifies single-nucleotide polymorphisms (SNP) or gene combinations that affect the flavor and quality of Korean cattle (Hanwoo) by using the SNP Harvester method. Methods Four economic traits (oleic acid [C18:1], saturated fatty acids), monounsaturated fatty acids, and marbling score) were adjusted for environmental factors in order to focus solely on genetic effects. The SNP Harvester method was used to investigate gene combinations (two-way gene interactions) associated with these economic traits. Further, a multifactor dimensionality reduction method was used to identify superior genotypes in gene combinations. Results Table 3 to 4 show the analysis results for differences between superior genotypes and others for selected major gene combinations using the multifactor dimensionality reduction method. Environmental factors were adjusted for in order to evaluate only the genetic effect. Table 5 shows the adjustment effect by comparing the accuracy before and after correction in two-way gene interactions. Conclusion The g.3977-325 T>C and (g.2988 A>G, g.3977-325 T>C) combinations of fatty acid-binding protein4 were the superior gene, and the superior genotype combinations across all economic traits were the CC genotype at g.3977-325 T>C and the AACC, GACC, GGCC genotypes of (g.2988 A>G, g.3977-325 T>C). PMID:27492349

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

    PubMed Central

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

    2016-01-01

    Fatty acid kinase (Fak) is a ubiquitous Gram-positive bacterial enzyme consisting of an ATP-binding protein (FakA) that phosphorylates the fatty acid bound to FakB. In Staphylococcus aureus, Fak is a global regulator of virulence factor transcription and is essential for the activation of exogenous fatty acids for incorporation into phospholipids. The 1.2-Å x-ray structure of S. aureus FakB2, activity assays, solution studies, site-directed mutagenesis, and in vivo complementation were used to define the functions of the five conserved residues that define the FakB protein family (Pfam02645). The fatty acid tail is buried within the protein, and the exposed carboxyl group is bound by a Ser-93-fatty acid carboxyl-Thr-61-His-266 hydrogen bond network. The guanidinium of the invariant Arg-170 is positioned to potentially interact with a bound acylphosphate. The reduced thermal denaturation temperatures of the T61A, S93A, and H266A FakB2 mutants illustrate the importance of the hydrogen bond network in protein stability. The FakB2 T61A, S93A, and H266A mutants are 1000-fold less active in the Fak assay, and the R170A mutant is completely inactive. All FakB2 mutants form FakA(FakB2)2 complexes except FakB2(R202A), which is deficient in FakA binding. Allelic replacement shows that strains expressing FakB2 mutants are defective in fatty acid incorporation into phospholipids and virulence gene transcription. These conserved residues are likely to perform the same critical functions in all bacterial fatty acid-binding proteins. PMID:26774272

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

    PubMed Central

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

    2012-01-01

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

  16. The endothelial cell binding determinant of human factor IX resides in the. gamma. -carboxyglutamic acid domain

    SciTech Connect

    Toomey, J.R.; Roberts, H.R.; Stafford, D.W. ); Smith, K.J. United Blood Services, Albuquerque, NM )

    1992-02-18

    The blood coagulation factor IX(a) binds specifically to a site on endothelial cells with a K{sub d} of 2.0-3.0 nM. A number of previous studies have attempted to define the region(s) of factor IX(a) that mediate this interaction. These studies suggested that there are two regions of factor IX(a), the {gamma}-carboxyglutamic acid (Gla) domain and the epidermal growth factor like (EGF-like) domains, that mediate high-affinity binding to endothelial cells. Recently, however, the participation of the EGF1 domain has been excluded from the interaction. This indicated that if there was an EGF component of factor IX contributing to the binding affinity, then it must be in the second EGF-like domain. In order to further evaluate this relationship, the authors performed competitive binding experiments between {sup 125}I plasma factor IX and a set of six chimeric proteins composed of portions of factor VII and factor IX. The data suggest that the high-affinity interaction between factor IX and the endothelial cell binding site is mediated by the factor IX Gla domain and that the factor IX EGF domains are not involved in binding specificity.

  17. 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.

  18. Cyclic Phosphopeptides to Rationalize the Role of Phosphoamino Acids in Uranyl Binding to Biological Targets.

    PubMed

    Starck, Matthieu; Laporte, Fanny A; Oros, Stephane; Sisommay, Nathalie; Gathu, Vicky; Solari, Pier Lorenzo; Creff, Gaëlle; Roques, Jérôme; Den Auwer, Christophe; Lebrun, Colette; Delangle, Pascale

    2017-02-05

    The specific molecular interactions responsible for uranium toxicity are not yet understood. The uranyl binding sites in high-affinity target proteins have not been identified yet and the involvement of phosphoamino acids is still an important question. Short cyclic peptide sequences, with three glutamic acids and one phosphoamino acid, are used as simple models to mimic metal binding sites in phosphoproteins and to help understand the mechanisms involved in uranium toxicity. A combination of peptide design and synthesis, analytical chemistry, extended X-ray absorption fine structure (EXAFS) spectroscopy, and DFT calculations demonstrates the involvement of the phosphate group in the uranyl coordination sphere together with the three carboxylates of the glutamate moieties. The affinity constants measured with a reliable analytical competitive approach at physiological pH are significantly enhanced owing to the presence of the phosphorous moiety. These findings corroborate the importance of phosphoamino acids in uranyl binding in proteins and the relevance of considering phosphoproteins as potential uranyl targets in vivo.

  19. Molecular Dynamics Simulation of Ligand Dissociation from Liver Fatty Acid Binding Protein

    PubMed Central

    Long, Dong; Mu, Yuguang; Yang, Daiwen

    2009-01-01

    The mechanisms of how ligands enter and leave the binding cavity of fatty acid binding proteins (FABPs) have been a puzzling question over decades. Liver fatty acid binding protein (LFABP) is a unique family member which accommodates two molecules of fatty acids in its cavity and exhibits the capability of interacting with a variety of ligands with different chemical structures and properties. Investigating the ligand dissociation processes of LFABP is thus a quite interesting topic, which however is rather difficult for both experimental approaches and ordinary simulation strategies. In the current study, random expulsion molecular dynamics simulation, which accelerates ligand motions for rapid dissociation, was used to explore the potential egress routes of ligands from LFABP. The results showed that the previously hypothesized “portal region” could be readily used for the dissociation of ligands at both the low affinity site and the high affinity site. Besides, one alternative portal was shown to be highly favorable for ligand egress from the high affinity site and be related to the unique structural feature of LFABP. This result lends strong support to the hypothesis from the previous NMR exchange studies, which in turn indicates an important role for this alternative portal. Another less favored potential portal located near the N-terminal end was also identified. Identification of the dissociation pathways will allow further mechanistic understanding of fatty acid uptake and release by computational and/or experimental techniques. PMID:19564911

  20. Regulation of GABA-modulin phosphorylation and GABA receptor binding by excitatory amino acids

    SciTech Connect

    Vaccarino, F.; Guidotti, A.

    1987-05-01

    Primary cultures of cerebellar granule cells phosphorylate numerous proteins including GABA-modulin (GM), which is a putative allosteric modulator of GABA receptors. Cell depolarization and treatment with dicarboxylic excitatory amino acids, which activate PI turnover, Ca/sup 2 +/ influx and guanylate cyclase in granule cells increase the phosphorylation of specific proteins. To determine GM phosphorylation by endogenous protein kinases in living granule cell cultures, GM was isolated by immunoprecipitation and reverse-phase HPLC. High K/sup +/, veratridine, glutamate and NMDA treatment stimulated GM phosphorylation over 2-fold. This increase was abolished by the absence of extracellular Ca/sup 2 +/ and was antagonized by Mg/sup 2 +/ ions and by AVP. The excitatory amino acid action was mimicked by phorbol esters but not by forskolin or by cGMP, and thus may be mediated by an activation of protein kinase C (PKC). Moreover, excitatory amino acids increase /sup 3/H-labelled phorbol ester binding sites in granule cell membrane. The same cultures, treated with glutamate or kainate, showed a 50-fold greater efficacy of muscimol for the stimulation of benzodiazepine (BZ) binding. These data-suggest that excitatory amino acid stimulation of neurons triggers PKC translocation and the activated enzyme phosphorylates GM. The extent of GM phosphorylation may regulate the coupling between GABA and BZ binding sites.

  1. Arsenic exposure disrupts the normal function of the FA/BRCA repair pathway.

    PubMed

    Peremartí, Jana; Ramos, Facundo; Marcos, Ricard; Hernández, Alba

    2014-11-01

    Chronic arsenic exposure is known to enhance the genotoxicity/carcinogenicity of other DNA-damaging agents by inhibiting DNA repair activities. Interference with nucleotide excision repair and base excision repair are well documented, but interactions with other DNA repair pathways are poorly explored so far. The Fanconi anemia FA/BRCA pathway is a DNA repair mechanism required for maintaining genomic stability and preventing cancer. Here, interactions between arsenic compounds and the FA/BRCA pathway were explored by using isogenic FANCD2(-/-) (FA/BRCA-deficient) and FANCD2(+/+) (FA/BRCA-corrected) human fibroblasts. To study whether arsenic disrupts the normal FA/BRCA function, FANCD2(+/+) cells were preexposed to subtoxic concentrations of the trivalent arsenic compounds methylarsonous acid (MMA(III)) and arsenic trioxide (ATO) for 2 weeks. The cellular response to mitomicin-C, hydroxyurea, or diepoxybutane, typical inducers of the studied pathway, was then evaluated and compared to that of FANCD2(-/-) cells. Our results show that preexposure to the trivalent arsenicals MMA(III) and ATO induces in corrected cells, a cellular FA/BRCA-deficient phenotype characterized by hypersensitivity, enhanced accumulation in the G2/M compartment and increased genomic instability--measured as micronuclei. Overall, our data demonstrate that environmentally relevant arsenic exposures disrupt the normal function of the FA/BRCA activity, supporting a novel source of arsenic co- and carcinogenic effects. This is the first study linking arsenic exposure with the FA/BRCA DNA repair pathway.

  2. The binding of strontium and europium by an aquatic fulvic acid--ion exchange distribution and ultrafiltration studies.

    PubMed

    Nordén, M; Ephhraim, J H; Allard, B

    1993-09-01

    The complexation of an aquatic fulvic acid, FA, with Sr(2+) and Eu(3+) was studied at 0.10 and O1.O1M NaClO(4) using trace levels of metal ([Sr(2+)] = 10(-9)M and [Eu(3+)] = 10(-11)M) and a constant FA concentration (0.12 g/l) by an ultrafiltration technique (UF) and an ion exchange distribution method (IEDS). The overall complex formation function, beta(OV) for the two metals was calculated and its dependence on pH, ionic strength and method was investigated. The absolute value of log beta(OV), the pH dependence and the influence of the ionic strength on the complexation differed depending on the metal ion and experimental technique employed. By considering the functional group heterogeneity of the FA molecule, it was possible to predict the most predominantly bound site (keto-enol) and resolve the complex formation function for this site and EU(3+) (IEDS: 9.43 +/- 0.29 l/eq at 0.10M and 10.58 +/- 0.72 l/eq at 0.01M; UF: 7.19 +/- 1.51 l/eq at 0.01M and 6.88 +/- 0.91 l/eq at 0.01M). The results are discussed in the light of possible intrinsic problems of the two experimental methods.

  3. 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

  4. TRIM.FaTE Evaluation Report

    EPA Pesticide Factsheets

    The TRIM.FaTE Evaluation Report is composed of three volumes. Volume I presents conceptual, mechanistic, and structural complexity evaluations of various aspects of the model. Volumes II and III present performance evaluation.

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

    SciTech Connect

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

    2016-01-01

    The development of technology to harvest the uranium dissolved in seawater would enable access to vast quantities of this critical metal for nuclear power generation. Amidoxime polymers are the most promising platforms for achieving this separation, yet the design of advanced adsorbents is hindered by uncertainty regarding the uranium binding mode. In this work we use XAFS to investigate the uranium coordination environment in an amidoxime–phosphonic acid copolymer adsorbent. In contrast to the binding mode predicted computationally and from small molecule studies, a cooperative chelating model is favoured, attributable to emergent behavior resulting from inclusion of amidoxime in the polymer. Samples exposed to seawater also display a feature consistent with a μ2-oxo-bridged transition metal, suggesting the formation of an in situ specific binding site. These findings challenge long held assumptions and provide new opportunities for the design of advanced adsorbent materials.

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

    DOE PAGES

    Abney, C. W.; Das, S.; Mayes, R. T.; ...

    2016-08-01

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

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

    SciTech Connect

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

    2016-08-01

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

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

    PubMed Central

    Giustiniano, Mariateresa; Agamennone, Mariangela; Rossello, Armando; Gomez-Monterrey, Isabel; Novellino, Ettore; Campiglia, Pietro; Vernieri, Ermelinda; Bertamino, Alessia; Carotenuto, Alfonso

    2013-01-01

    A number of matrix metalloproteinases (MMPs) are important medicinal targets for conditions ranging from rheumatoid arthritis to cardiomyopathy, periodontal disease, liver cirrhosis, multiple sclerosis, and cancer invasion and metastasis, where they showed to have a dual role, inhibiting or promoting important processes involved in the pathology. MMPs contain a zinc (II) ion in the protein active site. Small-molecule inhibitors of these metalloproteins are designed to bind directly to the active site metal ions. In an effort to devise new approaches to selective inhibitors, in this paper, we describe the synthesis and preliminary biological evaluation of amino acid derivatives as new zinc binding groups (ZBGs). The incorporation of selected metal-binding functions in more complex biphenyl sulfonamide moieties allowed the identification of one compound able to interact selectively with different MMP enzymatic isoforms. PMID:23555050

  11. Identification of cytosolic and microsomal bile acid-binding proteins in rat ileal enterocytes

    SciTech Connect

    Lin, M.C.; Kramer, W.; Wilson, F.A. )

    1990-09-05

    Studies were performed to determine the subcellular fractions and proteins involved in the intracellular transport of bile acids in rat ileal cells. The photolabile derivative 7,7-azo-taurocholate inhibited the Na(+)-dependent uptake of taurocholate into rat ileal enterocytes reversibly in the dark and irreversibly following photolysis. When photolabeled cells were submitted to subcellular fractionation, greatest radioactivity was found in the soluble protein (SP) fraction with decreasing radioactivity in the brush-border-(BBM), basolateral-(BLM), mitochondria-(MT), microsome-(MC), and Golgi-(GO) enriched fractions. Following trichloroacetic acid precipitation, delipidation, and correction for loss of marker enzyme activity, protein bound radioactivity was in SP greater than BBM greater than MC greater than BLM greater than GO greater than MT. When photolabeled cells were first fractionated and then submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a 99-kDa polypeptide was associated with BBM, 54- and 59-kDa polypeptides with BLM, 14-, 35-, 43-, 59-, and 68-kDa polypeptides with SP and a 20-kDa polypeptide with MC fractions. Immunoprecipitation with known antisera identified the 68-kDa polypeptide as albumin and the 43-kDa polypeptide as actin. No precipitation on the 14-kDa polypeptide was noted with anti-hepatic and anti-intestinal fatty acid-binding proteins. No precipitation of the 35-kDa polypeptide occurred with antibody to the hepatic cytosolic bile acid-binding protein. These studies reveal a previously unrecognized 20-kDa microsomal, and 14- and 35-kDa cytosolic bile acid-binding polypeptides which may be involved in the transcellular movement of bile acids.

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

    NASA Astrophysics Data System (ADS)

    Tofoleanu, Florentina; Lee, Juyong; Pickard, Frank C., IV; König, Gerhard; Huang, Jing; Baek, Minkyung; Seok, Chaok; Brooks, Bernard R.

    2017-01-01

    As part of the SAMPL5 blind prediction challenge, we calculate the absolute binding free energies of six guest molecules to an octa-acid (OAH) and to a methylated octa-acid (OAMe). We use the double decoupling method via thermodynamic integration (TI) or Hamiltonian replica exchange in connection with the Bennett acceptance ratio (HREM-BAR). We produce the binding poses either through manual docking or by using GalaxyDock-HG, a docking software developed specifically for this study. The root mean square deviations for our most accurate predictions are 1.4 kcal mol-1 for OAH with TI and 1.9 kcal mol-1 for OAMe with HREM-BAR. Our best results for OAMe were obtained for systems with ionic concentrations corresponding to the ionic strength of the experimental solution. The most problematic system contains a halogenated guest. Our attempt to model the σ-hole of the bromine using a constrained off-site point charge, does not improve results. We use results from molecular dynamics simulations to argue that the distinct binding affinities of this guest to OAH and OAMe are due to a difference in the flexibility of the host. We believe that the results of this extensive analysis of host-guest complexes will help improve the protocol used in predicting binding affinities for larger systems, such as protein-substrate compounds.

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

    PubMed

    Tarushi, Alketa; Psomas, George; Raptopoulou, Catherine P; Kessissoglou, Dimitris P

    2009-06-01

    The neutral mononuclear zinc complexes with the quinolone antibacterial drug oxolinic acid in the absence or presence of a nitrogen donor heterocyclic ligand 2,2'-bipyridine or 1,10-phenanthroline have been synthesized and characterized. The experimental data suggest that oxolinic acid is on deprotonated mode acting as a bidentate ligand coordinated to the metal ion through the ketone and one carboxylato oxygen atoms. The crystal structures of (chloro)(oxolinato)(2,2'-bipyridine)zinc(II), 2, and bis(oxolinato)(1,10-phenanthroline)zinc(II), 3, have been determined with X-ray crystallography. The biological activity of the complexes has been evaluated by examining their ability to bind to calf-thymus DNA (CT DNA) with UV and fluorescence spectroscopies. UV studies of the interaction of the complexes with DNA have shown that they can bind to CT DNA and the DNA-binding constants have been calculated. Competitive studies with ethidium bromide (EB) have shown that complex 3 exhibits the ability to displace the DNA-bound EB indicating that it binds to DNA in strong competition with EB.

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

    PubMed Central

    Briata, Paola; Bordo, Domenico; Puppo, Margherita; Gorlero, Franco; Rossi, Martina; Bizzozzero, Nora Perrone; Gherzi, Roberto

    2015-01-01

    The single-stranded nucleic acid binding protein KHSRP (KH-Type Splicing Regulatory Protein) modulates RNA life and gene expression at various levels. KHSRP controls important cellular functions as different as proliferation, differentiation, metabolism and response to infectious agents. We summarize and discuss experimental evidence providing a potential link between changes in KHSRP expression/function and human diseases including neuromuscular disorders, obesity, type II diabetes, and cancer. PMID:26708421

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

    PubMed

    Briata, Paola; Bordo, Domenico; Puppo, Margherita; Gorlero, Franco; Rossi, Martina; Perrone-Bizzozero, Nora; Gherzi, Roberto

    2016-01-01

    The single-stranded nucleic acid-binding protein KHSRP (KH-type splicing regulatory protein) modulates RNA life and gene expression at various levels. KHSRP controls important cellular functions as different as proliferation, differentiation, metabolism, and response to infectious agents. We summarize and discuss experimental evidence providing a potential link between changes in KHSRP expression/function and human diseases including neuromuscular disorders, obesity, type II diabetes, and cancer.

  16. 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.

  17. Correlation between carbohydrate-binding specificity and amino acid sequence of carbohydrate-binding regions of Cytisus-type anti-H(O) lectins.

    PubMed

    Konami, Y; Yamamoto, K; Osawa, T; Irimura, T

    1992-06-15

    A carbohydrate-binding peptide of the di-N-acetylchitobiose-binding Cytisus sessilifolius anti-H(O) lectin I (CSA-I) was isolated from the endoproteinase Asp-N digest of CSA-I by affinity chromatography on a column of N-acetyl-D-glucosamine oligomer-Sepharose (GlcNAc oligomer-Sepharose). The amino acid sequence of the carbohydrate-binding peptide of CSA-I was determined to be DTYFGKTYNPW using a gas-phase protein sequencer. This sequence corresponds to the sequence from Asp-129 to Trp-139 based on the primary structure of CSA-I, and shows a high degree of homology to those of the putative carbohydrate-binding peptide of the Laburnum alpinum lectin I (LAA-I) (DTYFGKAYNPW) and of the Ulex europaeus lectin II (UEA-II) (DSYFGKTYNPW). The binding of these three anti-H(O) lectins is known to be inhibited by di-N-acetylchitobiose but not by L-fucose. These results strongly suggest that there is a good correlation between the carbohydrate-binding specificity and the amino acid sequence of the carbohydrate-binding regions of di-N-acetylchitobiose-binding lectins.

  18. Identification and characterization of PshB, the dicluster ferredoxin that harbors the terminal electron acceptors F(A) and F(B) in Heliobacterium modesticaldum.

    PubMed

    Heinnickel, Mark; Shen, Gaozhong; Golbeck, John H

    2007-03-06

    The Type I homodimeric photosynthetic reaction center found in anaerobic gram-positive bacteria of the genus Heliobacteriaceae incorporates FA- and FB-like iron-sulfur clusters similar to those found in Photosystem I as terminal electron acceptors. We recently isolated the PshB protein that harbors the iron-sulfur clusters from the reaction centers of Heliobacterium modesticaldum. Here, we report the cloning of a candidate gene and the properties of its product. Genuine PshB was dissociated from the reaction center with 1 M NaCl and purified using an affinity strategy. After acquiring its N-terminal amino acid sequence, an fd2-like gene encoding a 5.5-kDa dicluster ferredoxin was identified as a candidate for PshB. The Fd2-like apoprotein was expressed in Escherichia coli with a His tag, and the Fe/S clusters were inserted using inorganic reagents. The optical absorbance and EPR spectra of the Fd2-like holoprotein were similar to those of genuine PshB. The Fd2-like holoprotein was coeluted with P798-FX cores on both G-75 gel filtration and Ni affinity columns. Consistent with binding, the EPR resonances at g = 2.067, 1.933, and 1.890 from [FA/FB]- were restored after illumination at 15 K, and the long-lived, room-temperature charge recombination kinetics between P798+ and [FA/FB]- reappeared on a laser flash. These characteristics indicate that the long-sought gene and polypeptide harboring the FA- and FB-like clusters in heliobacteria have been identified. The amino acid sequence of PshB indicates an entirely different mode of binding with the reaction center core than PsaC, its counterpart in Photosystem I.

  19. 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.

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

    PubMed

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

    2015-04-01

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

  1. 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.

  2. 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.

  3. 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.

  4. Binding modes of zaragozic acid A to human squalene synthase and staphylococcal dehydrosqualene synthase.

    PubMed

    Liu, Chia-I; Jeng, Wen-Yih; Chang, Wei-Jung; Ko, Tzu-Ping; Wang, Andrew H-J

    2012-05-25

    Zaragozic acids (ZAs) belong to a family of fungal metabolites with nanomolar inhibitory activity toward squalene synthase (SQS). The enzyme catalyzes the committed step of sterol synthesis and has attracted attention as a potential target for antilipogenic and antiinfective therapies. Here, we have determined the structure of ZA-A complexed with human SQS. ZA-A binding induces a local conformational change in the substrate binding site, and its C-6 acyl group also extends over to the cofactor binding cavity. In addition, ZA-A effectively inhibits a homologous bacterial enzyme, dehydrosqualene synthase (CrtM), which synthesizes the precursor of staphyloxanthin in Staphylococcus aureus to cope with oxidative stress. Size reduction at Tyr(248) in CrtM further increases the ZA-A binding affinity, and it reveals a similar overall inhibitor binding mode to that of human SQS/ZA-A except for the C-6 acyl group. These structures pave the way for further improving selectivity and development of a new generation of anticholesterolemic and antimicrobial inhibitors.

  5. Phosphatidic Acid Binds to Cytosolic Glyceraldehyde-3-phosphate Dehydrogenase and Promotes Its Cleavage in Arabidopsis *

    PubMed Central

    Kim, Sang-Chul; Guo, Liang; Wang, Xuemin

    2013-01-01

    Phosphatidic acid (PA) is a class of lipid messengers involved in a variety of physiological processes. To understand how PA mediates cell functions in plants, we used a PA affinity membrane assay to isolate PA-binding proteins from Camelina sativa followed by mass spectrometric sequencing. A cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC) was identified to bind to PA, and detailed analysis was carried out subsequently using GAPC1 and GAPC1 from Arabidopsis. The PA and GAPC binding was abolished by the cation zinc whereas oxidation of GAPCs promoted the PA binding. PA had little impact on the GAPC catalytic activity in vitro, but the PA treatment of Arabidopsis seedlings induced proteolytic cleavage of GAPC2 and inhibited Arabidopsis seedling growth. The extent of PA inhibition was greater in GAPC-overexpressing than wild-type seedlings, but the greater PA inhibition was abolished by application of zinc to the seedling. The PA treatment also reduced the expression of genes involved in PA synthesis and utilization, and the PA-reduced gene expression was partially recovered by zinc treatment. These data suggest that PA binds to oxidized GAPDH and promotes its cleavage and that the PA and GAPC interaction may provide a signaling link coordinating carbohydrate and lipid metabolism. PMID:23504314

  6. Study on the binding of chlorogenic acid to pepsin by spectral and molecular docking.

    PubMed

    Zeng, Hua-jin; Liang, Hui-li; You, Jing; Qu, Ling-bo

    2014-11-01

    The interaction of pepsin with chlorogenic acid (CHA) was investigated using fluorescence, UV/vis spectroscopy and molecular modeling methods. Stern-Volmer analysis indicated that the fluorescence quenching of pepsin by CHA resulted from a static mechanism, and the binding constant was 1.1846 × 10(5) and 1.1587 × 10(5) L/mol at 288 and 310 K, respectively. The distance between donor (pepsin) and acceptor (CHA) was calculated to be 2.39 nm and the number of binding sites for CHA binding on pepsin was ~ 1. The results of synchronous fluorescence and three-dimensional fluorescence showed that binding of CHA to pepsin could induce conformational changes in pepsin. Molecular docking experiments found that CHA bonded with pepsin in the area of the hydrophobic cavity with Van der Waals' forces or hydrogen bonding interaction, which were consistent with the results obtained from the thermodynamic parameter analysis. Furthermore, the binding of CHA can inhibit pepsin activity in vitro.

  7. 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.

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

    PubMed

    Meng, Junxiu; Yu, Shaoqing; Wan, Shengbiao; Ren, Sumei; Jiang, Tao

    2011-11-01

    A facile synthesis of a series of saccharide-binding arylboronic acid derivatives of indoloquinoline was described. The key synthetic steps were polyphosphoric acid-mediated cyclization, chlorinative aromatization, and amidation. Mass spectrometry experiments revealed these synthetic arylboronic acid derivatives of indoquinolines could bind to biologically important carbohydrates (sialic acid, fucose, glucose, and galactose) by forming boronate di-esters in alkaline aqueous solution. Most of the arylboronic acid derivatives of indoquinolines inhibited human breast cancer cell (MDA-231) proliferation at a concentration of 5 μm, whereas the compound 17 exhibited highest percentages (76.74%) of the cancer cell proliferation inhibition.

  9. Mutational Insights into the Roles of Amino Acid Residues in Ligand Binding for Two Closely Related Family 16 Carbohydrate Binding Modules

    SciTech Connect

    Su, Xiaoyun; Agarwal, Vinayak; Dodd, Dylan; Bae, Brian; Mackie, Roderick I.; Nair, Satish K.; Cann, Isaac K.O.

    2010-11-22

    Carbohydrate binding modules (CBMs) are specialized proteins that bind to polysaccharides and oligosaccharides. Caldanaerobius polysaccharolyticus Man5ACBM16-1/CBM16-2 bind to glucose-, mannose-, and glucose/mannose-configured substrates. The crystal structures of the two proteins represent the only examples in CBM family 16, and studies that evaluate the roles of amino acid residues in ligand binding in this family are lacking. In this study, we probed the roles of amino acids (selected based on CBM16-1/ligand co-crystal structures) on substrate binding. Two tryptophan (Trp-20 and Trp-125) and two glutamine (Gln-81 and Gln-93) residues are shown to be critical in ligand binding. Additionally, several polar residues that flank the critical residues also contribute to ligand binding. The CBM16-1 Q121E mutation increased affinity for all substrates tested, whereas the Q21G and N97R mutants exhibited decreased substrate affinity. We solved CBM/substrate co-crystal structures to elucidate the molecular basis of the increased substrate binding by CBM16-1 Q121E. The Gln-121, Gln-21, and Asn-97 residues can be manipulated to fine-tune ligand binding by the Man5A CBMs. Surprisingly, none of the eight residues investigated was absolutely conserved in CBM family 16. Thus, the critical residues in the Man5A CBMs are either not essential for substrate binding in the other members of this family or the two CBMs are evolutionarily distinct from the members available in the current protein database. Man5A is dependent on its CBMs for robust activity, and insights from this study should serve to enhance our understanding of the interdependence of its catalytic and substrate binding modules.

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

    PubMed

    Vidali, Roza; Remoundaki, Emmanouela; Tsezos, Marios

    2009-11-15

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

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

    PubMed

    Brody, A R; George, G; Hill, L H

    1983-10-01

    Chrysotile and crocidolite are commonly used forms of asbestos. Hemolysis has been widely used as a test of membrane injury, and it has been shown previously that chrysotile causes rapid breakdown of red blood cells (RBCs), whereas crocidolite is only weakly hemolytic. A reasonable hypothesis set forth to explain the cytotoxic effects of chrysotile maintains that positively charged chrysotile fibers bind to negatively charged sialic acid residues on RBC membranes causing clustering of membrane proteins and increased cell permeability to Na and K ions. Our studies presented here provide two lines of evidence in direct support of this hypothesis. (a) Morphologic--Ultrastructural techniques showed that both chrysotile and crocidolite asbestos bind to and distort more than 85% of RBCs treated for 15 minutes. The distorting effects of chrysotile, but not crocidolite, were almost totally ablated by pretreating the cells with neuraminidase. In addition, gold-conjugated wheat germ agglutinin was used to label the distribution of sialic acid groups on RBC membranes. Pretreatment of the RBCs with chrysotile, but not crocidolite, reduced the number of gold-conjugated wheat germ agglutinin-labeled sites to less than 30% of the control level. (b) Biochemical--The thiobarbituric acid assay was used to determine the percentage of sialic acid that remained with the cell pellet after neuraminidase and/or asbestos treatment. Asbestos treatment alone caused no release of sialic acid from the cells. Neuraminidase treatment for 3.5 hours removed more than 80% of the sialic acid from cell surfaces. Chrysotile, but not crocidolite, asbestos prevented neuraminidase-mediated removal of sialic acid from RBCs. In addition, x-ray energy spectrometry of freeze-dried cells showed that RBCs distorted by chrysotile, but not by crocidolite, exhibited significant alterations in intracellular Na:K ratios. The morphologic and biochemical data strongly support the hypothesis that chrysotile asbestos

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

    PubMed

    Viani, A; Cappiello, M; Pacifici, G M

    1991-01-01

    Albumin was isolated from pooled fetal serum obtained at normal delivery at term and from pooled adult plasma. Albumin isolation was carried out by means of PEG precipitation followed by ion exchange chromatography on DEAE-Sephadex A 50 and then on SP-Sephadex C 50. The binding of diazepam (1 microM), salicylic acid (2 mM) and digitoxin (6 nM) to albumin (40 g/l) was measured by equilibrium dialysis at 37 degrees C. The unbound fraction (mean +/- SD) for fetal and adult albumin of diazepam was 1.86 +/- 0.24 and 1.82 +/- 0.15% (NS), that of digitoxin was 3.18 +/- 0.27 and 3.36 +/- 0.04% (NS) and that of salicylic acid was 11.65 +/- 0.99 and 9.47 +/- 0.75% (p less than 0.05), respectively. With both fetal and adult albumin, a single class of binding sites was observed for diazepam and digitoxin, whereas two classes of binding sites were observed for salicylic acid. The number of binding sites (n, moles of drug per mole of albumin) for fetal and adult albumin was 0.83 and 1.02 for diazepam and 0.014 and 0.018 for digitoxin, respectively. For salicylic acid, n was 1.45 (fetal albumin) and 1.55 (adult albumin) for the higher affinity site, and 3.06 (fetal albumin) and 3.27 (adult albumin) for the lower affinity site. The association constant (Ka, M-1) for diazepam was 1.36 x 10(5) (fetal albumin) and 1.00 x 10(5) (adult albumin) and that for digitoxin was 4.12 x 10(6) (fetal albumin) and 2.7 x 10(6) (adult albumin). For salicylic acid, Ka was 38.4 x 10(3) (fetal albumin) and 35.8 x 10(3) (adult albumin) for the higher affinity site, and 2.7 x 10(3) (fetal albumin) and 4.3 x 10(3) (adult albumin) for the lower affinity site. This work shows that fetal and adult albumin have similar binding properties and corroborates our previous findings with furosemide.

  13. Regulation of AMPK Activation by CD36 Links Fatty Acid Uptake to β-Oxidation

    PubMed Central

    Sun, Jingyu; Pietka, Terri; Gross, Richard W.; Eckel, Robert H.; Su, Xiong; Stahl, Philip D.

    2015-01-01

    Increases in muscle energy needs activate AMPK and induce sarcolemmal recruitment of the fatty acid (FA) translocase CD36. The resulting rises in FA uptake and FA oxidation are tightly correlated, suggesting coordinated regulation. We explored the possibility that membrane CD36 signaling might influence AMPK activation. We show, using several cell types, including myocytes, that CD36 expression suppresses AMPK, keeping it quiescent, while it mediates AMPK activation by FA. These dual effects reflect the presence of CD36 in a protein complex with the AMPK kinase LKB1 (liver kinase B1) and the src kinase Fyn. This complex promotes Fyn phosphorylation of LKB1 and its nuclear sequestration, hindering LKB1 activation of AMPK. FA interaction with CD36 dissociates Fyn from the protein complex, allowing LKB1 to remain cytosolic and activate AMPK. Consistent with this, CD36−/− mice have constitutively active muscle and heart AMPK and enhanced FA oxidation of endogenous triglyceride stores. The molecular mechanism described, whereby CD36 suppresses AMPK, with FA binding to CD36 releasing this suppression, couples AMPK activation to FA availability and would be important for the maintenance of cellular FA homeostasis. Its dysfunction might contribute to the reported association of CD36 variants with metabolic complications of obesity in humans. PMID:25157091

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

    PubMed

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

    2017-03-16

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

  15. 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.

  16. From keys to bulldozers: expanding roles for winged helix domains in nucleic-acid-binding proteins.

    PubMed

    Harami, Gábor M; Gyimesi, Máté; Kovács, Mihály

    2013-07-01

    The winged helix domain (WHD) is a widespread nucleic-acid-binding protein structural element found in all kingdoms of life. Although the overall structure of the WHD is conserved, its functional properties and interaction profiles are extremely versatile. WHD-containing proteins can exploit nearly the full spectrum of nucleic acid structural features for recognition and even covalent modification or noncovalent rearrangement of target molecules. WHD functions range from sequence-recognizing keys in transcription factors and bulldozer-like strand-separating wedges in helicases to mediators of protein-protein interactions (PPIs). Further investigations are needed to understand the contribution of WHD structural dynamics to nucleic-acid-modifying enzymatic functions.

  17. 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

  18. Nucleic acid sensing with enzyme-DNA binding protein conjugates cascade and simple DNA nanostructures.

    PubMed

    Aktas, Gülsen Betül; Skouridou, Vasso; Masip, Lluis

    2017-03-22

    A versatile and universal DNA sensing platform is presented based on enzyme-DNA binding protein tags conjugates and simple DNA nanostructures. Two enzyme conjugates were thus prepared, with horseradish peroxidase linked to the dimeric single-chain bacteriophage Cro repressor protein (HRP-scCro) and glucose oxidase linked to the dimeric headpiece domain of Escherichia coli LacI repressor protein (GOx-dHP), and used in conjunction with a hybrid ssDNA-dsDNA detection probe. This probe served as a simple DNA nanostructure allowing first for target recognition through its target-complementary single-stranded DNA (ssDNA) part and then for signal generation after conjugate binding on the double-stranded DNA (dsDNA) containing the specific binding sites for the dHP and scCro DNA binding proteins. The DNA binding proteins chosen in this work have different sequence specificity, high affinity, and lack of cross-reactivity. The proposed sensing system was validated for the detection of model target ssDNA from high-risk human papillomavirus (HPV16) and the limits of detection of 45, 26, and 21 pM were achieved using the probes with scCro/dHP DNA binding sites ratio of 1:1, 2:1, and 1:2, respectively. The performance of the platform in terms of limit of detection was comparable to direct HRP systems using target-specific oligonucleotide-HRP conjugates. The ratio of the two enzymes can be easily manipulated by changing the number of binding sites on the detection probe, offering further optimization possibilities of the signal generation step. Moreover, since the signal is obtained in the absence of externally added hydrogen peroxide, the described platform is compatible with paper-based assays for molecular diagnostics applications. Finally, just by changing the ssDNA part of the detection probe, this versatile nucleic acid platform can be used for the detection of different ssDNA target sequences or in a multiplex detection configuration without the need to change any of the

  19. Single aromatic residue location alters nucleic acid binding and chaperone function of FIV nucleocapsid protein

    PubMed Central

    Wu, Hao; Wang, Wei; Naiyer, Nada; Fichtenbaum, Eric; Qualley, Dominic F.; McCauley, Micah J.; Gorelick, Robert J.; Rouzina, Ioulia; Musier-Forsyth, Karin; Williams, Mark C.

    2014-01-01

    Feline immunodeficiency virus (FIV) is a retrovirus that infects domestic cats, and is an excellent animal model for human immunodeficiency virus type 1 (HIV-1) pathogenesis. The nucleocapsid (NC) protein is critical for replication in both retroviruses. FIV NC has several structural features that differ from HIV-1 NC. While both NC proteins have a single conserved aromatic residue in each of the two zinc fingers, the aromatic residue on the second finger of FIV NC is located on the opposite C-terminal side relative to its location in HIV-1 NC. In addition, whereas HIV-1 NC has a highly charged cationic N-terminal tail and a relatively short C-terminal extension, the opposite is true for FIV NC. To probe the impact of these differences on the nucleic acid (NA) binding and chaperone properties of FIV NC, we carried out ensemble and single-molecule assays with wild-type (WT) and mutant proteins. The ensemble studies show that FIV NC binding to DNA is strongly electrostatic, with a higher effective charge than that observed for HIV-1 NC. The C-terminal basic domain contributes significantly to the NA binding capability of FIV NC. In addition, the non-electrostatic component of DNA binding is much weaker for FIV NC than for HIV-1 NC. Mutation of both aromatic residues in the zinc fingers to Ala (F12A/W44A) further increases the effective charge of FIV NC and reduces its non-electrostatic binding affinity. Interestingly, switching the location of the C-terminal aromatic residue to mimic the HIV-1 NC sequence (N31W/W44A) reduces the effective charge of FIV NC and increases its non-electrostatic binding affinity to values similar to HIV-1 NC. Consistent with the results of these ensemble studies, single-molecule DNA stretching studies show that while WT FIV NC has reduced stacking capability relative to HIV-1 NC, the aromatic switch mutant recovers the ability to intercalate between the DNA bases. Our results demonstrate that altering the position of a single aromatic

  20. Thermodynamic and solution state NMR characterization of the binding of secondary and conjugated bile acids to STARD5.

    PubMed

    Létourneau, Danny; Lorin, Aurélien; Lefebvre, Andrée; Cabana, Jérôme; Lavigne, Pierre; LeHoux, Jean-Guy

    2013-11-01

    STARD5 is a member of the STARD4 sub-family of START domain containing proteins specialized in the non-vesicular transport of lipids and sterols. We recently reported that STARD5 binds primary bile acids. Herein, we report on the biophysical and structural characterization of the binding of secondary and conjugated bile acids by STARD5 at physiological concentrations. We found that the absence of the 7α-OH group and its epimerization increase the affinity of secondary bile acids for STARD5. According to NMR titration and molecular modeling, the affinity depends mainly on the number and positions of the steroid ring hydroxyl groups and to a lesser extent on the presence or type of bile acid side-chain conjugation. Primary and secondary bile acids have different binding modes and display different positioning within the STARD5 binding pocket. The relative STARD5 affinity for the different bile acids studied is: DCA>LCA>CDCA>GDCA>TDCA>CA>UDCA. TCA and GCA do not bind significantly to STARD5. The impact of the ligand chemical structure on the thermodynamics of binding is discussed. The discovery of these new ligands suggests that STARD5 is involved in the cellular response elicited by bile acids and offers many entry points to decipher its physiological role.

  1. ATP binding cassette modulators control abscisic acid-regulated slow anion channels in guard cells

    PubMed Central

    Leonhardt, N; Vavasseur, A; Forestier, C

    1999-01-01

    In animal cells, ATP binding cassette (ABC) proteins are a large family of transporters that includes the sulfonylurea receptor and the cystic fibrosis transmembrane conductance regulator (CFTR). These two ABC proteins possess an ion channel activity and bind specific sulfonylureas, such as glibenclamide, but homologs have not been identified in plant cells. We recently have shown that there is an ABC protein in guard cells that is involved in the control of stomatal movements and guard cell outward K+ current. Because the CFTR, a chloride channel, is sensitive to glibenclamide and able to interact with K+ channels, we investigated its presence in guard cells. Potent CFTR inhibitors, such as glibenclamide and diphenylamine-2-carboxylic acid, triggered stomatal opening in darkness. The guard cell protoplast slow anion current that was recorded using the whole-cell patch-clamp technique was inhibited rapidly by glibenclamide in a dose-dependent manner; the concentration producing half-maximum inhibition was at 3 &mgr;M. Potassium channel openers, which bind to and act through the sulfonylurea receptor in animal cells, completely suppressed the stomatal opening induced by glibenclamide and recovered the glibenclamide-inhibited slow anion current. Abscisic acid is known to regulate slow anion channels and in our study was able to relieve glibenclamide inhibition of slow anion current. Moreover, in epidermal strip bioassays, the stomatal closure triggered by Ca2+ or abscisic acid was reversed by glibenclamide. These results suggest that the slow anion channel is an ABC protein or is tightly controlled by such a protein that interacts with the abscisic acid signal transduction pathway in guard cells. PMID:10368184

  2. Pharmacokinetics, tissue distribution, and plasma protein binding study of chicoric acid by HPLC-MS/MS.

    PubMed

    Wang, Yutang; Xie, Guo; Liu, Qian; Duan, Xiang; Liu, Zhigang; Liu, Xuebo

    2016-09-15

    Chicoric acid is a major active constituent of Echinacea purpurea and has a variety of biological functions. In this study, a liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) approach was developed and validated for the determination of chicoric acid in rat plasma and various tissues using ferulic acid as an internal standard (IS). This method was successfully applied to pharmacokinetics, tissue distribution, and plasma protein binding (PPB) study of chicoric acid in Sprague-Dawley (SD) rats dosed with 50mg/kg by gastric gavage. The pharmacokinetic parameters were determined and showed a half-life (t1/2) of 4.53±1.44h, an apparent volume of mean residual time (MRT) of 18.58±4.43h, and an area under the curve (AUC) of 26.14 mghL(-1). The tissue distribution of chicoric acid in rats after gavage administration showed a decreasing tendency in different tissues (liver>lung>kidney>heart>spleen>brain). The PPB rates in rat plasma, human plasma, and bovine serum albumin were 98.3, 96.9, and 96.6%, respectively. These results provide insight for the further pharmacological investigation of chicoric acid.

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

    PubMed

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

    2017-03-13

    The excellent performance of functionalized iron oxide nanoparticles (IONPs) in nanomaterial and biomedical applications often relies on achieving the attachment of ligands to the iron oxide surface both in sufficient number and with proper orientation. Toward this end, we determine relationships between the ligand chemical structure and surface binding on magnetic IONPs for a series of related benzoic acid and catechol derivatives. Ligand exchange was used to introduce the model ligands, and the resultant nanoparticles were characterized using Fourier transform infrared-attenuated internal reflectance spectroscopy, transmission electron microscopy, and nanoparticle solubility behavior. An in-depth analysis of ligand electronic effects and reaction conditions reveals that the nature of ligand binding does not solely depend on the presence of functional groups known to bind to IONPs. The structure of the resultant ligand-surface complex was primarily influenced by the relative positioning of hydroxyl and carboxylic acid groups within the ligand and whether or not HCl(aq) was added to the ligand-exchange reaction. Overall, this study will help guide future ligand-design and ligand-exchange strategies toward realizing truly custom-built IONPs.

  4. Evidence for pentagalloyl glucose binding to human salivary alpha-amylase through aromatic amino acid residues.

    PubMed

    Gyémánt, Gyöngyi; Zajácz, Agnes; Bécsi, Bálint; Ragunath, Chandran; Ramasubbu, Narayanan; Erdodi, Ferenc; Batta, Gyula; Kandra, Lili

    2009-02-01

    We demonstrate here that pentagalloyl glucose (PGG), a main component of gallotannins, was an effective inhibitor of HSA and it exerted similar inhibitory potency to Aleppo tannin used in this study. The inhibition of HSA by PGG was found to be non-competitive and inhibitory constants of K(EI)=2.6 microM and K(ESI)=3.9 microM were determined from Lineweaver-Burk secondary plots. PGG as a model compound for gallotannins was selected to study the inhibitory mechanism and to characterize the interaction of HSA with this type of molecules. Surface plasmon resonance (SPR) binding experiments confirmed the direct interaction of HSA and PGG, and it also established similar binding of Aleppo tannin to HSA. Saturation transfer difference (STD) experiment by NMR clearly demonstrated the aromatic rings of PGG may be involved in the interaction suggesting a possible stacking with the aromatic side chains of HSA. The role of aromatic amino acids of HSA in PGG binding was reinforced by kinetic studies with the W58L and Y151M mutants of HSA: the replacement of the active site aromatic amino acids with aliphatic ones decreased the PGG inhibition dramatically, which justified the importance of these residues in the interaction.

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

    PubMed

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

    2016-01-01

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

  6. Analysis of amino acid sequence variations and immunoglobulin E-binding epitopes of German cockroach tropomyosin.

    PubMed

    Jeong, Kyoung Yong; Lee, Jongweon; Lee, In-Yong; Ree, Han-Il; Hong, Chein-Soo; Yong, Tai-Soon

    2004-09-01

    The allergenicities of tropomyosins from different organisms have been reported to vary. The cDNA encoding German cockroach tropomyosin (Bla g 7) was isolated, expressed, and characterized previously. In the present study, the amino acid sequence variations in German cockroach tropomyosin were analyzed in order to investigate its influence on allergenicity. We also undertook the identification of immunodominant peptides containing immunoglobulin E (IgE) epitopes which may facilitate the development of diagnostic and immunotherapeutic strategies based on the recombinant proteins. Two-dimensional gel electrophoresis and immunoblot analysis with mouse anti-recombinant German cockroach tropomyosin serum was performed to investigate the isoforms at the protein level. Reverse transcriptase PCR (RT-PCR) was applied to examine the sequence diversity. Eleven different variants of the deduced amino acid sequences were identified by RT-PCR. German cockroach tropomyosin has only minor sequence variations that did not seem to affect its allergenicity significantly. These results support the molecular basis underlying the cross-reactivities of arthropod tropomyosins. Recombinant fragments were also generated by PCR, and IgE-binding epitopes were assessed by enzyme-linked immunosorbent assay. Sera from seven patients revealed heterogeneous IgE-binding responses. This study demonstrates multiple IgE-binding epitope regions in a single molecule, suggesting that full-length tropomyosin should be used for the development of diagnostic and therapeutic reagents.

  7. Inosine 5'-monophosphate dehydrogenase binds nucleic acids in vitro and in vivo.

    PubMed Central

    McLean, Jeremy E; Hamaguchi, Nobuko; Belenky, Peter; Mortimer, Sarah E; Stanton, Martin; Hedstrom, Lizbeth

    2004-01-01

    Inosine 5'-monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in the de novo biosynthesis of guanine nucleotides. In addition to the catalytic domain, IMPDH contains a subdomain of unknown function composed of two cystathione beta-synthase domains. Our results, using three different assays, show that IMPDHs from Tritrichomonas foetus, Escherichia coli, and both human isoforms bind single-stranded nucleic acids with nanomolar affinity via the subdomain. Approx. 100 nucleotides are bound per IMPDH tetramer. Deletion of the subdomain decreases affinity 10-fold and decreases site size to 60 nucleotides, whereas substitution of conserved Arg/Lys residues in the subdomain with Glu decreases affinity by 20-fold. IMPDH is found in the nucleus of human cells, as might be expected for a nucleic-acid-binding protein. Lastly, immunoprecipitation experiments show that IMPDH binds both RNA and DNA in vivo. These experiments indicate that IMPDH has a previously unappreciated role in replication, transcription or translation that is mediated by the subdomain. PMID:14766016

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

    PubMed

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

    2016-03-01

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

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

    PubMed

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

    2015-09-11

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

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

    PubMed

    Kizilbash, Nadeem A; Hai, Abdul; Alruwaili, Jamal

    2013-01-01

    The β-sheet of muscle fatty acid binding protein of Locusta migratoria (Lm-FABP) was modeled by employing 2-D NMR data and the Rigid Body Assembly method. The model shows the β-sheet to comprise ten β-strands arranged anti-parallel to each other. There is a β-bulge between Ser 13 and Gln 14 which is a difference from the published structure of β-sheet of bovine heart Fatty Acid Binding Protein. Also, a hydrophobic patch consisting of Ile 45, Phe 51, Phe 64 and Phe 66 is present on the surface which is characteristic of most Fatty Acid Binding Proteins. A "gap" is present between βD and βE that provides evidence for the presence of a portal or opening between the polypeptide chains which allows ligand fatty acids to enter the protein cavity and bind to the protein.

  11. 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.

  12. Isolation of dicarboxylic acid- and glucose-binding proteins from Pseudomonas aeruginosa.

    PubMed Central

    Stinson, M W; Cohen, M A; Merrick, J M

    1976-01-01

    Inducible binding proteins for C4-dicarboxylic acids (DBP) and glucose (GBP) were isolated from Pseudomonas aeruginosa by extraction of exponential-phase cells with 0.2 M MgC12 (pH 8.5) and by an osmotic shock procedure without affecting cell viability. DBP synthesis was induced by growth on aspartate, alpha-ketoglutarate, succinate, fumarate, malate, and malonate but not by growth on acetate, citrate, pyruvate, or glucose. Binding of succinate by DBP was competitively inhibited by 10-fold concentrations of fumarate and malate but not by a variety of related substances. GBP synthesis and transport of methyl alpha-glucoside by whole cells were induced by growth on glucose or pyruvate plus galactose, 2-deoxyglucose, or methyl alpha-glucoside but not by growth on gluconate, succinate, acetate, or pyruvate. The binding of radioactive glucose by GBP was significantly inhibited by 10-fold concentrations of glucose, galactose, and glucose-1-phosphate but not by the other carbohydrates tested. The binding of glucose by GBP or succinate by DBP did not result in any chemical alteration of the substrates. PMID:824281

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

    PubMed

    Soskić, Milan; Magnus, Volker

    2007-07-01

    The plant hormone, indole-3-acetic acid (IAA), and its ring-substituted derivatives have recently attracted attention as promising pro-drugs in cancer therapy. Here we present relative binding constants to human serum albumin for IAA and 34 of its derivatives, as obtained using the immobilized protein bound to a support suitable for high-performance liquid chromatography. We also report their octanol-water partition coefficients (logK(ow)) computed from retention data on a C(18) coated silica gel column. A four-parameter QSPR (quantitative structure-property relationships) model, based on physico-chemical properties, is put forward, which accounts for more than 96% of the variations in the binding affinities of these compounds. The model confirms the importance of lipophilicity as a global parameter governing interaction with serum albumin, but also assigns significant roles to parameters specifically related to the molecular topology of ring-substituted IAAs. Bulky substituents at ring-position 6 increase affinity, those at position 2 obstruct binding, while no steric effects were noted at other ring-positions. Electron-withdrawing substituents at position 5 enhance binding, but have no obvious effect at other ring positions.

  14. Binding of thyroglobulin to bovine thyroid membranes. Role of specific amino acids in receptor recognition.

    PubMed

    Shifrin, S; Kohn, L D

    1981-10-25

    Bovine thyroglobulin was treated with increasing ratios of succinic anhydride, trinitrobenzene sulfonic acid, tetranitromethane, and N-acetylimidazole in an attempt to assess the role of lysine or tyrosine residues in binding to thyroid membrane receptors. Extensive succinylation results in dissociation to 12 S thyroglobulin with retention of a considerable portion of the three-dimensional structure. Only 25% of the lysine residues can be modified by trinitrophenylation without affecting inter-subunit interactions. Succinylation as well as trinitrophenylation increases the affinity of thyroglobulin for the membrane receptor by a factor of 2. The binding of thyroglobulin to the membrane was reduced after nitration of 30% of the tyrosyl residues with tetranitromethane. O-Acetylation of 40-70% of the tyrosyl residues by N-acetylimidazole nearly abolished the ability of thyroglobulin to bind to the membrane. Removal of the O-acetyl group with hydroxylamine restored the binding properties. The results indicate that tyrosyl residues play an important role in thyroglobulin interactions with thyroid membranes.

  15. 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.

  16. 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.

  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. 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.

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

    PubMed Central

    2011-01-01

    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. PMID:22027239

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

    SciTech Connect

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

    1991-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Marsac, Rémi; Davranche, Mélanie; Gruau, Gérard; Dia, Aline

    2010-03-01

    The effect of metal loading on the binding of rare earth elements (REE) to humic acid (HA) was studied by combining ultrafiltration and Inductively Coupled Plasma Mass Spectrometry techniques. REE-HA complexation experiments were performed at pH 3 for REE/C molar ratios ranging from ca 4 × 10 -4 to 2.7 × 10 -2. Results show that the relative amount of REE bound to HA strongly increases with decreasing REE/C. A middle-REE (MREE) downward concavity is shown by patterns at high metal loading, whereas patterns at low metal loading display a regular increase from La to Lu. Humic Ion Model VI modelling are close to the experimental data variations, provided that (i) the ΔLK 2 parameter (i.e. the Model VI parameter taken into account the presence of strong but low density binding sites) is allowed to increase regularly from La to Lu (from 1.1 to 2.1) and (ii) the published log KMA values (i.e. the REE-HA binding constants specific to Model VI) are slightly modified, in particular with respect to heavy REE. Modelling approach provided evidence that logKdREE patterns with varying REE/C likely arises because REE binding to HA occurs through two types of binding sites in different density: (i) a few strong sites that preferentially complex the heavy REE and thus control the logKdREE atterns at low REE/C; (ii) a larger amount of weaker binding sites that preferentially complex the middle-REE and thus control the logKdREE pattern at high REE/C. Hence, metal loading exerts a major effect on HA-mediated REE binding, which could explain the diversity of published conditional constants for REE binding with HA. A literature survey suggests that the few strong sites activated at low REE/C could be multidentate carboxylic sites, or perhaps N-, or P-functional groups. Finally, an examination of the literature field data proposed that the described loading effect could account for much of the variation in REE patterns observed in natural organic-rich waters (DOC > 5 mg L -1 and 4

  2. Amino acid-based ionic liquids: using XPS to probe the electronic environment via binding energies.

    PubMed

    Hurisso, Bitu Birru; Lovelock, Kevin R J; Licence, Peter

    2011-10-21

    Here we report the synthesis and characterisation by X-ray photoelectron spectroscopy (XPS) of eight high purity amino acid-based ionic liquids (AAILs), each containing the 1-octyl-3-methylimidazolium, [C(8)C(1)Im](+), as a standard reference cation. All expected elements were observed and the electronic environments of these elements identified. A fitting model for the carbon 1s region of the AAILs is reported; the C aliphatic component of the cation was used as an internal reference to obtain a series of accurate and reproducible binding energies. Comparisons are made between XP spectra of the eight AAILs and selected non-functionalised ionic liquids. 1-octyl-3-methylimidazolium acetate was also studied as a model of the carboxyl containing amino acid anion. The influence of anionic substituent groups on the measured binding energies of all elements is presented, and communication between anion and cation is investigated. This data is interpreted in terms of hard and soft anions and compared to the Kamlet-Taft hydrogen bond acceptor ability, β, for the ionic liquids. A linear correlation is presented which suggests that the functional side chain, or R group, of the amino acid has little impact upon the electronic environment of the charge-bearing moieties within the anions and cations studied.

  3. cDNA structure, expression and nucleic acid-binding properties of three RNA-binding proteins in tobacco: occurrence of tissue-specific alternative splicing.

    PubMed Central

    Hirose, T; Sugita, M; Sugiura, M

    1993-01-01

    Three cDNAs encoding RNA-binding proteins were isolated from a tobacco (Nicotiana sylvestris) cDNA library. The predicted proteins (RGP-1) are homologous to each other and consist of a consensus-sequence type RNA-binding domain of 80 amino acids in the N-terminal half and a glycine-rich domain of 61-78 amino acids in the C-terminal half. Nucleic acid-binding assay using the in vitro synthesized RGP-1 protein confirmed that it is an RNA-binding protein. Based on its strong affinity for poly(G) and poly(U), the RGP-1 proteins are suggested to bind specifically to G and/or U rich sequences. All three genes are expressed in leaves, roots, flowers and cultured cells, however, the substantial amount of pre-mRNAs are accumulated especially in roots. Sequence analysis and ribonuclease protection assay indicated that significant amounts of alternatively spliced mRNAs, which are produced by differential selection of 5' splice sites, are also present in various tissues. Tissue-specific alternative splicing was found in two of the three genes. The alternatively spliced mRNAs are also detected in polysomal fractions and are suggested to produce truncated polypeptides. A possible role of this alternative splicing is discussed. Images PMID:8371974

  4. 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.

  5. Screening nucleotide binding to amino acid-coated supports by surface plasmon resonance and nuclear magnetic resonance.

    PubMed

    Cruz, Carla; Cabrita, Eurico J; Queiroz, João A

    2011-08-01

    Here, we describe a rapid and efficient screening method using surface plasmon resonance (SPR) and saturation transfer difference-nuclear magnetic resonance (STD-NMR) spectroscopy to yield information regarding the residues involved in nucleotide binding to amino acid-coated supports. The aim of this work was to explore the use of these spectroscopic techniques to study amino acid-nucleotide interactions in order to improve the binding specificity of the amino acid ligands used to purify plasmid DNA. For SPR, we present a strategy that immobilizes arginine and lysine on a surface as model supports, and we analyze binding responses when synthetic homo-deoxyoligonucleotides are injected over the amino acid surface. The binding responses are detectable and reproducible despite the small size of the immobilized amino acids. Using STD-NMR, we performed epitope mapping of homo-deoxyoligonucleotides bound to L-arginine-bisoxyran-Sepharose and L-lysine-Sepharose supports. Polynucleotide binding preferences differed; for example, polyC interacted preferentially through its backbone with the two supports, whereas polyT bound the supports through its thymine moiety. STD-NMR combined with SPR measurements was successfully used to screen amino acid-nucleotide interactions and determine the binding affinities of the complexes.

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

    PubMed

    Albani, Jihad R

    2004-02-25

    Binding of hemin to alpha1-acid glycoprotein has been investigated. Hemin binds to the hydrophobic pocket of hemoproteins. The fluorescent probe 2-(p-toluidino)-6-naphthalenesulfonate (TNS) binds to a hydrophobic domain in alpha1-acid glycoprotein with a dissociation constant equal to 60 microM. Addition of hemin to an alpha1-acid glycoprotein-TNS complex induces the displacement of TNS from its binding site. At saturation (1 hemin for 1 protein) all the TNS has been displaced from its binding site. The dissociation constant of hemin-alpha1-acid glycoprotein was found equal to 2 microM. Thus, TNS and hemin bind to the same hydrophobic site: the pocket of alpha1-acid glycoprotein. Energy-transfer studies performed between the Trp residues of alpha1-acid glycoprotein and hemin indicated that efficiency (E) of Trp fluorescence quenching was equal to 80% and the Förster distance, R0 at which the efficiency of energy transfer is 50% was calculated to be 26 A, revealing a very high energy transfer.

  7. Catalytic and ligand binding properties of the FK506 binding protein FKBP12: effects of the single amino acid substitution of Tyr82 to Leu.

    PubMed Central

    Bossard, M J; Bergsma, D J; Brandt, M; Livi, G P; Eng, W K; Johnson, R K; Levy, M A

    1994-01-01

    The binding of FK506 and rapamycin to their cytosolic receptor FKBP12 is an intermediate step in the paths leading to their potent immunosuppressive properties. One of the amino acids defining the hydrophobic binding cleft for the macrocycles is Tyr82, which is thought to form a hydrogen bond with the amide oxygens of the common pipecolyl structural element within the two macrolides. To understand better the influence of this amino acid residue in catalytic activity (cis-trans peptidyl prolyl isomerization) and ligand binding properties, a Tyr82 to Leu site-specific modification of FKBP12 was prepared, purified and characterized. Kinetic experiments have demonstrated that the Tyr82 to Leu modification has a greater effect on catalytic properties than on ligand binding affinities, a result which indicates that these inhibitors may not be binding as true transition-state analogues. In an additional test for cellular function, expression of both wild-type and mutant human FKBP12 in a strain of Saccharomyces cerevisiae rendered resistant to rapamycin by deletion of the gene encoding a cytosolic rapamycin binding protein (RPB1), the yeast homologue of FKBP12, restored wild-type drug sensitivity. PMID:7507662

  8. d-Amino Acid Probes for Penicillin Binding Protein-based Bacterial Surface Labeling*

    PubMed Central

    Fura, Jonathan M.; Kearns, Daniel; Pires, Marcos M.

    2015-01-01

    Peptidoglycan is an essential and highly conserved mesh structure that surrounds bacterial cells. It plays a critical role in retaining a defined cell shape, and, in the case of pathogenic Gram-positive bacteria, it lies at the interface between bacterial cells and the host organism. Intriguingly, bacteria can metabolically incorporate unnatural d-amino acids into the peptidoglycan stem peptide directly from the surrounding medium, a process mediated by penicillin binding proteins (PBPs). Metabolic peptidoglycan remodeling via unnatural d-amino acids has provided unique insights into peptidoglycan biosynthesis of live bacteria and has also served as the basis of a synthetic immunology strategy with potential therapeutic implications. A striking feature of this process is the vast promiscuity displayed by PBPs in tolerating entirely unnatural side chains. However, the chemical space and physical features of this side chain promiscuity have not been determined systematically. In this report, we designed and synthesized a library of variants displaying diverse side chains to comprehensively establish the tolerability of unnatural d-amino acids by PBPs in both Gram-positive and Gram-negative organisms. In addition, nine Bacillus subtilis PBP-null mutants were evaluated with the goal of identifying a potential primary PBP responsible for unnatural d-amino acid incorporation and gaining insights into the temporal control of PBP activity. We empirically established the scope of physical parameters that govern the metabolic incorporation of unnatural d-amino acids into bacterial peptidoglycan. PMID:26499795

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

    PubMed

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

    2013-10-01

    The terminal monosaccharide of glycoconjugates on a eukaryotic cell surface is typically a sialic acid (Neu5Ac). Increased sialylation usually indicates progression and poor prognosis of most carcinomas. Here, we utilize two human mammary epithelial cell lines, HB4A (breast normal cells) and T47D (breast cancer cells), as a model system to demonstrate differential surface glycans when treated with sialic acid under nutrient deprivation. Under a starved condition, sialic acid treatment of both cells resulted in increased activities of α2→3/6 sialyltransferases as demonstrated by solid phase assay using lectin binding. However, a very strong Maackia amurensis agglutinin I (MAL-I) staining on the membrane of sialic acid-treated T47D cells was observed, indicating an increase of Neu5Acα2→3Gal on the cell surface. To our knowledge, this is a first report showing the utility of lectins, particularly MAL-I, as a means to discriminate between normal and cancer cells after sialic acid treatment under nutrient deprivation. This method is sensitive and allows selective detection of glycan sialylation on a cancer cell surface.

  10. Betulin binds to gamma-aminobutyric acid receptors and exerts anticonvulsant action in mice.

    PubMed

    Muceniece, Ruta; Saleniece, Kristine; Rumaks, Juris; Krigere, Liga; Dzirkale, Zane; Mezhapuke, Rudolfs; Zharkova, Olga; Klusa, Vija

    2008-10-01

    The lupane type pentacyclic triterpenes: lupeol, betulin, and betulinic acid are widely distributed natural compounds. Recently, pharmaceutical compositions from plant extracts (family Marcgraviaceae) containing betulinic acid, have been patented as anxiolytic remedies. To extend our knowledge of the CNS effects of the triterpenes, we suggest here that the chemically related lupeol, betulin and betulinic acid may interact with the brain neurotransmitter gamma-aminobutyric acid (GABA) receptors in vitro and in vivo. Using radioligand receptor-binding assay, we showed that only betulin bound to the GABA(A)-receptor sites in mice brain in vitro and antagonised the GABA(A)-receptor antagonist bicuculline-induced seizures in mice after intracisternal and intraperitoneal administration. Neither betulinic acid nor lupeol bound to GABA(A) receptor nor did they inhibit bicuculline-induced seizures in vivo. These findings demonstrate for the first time the CNS effects of betulin in vivo, and they also show distinct GABA(A)-receptor-related properties of lupane type triterpenes. These findings may open new avenues in understanding the central effects of betulin, and they also indicate possibilities for novel drug design on the basis of betulin structure.

  11. In situ detection of salicylic acid binding sites in plant tissues.

    PubMed

    Liu, Jing-Wen; Deng, Da-Yi; Yu, Ying; Liu, Fang-Fei; Lin, Bi-Xia; Cao, Yu-Juan; Hu, Xiao-Gang; Wu, Jian-Zhong

    2015-02-01

    The determination of hormone-binding sites in plants is essential in understanding the mechanisms behind hormone function. Salicylic acid (SA) is an important plant hormone that regulates responses to biotic and abiotic stresses. In order to label SA-binding sites in plant tissues, a quantum dots (QDs) probe functionalized with a SA moiety was successfully synthesized by coupling CdSe QDs capped with 3-mercaptopropionic acid (MPA) to 4-amino-2-hydroxybenzoic acid (PAS), using 1-ethyl-3-(3-dimethyllaminopropyl) carbodiimide (EDC) as the coupling agent. The probe was then characterized by dynamic light scattering and transmission electron microscopy, as well as UV/vis and fluorescence spectrophotometry. The results confirmed the successful conjugation of PAS to CdSe QDs and revealed that the conjugates maintained the properties of the original QDs, with small core diameters and adequate dispersal in solution. The PAS-CdSe QDs were used to detect SA-binding sites in mung bean and Arabidopsis thaliana seedlings in vitro and in vivo. The PAS-CdSe QDs were effectively transported into plant tissues and specifically bound to SA receptors in vivo. In addition, the effects of the PAS-CdSe QDs on cytosolic Ca(2+) levels in the tips of A. thaliana seedlings were investigated. Both SA and PAS-CdSe QDs had similar effects on the trend in cytosolic-free Ca(2+) concentrations, suggesting that the PAS-CdSe QDs maintained the bioactivity of SA. To summarize, PAS-CdSe QDs have high potential as a fluorescent probe for the in vitro/in vivo labeling and imaging of SA receptors in plants.

  12. 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.

  13. Structural analysis of site-directed mutants of cellular retinoic acid-binding protein II addresses the relationship between structural integrity and ligand binding

    SciTech Connect

    Vaezeslami, Soheila; Jia, Xiaofei; Vasileiou, Chrysoula; Borhan, Babak; Geiger, James H.

    2008-12-01

    A water network stabilizes the structure of cellular retionic acid binding protein II. The structural integrity of cellular retinoic acid-binding protein II (CRABPII) has been investigated using the crystal structures of CRABPII mutants. The overall fold was well maintained by these CRABPII mutants, each of which carried multiple different mutations. A water-mediated network is found to be present across the large binding cavity, extending from Arg111 deep inside the cavity to the α2 helix at its entrance. This chain of interactions acts as a ‘pillar’ that maintains the integrity of the protein. The disruption of the water network upon loss of Arg111 leads to decreased structural integrity of the protein. A water-mediated network can be re-established by introducing the hydrophilic Glu121 inside the cavity, which results in a rigid protein with the α2 helix adopting an altered conformation compared with wild-type CRABPII.

  14. Binding of Autotaxin to Integrins Localizes Lysophosphatidic Acid Production to Platelets and Mammalian Cells*

    PubMed Central

    Fulkerson, Zachary; Wu, Tao; Sunkara, Manjula; Kooi, Craig Vander; Morris, Andrew J.; Smyth, Susan S.

    2011-01-01

    Autotaxin (ATX) is a secreted lysophospholipase D that generates the bioactive lipid mediator lysophosphatidic acid (LPA). We and others have reported that ATX binds to integrins, but the function of ATX-integrin interactions is unknown. The recently reported crystal structure of ATX suggests a role for the solvent-exposed surface of the N-terminal tandem somatomedin B-like domains in binding to platelet integrin αIIbβ3. The opposite face of the somatomedin B-like domain interacts with the catalytic phosphodiesterase (PDE) domain to form a hydrophobic channel through which lysophospholipid substrates enter and leave the active site. Based on this structure, we hypothesize that integrin-bound ATX can access cell surface substrates and deliver LPA to cell surface receptors. To test this hypothesis, we investigated the integrin selectivity and signaling pathways that promote ATX binding to platelets. We report that both platelet β1 and β3 integrins interact in an activation-dependent manner with ATX via the SMB2 domain. ATX increases thrombin-stimulated LPA production by washed platelets ∼10-fold. When incubated under conditions to promote integrin activation, ATX generates LPA from CHO cells primed with bee venom phospholipase A2, and ATX-mediated LPA production is enhanced more than 2-fold by CHO cell overexpression of integrin β3. The effects of ATX on platelet and cell-associated LPA production, but not hydrolysis of small molecule or detergent-solubilized substrates, are attenuated by point mutations in the SMB2 that impair integrin binding. Integrin binding therefore localizes ATX activity to the cell surface, providing a mechanism to generate LPA in the vicinity of its receptors. PMID:21832043

  15. Molecular Characterization of a Strawberry FaASR Gene in Relation to Fruit Ripening

    PubMed Central

    Jiang, Yue-ming; Zhao, Ming-lei; Shan, Wei; Kuang, Jian-fei; Lu, Wang-jin

    2011-01-01

    Background ABA-, stress- and ripening-induced (ASR) proteins have been reported to act as a downstream component involved in ABA signal transduction. Although much attention has been paid to the roles of ASR in plant development and stress responses, the mechanisms by which ABA regulate fruit ripening at the molecular level are not fully understood. In the present work, a strawberry ASR gene was isolated and characterized (FaASR), and a polyclonal antibody against FaASR protein was prepared. Furthermore, the effects of ABA, applied to two different developmental stages of strawberry, on fruit ripening and the expression of FaASR at transcriptional and translational levels were investigated. Methodology/Principal Findings FaASR, localized in the cytoplasm and nucleus, contained 193 amino acids and shared common features with other plant ASRs. It also functioned as a transcriptional activator in yeast with trans-activation activity in the N-terminus. During strawberry fruit development, endogenous ABA content, levels of FaASR mRNA and protein increased significantly at the initiation of ripening at a white (W) fruit developmental stage. More importantly, application of exogenous ABA to large green (LG) fruit and W fruit markedly increased endogenous ABA content, accelerated fruit ripening, and greatly enhanced the expression of FaASR transcripts and the accumulation of FaASR protein simultaneously. Conclusions These results indicate that FaASR may be involved in strawberry fruit ripening. The observed increase in endogenous ABA content, and enhanced FaASR expression at transcriptional and translational levels in response to ABA treatment might partially contribute to the acceleration of strawberry fruit ripening. PMID:21915355

  16. Clinical benefit using sperm hyaluronic acid binding technique in ICSI cycles: a systematic review and meta-analysis.

    PubMed

    Beck-Fruchter, Ronit; Shalev, Eliezer; Weiss, Amir

    2016-03-01

    The human oocyte is surrounded by hyaluronic acid, which acts as a natural selector of spermatozoa. Human sperm that express hyaluronic acid receptors and bind to hyaluronic acid have normal shape, minimal DNA fragmentation and low frequency of chromosomal aneuploidies. Use of hyaluronic acid binding assays in intracytoplasmic sperm injection (ICSI) cycles to improve clinical outcomes has been studied, although none of these studies had sufficient statistical power. In this systematic review and meta-analysis, electronic databases were searched up to June 2015 to identify studies of ICSI cycles in which spermatozoa able to bind hyaluronic acid was selected. The main outcomes were fertilization rate and clinical pregnancy rate. Secondary outcomes included cleavage rate, embryo quality, implantation rate, spontaneous abortion and live birth rate. Seven studies and 1437 cycles were included. Use of hyaluronic acid binding sperm selection technique yielded no improvement in fertilization and pregnancy rates. A meta-analysis of all available studies showed an improvement in embryo quality and implantation rate; an analysis of prospective studies only showed an improvement in embryo quality. Evidence does not support routine use of hyaluronic acid binding assays in all ICSI cycles. Identification of patients that might benefit from this technique needs further study.

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

    DOE PAGES

    Kashyap, Sanjay; Woehl, Taylor; Valverde-Tercedor, Carmen; ...

    2014-01-01

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

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

    PubMed

    Zhang, Renshuai; Chen, Shaopeng; Wang, Xueting; Yu, Rilei; Li, Mingjing; Ren, Sumei; Jiang, Tao

    2016-06-24

    Novel DNA cyclo-intercalators, which incorporated two intercalator subunits linked by two bridges, were synthesized. Binding of the compounds to calf-thymus DNA was studied by fluorescence spectroscopy, and docking simulations were used to predict the binding modes of these cyclic compounds. The spectral data demonstrated that all of these compounds can interact with CT-DNA. The sugar moiety played an important role in the process of binding between the intercalators containing glucuronic acid and DNA. The length and flexibility of the connecting bridges affected the binding affinity of the resultant cyclo-intercalators. Docking simulations showed that compounds 7 and 8 interact with DNA as mono-intercalators.

  19. 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...

  20. Expression of fatty acid binding proteins is altered in aged mouse brain.

    PubMed

    Pu, L; Igbavboa, U; Wood, W G; Roths, J B; Kier, A B; Spener, F; Schroeder, F

    1999-08-01

    Brain membrane lipid fatty acid composition and consequently membrane fluidity change with increasing age. Intracellular fatty acid binding proteins (FABPs) such as heart H-FABP and the brain specific B-FABP, detected by immunoblotting of brain tissue, are thought to be involved in fatty acid uptake, metabolism, and differentiation in brain. Yet, almost nothing is known regarding the effect of age on the expression of the cytosolic fatty acid binding proteins (FABPs) or their content in brain subfractions. Electrophoresis and quantitative immunoblotting were used to examine the content of these FABPs in synaptosomes in brains from 4, 15, and 25 month old C57BL/6NNia male mice. Brain H-FABP and B-FABP were differentially expressed in mouse brain subcellular fractions. Brain H-FABP was highly concentrated in synaptosomal cytosol. The level of brain H-FABP in synaptosomes, synaptosomal cytosol, and intrasynaptosomal membranes was decreased 33, 35, and 43%, respectively, in 25 month old mice. B-FABP was detected in lower quantity than H-FABP. More important, B-FABP decreased in synaptosomes, synaptic plasma membranes, and synaptosomal cytosol from brains of 25 month old mice. In contrast to H-FABP, B-FABP was not detectable in the intrasynaptosomal membranes in any of the three age groups of mice. In conclusion, expression of both H-FABP and B-FABP was markedly reduced in aged mouse brain. Age differences in brain H-FABP and B-FABP levels in synaptosomal plasma membranes and synaptosomal cytosol may be important factors modulating neuronal differentiation and function.

  1. 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.

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

    PubMed

    Leontiev, V V; Uversky, V N; Permyakov, E A; Murzin, A G

    1993-03-05

    The 51-62 loop of T4 phage lysozyme was altered by site-directed mutagenesis to obtain maximal homology with the typical EF-hand motif. A Ca(2+)-binding site was designed and created by replacing both Gly-51 and Asn-53 with aspartic acid. The mutant T4 lysozyme (G51D/N53D) was expressed in Escherichia coli. The activity of the G51D/N53D-mutant was about 60% of that of the wild-type protein. This mutant can bind Ca2+ ions specifically, while the effective dissociation constant was essentially greater than that of the EF-hand proteins. Stability of the G51D/N53D-mutant apo-form to urea- or temperature-induced denaturation was the same as that of the wild-type protein. In the presence of Ca2+ ions in solution the stability of the mutant T4 phage lysozyme was less than that of the wild-type protein. It is suggested that the binding of Ca2+ by the mutant is accompanied by the considerable conformational changes in the 'corrected' loop, which can lead to the Ca(2+)-induced destabilization of the protein.

  3. Competitive Binding to Cuprous Ions of Protein and BCA in the Bicinchoninic Acid Protein Assay

    PubMed Central

    Huang, Tao; Long, Mian; Huo, Bo

    2010-01-01

    Although Bicinchoninic acid (BCA) has been widely used to determine protein concentration, the mechanism of interaction between protein, copper ion and BCA in this assay is still not well known. Using the Micro BCA protein assay kit (Pierce Company), we measured the absorbance at 562 nm of BSA solutions with different concentrations of protein, and also varied the BCA concentration. When the concentration of protein was increased, the absorbance exhibited the known linear and nonlinear increase, and then reached an unexpected plateau followed by a gradual decrease. We introduced a model in which peptide chains competed with BCA for binding to cuprous ions. Formation of the well-known chromogenic complex of BCA-Cu1+-BCA was competed with the binding of two peptide bonds (NTPB) to cuprous ion, and there is the possibility of the existence of two new complexes. A simple equilibrium equation was established to describe the correlations between the substances in solution at equilibrium, and an empirical exponential function was introduced to describe the reduction reaction. Theoretical predictions of absorbance from the model were in good agreement with the measurements, which not only validated the competitive binding model, but also predicted a new complex of BCA-Cu1+-NTPB that might exist in the final solution. This work provides a new insight into understanding the chemical bases of the BCA protein assay and might extend the assay to higher protein concentration. PMID:21625379

  4. 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.

  5. Competitive Binding to Cuprous Ions of Protein and BCA in the Bicinchoninic Acid Protein Assay.

    PubMed

    Huang, Tao; Long, Mian; Huo, Bo

    2010-01-01

    Although Bicinchoninic acid (BCA) has been widely used to determine protein concentration, the mechanism of interaction between protein, copper ion and BCA in this assay is still not well known. Using the Micro BCA protein assay kit (Pierce Company), we measured the absorbance at 562 nm of BSA solutions with different concentrations of protein, and also varied the BCA concentration. When the concentration of protein was increased, the absorbance exhibited the known linear and nonlinear increase, and then reached an unexpected plateau followed by a gradual decrease. We introduced a model in which peptide chains competed with BCA for binding to cuprous ions. Formation of the well-known chromogenic complex of BCA-Cu(1+)-BCA was competed with the binding of two peptide bonds (NTPB) to cuprous ion, and there is the possibility of the existence of two new complexes. A simple equilibrium equation was established to describe the correlations between the substances in solution at equilibrium, and an empirical exponential function was introduced to describe the reduction reaction. Theoretical predictions of absorbance from the model were in good agreement with the measurements, which not only validated the competitive binding model, but also predicted a new complex of BCA-Cu(1+)-NTPB that might exist in the final solution. This work provides a new insight into understanding the chemical bases of the BCA protein assay and might extend the assay to higher protein concentration.

  6. JPRS Report Science & Technology Japan Future Prospects of FA-From FA to IMS

    DTIC Science & Technology

    2007-11-02

    STATEMENT A Approved lor public release; . Distribution Unlimited aa "lbAoa iaod sszs £0T 5S3D0ad sNiib 51? inzz 19980701 126 DTIC QUALITY INSPECTED...5 4. Tasks and Future Prospects of FA 13 5. Proposals 21 6. "Status of FA" Survey and Analysis 24 7.FA-related Vocabulary 31...came to about ¥ 13 trillion. This plant investment included such things as investment in new operations and R&D investment; investment for the purpose

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

    PubMed

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

    2016-11-01

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

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

    PubMed Central

    Maiti, Motilal; Kumar, Gopinatha Suresh

    2010-01-01

    Bioactive alkaloids occupy an important position in applied chemistry and play an indispensable role in medicinal chemistry. Amongst them, isoquinoline alkaloids like berberine, palmatine and coralyne of protoberberine group, sanguinarine of the benzophenanthridine group, and their derivatives represent an important class of molecules for their broad range of clinical and pharmacological utility. In view of their extensive occurrence in various plant species and significantly low toxicities, prospective development and use of these alkaloids as effective anticancer agents are matters of great current interest. This review has focused on the interaction of these alkaloids with polymorphic nucleic acid structures (B-form, A-form, Z-form, HL-form, triple helical form, quadruplex form) and their topoisomerase inhibitory activity reported by several research groups using various biophysical techniques like spectrophotometry, spectrofluorimetry, thermal melting, circular dichroism, NMR spectroscopy, electrospray ionization mass spectroscopy, viscosity, isothermal titration calorimetry, differential scanning calorimetry, molecular modeling studies, and so forth, to elucidate their mode and mechanism of action for structure-activity relationships. The DNA binding of the planar sanguinarine and coralyne are found to be stronger and thermodynamically more favoured compared to the buckled structure of berberine and palmatine and correlate well with the intercalative mechanism of sanguinarine and coralyne and the partial intercalation by berberine and palmatine. Nucleic acid binding properties are also interpreted in relation to their anticancer activity. PMID:20814427

  9. 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

  10. 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

  11. 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.

  12. A putative carbohydrate-binding domain of the lactose-binding Cytisus sessilifolius anti-H(O) lectin has a similar amino acid sequence to that of the L-fucose-binding Ulex europaeus anti-H(O) lectin.

    PubMed

    Konami, Y; Yamamoto, K; Osawa, T; Irimura, T

    1995-04-01

    The complete amino acid sequence of a lactose-binding Cytisus sessilifolius anti-H(O) lectin II (CSA-II) was determined using a protein sequencer. After digestion of CSA-II with endoproteinase Lys-C or Asp-N, the resulting peptides were purified by reversed-phase high performance liquid chromatography (HPLC) and then subjected to sequence analysis. Comparison of the complete amino acid sequence of CSA-II with the sequences of other leguminous seed lectins revealed regions of extensive homology. The amino acid sequence of a putative carbohydrate-binding domain of CSA-II was found to be similar to those of several anti-H(O) leguminous lectins, especially to that of the L-fucose-binding Ulex europaeus lectin I (UEA-I).

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

    PubMed

    Lü, Chenchen; Liu, Zhen

    2015-01-01

    The affinity of boronic acids toward cis-diol-containing biomolecules has found wide applications in many fields, such as sensing, separation, drug delivery, and functional materials. A sound understanding of the binding interactions will greatly facilitate exquisite applications of this chemistry. Traditional techniques are associated with some apparent drawbacks, so they are only applicable to a limited range of boronic acids and cis-diol-containing biomolecules. This chapter describes an affinity capillary electrophoresis (ACE) method for the characterization of the binding strengths between boronic acids and cis-diol-containing biomolecules. As compared with existing approaches, such as (11)B NMR, the ACE method exhibits several significant advantages: (1) possibility of simultaneous study of multiple interactions, (2) low requirement on the purity of the binding species, (3) widely applicable to almost all types of cis-diol-containing compounds and boronic acids, and (4) high accuracy and precision.

  14. 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.

  15. 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

  16. 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

  17. Dietary t10,c12-CLA but not c9,t11 CLA reduces adipocyte size in the absence of changes in the adipose renin-angiotensin system in fa/fa Zucker rats.

    PubMed

    DeClercq, Vanessa; Zahradka, Peter; Taylor, Carla G

    2010-11-01

    In obesity, increased activity of the local renin-angiotensin system (RAS) and enlarged adipocytes with altered adipokine production are linked to the development of obesity-related health problems and cardiovascular disease. Mixtures of conjugated linoleic acid (CLA) isomers have been shown to reduce adipocyte size and alter the production of adipokines. The objective of this study was to investigate the effects of feeding individual CLA isomers on adipocyte size and adipokines associated with the local adipose RAS. Male fa/fa Zucker rats received either (a) control, (b) cis(c)9,trans(t)11-CLA, or (c) t10,c12-CLA diet for 8 weeks. The t10,c12-CLA isomer reduced adipocyte size and increased cell number in epididymal adipose tissue. RT-PCR and Western blot analysis revealed that neither CLA isomer altered mRNA or protein levels of angiotensinogen or AngII receptors in adipose tissue. Likewise, levels of the pro-inflammatory cytokines TNF-α and IL-6 or the anti-inflammatory cytokine IL-10 were unchanged in adipose tissue. Similarly, neither CLA isomer had any effect on phosphorylation nor DNA binding of NF-κB. Our results suggest that although the t10,c12-CLA isomer had beneficial effects on reducing adipocyte size in obese rats, this did not translate into changes in the local adipose RAS or associated adipokines.

  18. 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

  19. Fatty acid binding protein facilitates sarcolemmal fatty acid transport but not mitochondrial oxidation in rat and human skeletal muscle

    PubMed Central

    Holloway, Graham P; Lally, Jamie; Nickerson, James G; Alkhateeb, Hakam; Snook, Laelie A; Heigenhauser, George J F; Calles-Escandon, Jorge; Glatz, Jan F C; Luiken, Joost J F P; Spriet, Lawrence L; Bonen, Arend

    2007-01-01

    The transport of long-chain fatty acids (LCFAs) across mitochondrial membranes is regulated by carnitine palmitoyltransferase I (CPTI) activity. However, it appears that additional fatty acid transport proteins, such as fatty acid translocase (FAT)/CD36, influence not only LCFA transport across the plasma membrane, but also LCFA transport into mitochondria. Plasma membrane-associated fatty acid binding protein (FABPpm) is also known to be involved in sacrolemmal LCFA transport, and it is also present on the mitochondria. At this location, it has been identified as mitochondrial aspartate amino transferase (mAspAT), despite being structurally identical to FABPpm. Whether this protein is also involved in mitochondrial LCFA transport and oxidation remains unknown. Therefore, we have examined the ability of FABPpm/mAspAT to alter mitochondrial fatty acid oxidation. Muscle contraction increased (P < 0.05) the mitochondrial FAT/CD36 content in rat (+22%) and human skeletal muscle (+33%). By contrast, muscle contraction did not alter the content of mitochondrial FABPpm/mAspAT protein in either rat or human muscles. Electrotransfecting rat soleus muscles, in vivo, with FABPpm cDNA increased FABPpm protein in whole muscle (+150%; P < 0.05), at the plasma membrane (+117%; P < 0.05) and in mitochondria (+80%; P < 0.05). In these FABPpm-transfected muscles, palmitate transport into giant vesicles was increased by +73% (P < 0.05), and fatty acid oxidation in intact muscle was increased by +18% (P < 0.05). By contrast, despite the marked increase in mitochondrial FABPpm/mAspAT protein content (+80%), the rate of mitochondrial palmitate oxidation was not altered (P > 0.05). However, electrotransfection increased mAspAT activity by +70% (P < 0.05), and the mitochondrial FABPpm/mAspAT protein content was significantly correlated with mAspAT activity (r= 0.75). It is concluded that FABPpm has two distinct functions depending on its subcellular location: (a) it contributes to

  20. A single amino acid change humanizes long-chain fatty acid binding and activation of mouse peroxisome proliferator-activated receptor α

    PubMed Central

    Oswal, Dhawal P.; Alter, Gerald M.; Rider, S. Dean; Hostetler, Heather A.

    2014-01-01

    Peroxisome proliferator-activated receptor α (PPARα) is an important regulator of hepatic lipid metabolism which functions through ligand binding. Despite high amino acid sequence identity (>90%), marked differences in PPARα ligand binding, activation and gene regulation have been noted across species. Similar to previous observations with synthetic agonists, we have recently reported differences in ligand affinities and extent of activation between human PPARα (hPPARα) and mouse PPARα (mPPARα) in response to long chain fatty acids (LCFA). The present study was aimed to determine if structural alterations could account for these differences. The binding of PPARα to LCFA was examined through in silico molecular modeling and docking simulations. Modeling suggested that variances at amino acid position 272 are likely to be responsible for differences in saturated LCFA binding to hPPARα and mPPARα. To confirm these results experimentally, LCFA binding, circular dichroism, and transactivation studies were performed using a F272I mutant form of mPPARα. Experimental data correlated with in silico docking simulations, further confirming the importance of amino acid 272 in LCFA binding. Although the driving force for evolution of species differences at this position are yet unidentified, this study enhances our understanding of ligand-induced regulation by PPARα and demonstrates the efficacy of molecular modeling and docking simulations. PMID:24858253

  1. 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.

  2. 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.

  3. 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.

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

    PubMed Central

    Amal, Ismail; Lutzing, Régis; Stote, Roland H.; Rochette-Egly, Cécile; Rochel, Natacha; Dejaegere, Annick

    2017-01-01

    Retinoic acid (RA) plays key roles in cell differentiation and growth arrest through nuclear retinoic acid receptors (RARs), which are ligand-dependent transcription factors. While the main trigger of RAR activation is the binding of RA, phosphorylation of the receptors has also emerged as an important regulatory signal. Phosphorylation of the RARγ N-terminal domain (NTD) is known to play a functional role in neuronal differentiation. In this work, we investigated the phosphorylation of RARγ ligand binding domain (LBD), and present evidence that the phosphorylation status of the LBD affects the phosphorylation of the NTD region. We solved the X-ray structure of a phospho-mimetic mutant of the LBD (RARγ S371E), which we used in molecular dynamics simulations to characterize the consequences of the S371E mutation on the RARγ structural dynamics. Combined with simulations of the wild-type LBD, we show that the conformational equilibria of LBD salt bridges (notably R387-D340) are affected by the S371E mutation, which likely affects the recruitment of the kinase complex that phosphorylates the NTD. The molecular dynamics simulations also showed that a conservative mutation in this salt bridge (R387K) affects the dynamics of the LBD without inducing large conformational changes. Finally, cellular assays showed that the phosphorylation of the NTD of RARγ is differentially regulated by retinoic acid in RARγWT and in the S371N, S371E and R387K mutants. This multidisciplinary work highlights an allosteric coupling between phosphorylations of the LBD and the NTD of RARγ and supports the importance of structural dynamics involving electrostatic interactions in the regulation of RARs activity. PMID:28125680

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

    PubMed

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

    2017-02-16

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

  6. Amino acids outside of the loops that define the agonist binding site are important for ligand binding to insect nicotinic acetylcholine receptors.

    PubMed

    Liu, Zewen; Han, Zhaojun; Liu, Shuhua; Zhang, Yixi; Song, Feng; Yao, Xiangmei; Gu, Jianhua

    2008-07-01

    Nicotinic acetylcholine (ACh) receptors (nAChRs) are the targets of several kinds of insecticides. Based on the mutagenesis studies of Torpedo californica nAChRs and solved structure of a molluscan, glial-derived soluble ACh-binding protein, a model of the agonist site was constructed with contributing amino acids from three distinct loops (A, B, and C) of the alpha subunits and another three loops (D, E, and F) of the non-alpha subunits. According to this model, most insect nAChR subunits can form the functional heteromeric or homomeric receptors. Actually, insect subunits themselves did not form any functional receptor at various combinations as yet, and only part of them can form the functional receptors with vertebrate non-alpha subunits. These findings suggested that the agonist binding for insect nAChRs was not only contributed by those key amino acids in six loops, but also some unidentified amino acids from other regions. In our previous studies on nAChRs for Nilaparvata lugens, a target-site mutation (Y151S) was found within two alpha subunits (Nlalpha1 and Nlalpha3). In Drosophila S2 cells and Xenopus oocytes, Nlalpha1 can form functional receptors with rat beta2 subunit. However, the same thing was not observed in Nlalpha3. In the present paper, by exchanging the corresponding regions between Nlalpha1 and Nlalpha3 to generate different chimeras, amino acid residues or residue clusters in the regions outside the six loops were found to play essential roles in agonist binding, especially for the amino acid clusters between loop B and C. This result indicated that the residues in the six loops could be necessary, but not enough for the activity of agonist binding.

  7. Uncoupling of gamma-aminobutyric acid B receptors from GTP-binding proteins by N-ethylmaleimide: effect of N-ethylmaleimide on purified GTP-binding proteins

    SciTech Connect

    Asano, T.; Ogasawara, N.

    1986-03-01

    Treatment of membranes from bovine cerebral cortex with N-ethylmaleimide (NEM) resulted in inhibition of gamma-aminobutyric acid (GABA) binding to GABAB receptors. The binding curve for increasing concentrations of agonist was shifted to the right by NEM treatment. Guanine nucleotide had little effect on the binding of GABA to NEM-treated membranes. The addition of purified GTP-binding proteins, which were the substrates of islet-activating protein (IAP), pertussis toxin, to the NEM-treated membranes caused a shift of the binding curve to the left, suggesting modification of GTP-binding proteins rather than receptors by NEM. The effect of NEM on two purified GTP-binding proteins, Gi (composed of three subunits with molecular weight of alpha, 41,000; beta, 35,000; gamma, 10,000) and Go (alpha, 39,000; beta, 35,000; gamma, 10,000) was studied. NEM did not significantly change guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) binding and GTPase activity of these two proteins. NEM-treated Gi and Go were not ADP-ribosylated by IAP and did not increase GABA binding to NEM-treated membranes. When alpha and beta gamma subunits were treated with NEM and then mixed with nontreated alpha and beta gamma to form Gi or Go, respectively, both oligomers with NEM-treated alpha-subunits lost their abilities to be IAP substrates and to couple to receptors. Results indicate that NEM uncoupled GTP-binding proteins from receptors by modifying alpha-subunits of GTP-binding proteins, and the site seemed to be on or near the site of ADP-ribosylation by IAP. When alpha and beta gamma subunits were treated with NEM and then mixed to form Gi or Go, GTP gamma S binding in the absence of Mg2+ and GTPase activity were changed, although they were not affected when oligomers were treated with NEM. Results suggest the existence of another sulfhydryl group which is protected from NEM by the association of subunits.

  8. Chemically binding carboxylic acids onto TiO2 nanoparticles with adjustable coverage by solvothermal strategy.

    PubMed

    Qu, Qiyun; Geng, Hongwei; Peng, Ruixiang; Cui, Qi; Gu, Xiaohong; Li, Fanqing; Wang, Mingtai

    2010-06-15

    This paper presents a solvothermal strategy for chemical modification of TiO(2) nanoparticles with carboxylic acids. Solvothermal reaction between the TiO(2) nanoparticles and carboxylic acid molecules in an autoclave at 100 degrees C provides carboxylic acid-modified TiO(2) particles with a modification efficiency much higher than the conventional immersion method. TiO(2) nanoparticles were prepared by hydrolysis of titanium isopropoxide in nitric acid solution; the modified nanoparticles were characterized by powder X-ray diffraction pattern, scanning electron microscopy, absorption and Fourier transform infrared spectra, and thermogravimetric analysis. Results show that the binding form of the modifier molecules on TiO(2) surface is in a bidentate chelating mode, the crystalline phase composition and morphological structure of the preformed TiO(2) nanoparticles are not affected by the solvothermal reaction, and the surface coverage of the modifier molecules can be adjusted by the weight ratio of modifier/TiO(2) during feeding. It is evident that the reaction processes in the solvothermal strategy involve the formation of double hydrogen bondings between carboxylic acid molecule and TiO(2) at the same Ti site and the coordination at solvothermal temperature by dehydration from the hydrogen bondings. The solvothermal strategy for modifying TiO(2) nanoparticles is expected to find potential applications in many fields; for example, our results demonstrate that the photovoltaic performance of the TiO(2) nanoparticles can be improved by the solvothermal modification even with an insulating modifier and controlled by the modifier coverage.

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

    PubMed Central

    Shubeita, H E; Sambrook, J F; McCormick, A M

    1987-01-01

    A recombinant cDNA clone, pCRABP-HS1, encoding cellular retinoic acid-binding protein was isolated from a bovine adrenal cDNA library. COS-7 cells transfected with pCRABP-HS1 produced a biologically active retinoic acid-binding protein molecule of the expected molecular mass (15.5 kDa). RNA blot hybridization analysis using pCRABP-HS1 as a probe revealed a single 1050-nucleotide mRNA species in bovine adrenal, uterus, and testis, tissues that contain the highest levels of retinoic acid-binding activity. No hybridization was detected in RNA extracted from ovary, spleen, kidney, or liver, which contain relatively low levels of cellular retinoic acid-binding protein activity. Analysis of genomic clones isolated from an EcoRI bovine genomic library demonstrated that the bovine cellular retinoic acid-binding protein gene is composed of four exons and three introns. Two putative promoter sequences were identified in the cloned 5' sequence of the gene. Images PMID:3039499

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

    PubMed

    Shubeita, H E; Sambrook, J F; McCormick, A M

    1987-08-01

    A recombinant cDNA clone, pCRABP-HS1, encoding cellular retinoic acid-binding protein was isolated from a bovine adrenal cDNA library. COS-7 cells transfected with pCRABP-HS1 produced a biologically active retinoic acid-binding protein molecule of the expected molecular mass (15.5 kDa). RNA blot hybridization analysis using pCRABP-HS1 as a probe revealed a single 1050-nucleotide mRNA species in bovine adrenal, uterus, and testis, tissues that contain the highest levels of retinoic acid-binding activity. No hybridization was detected in RNA extracted from ovary, spleen, kidney, or liver, which contain relatively low levels of cellular retinoic acid-binding protein activity. Analysis of genomic clones isolated from an EcoRI bovine genomic library demonstrated that the bovine cellular retinoic acid-binding protein gene is composed of four exons and three introns. Two putative promoter sequences were identified in the cloned 5' sequence of the gene.

  11. Selective binding of C-6 OH sulfated hyaluronic acid to the angiogenic isoform of VEGF(165).

    PubMed

    Lim, Dong-Kwon; Wylie, Ryan G; Langer, Robert; Kohane, Daniel S

    2016-01-01

    Vascular endothelial growth factor 165 (VEGF165) is an important extracellular protein involved in pathological angiogenesis in diseases such as cancer, wet age-related macular degeneration (wet-AMD) and retinitis pigmentosa. VEGF165 exists in two different isoforms: the angiogenic VEGF165a, and the anti-angiogenic VEGF165b. In some angiogenic diseases the proportion of VEGF165b may be equal to or higher than that of VEGF165a. Therefore, developing therapeutics that inhibit VEGF165a and not VEGF165b may result in greater anti-angiogenic activity and therapeutic benefit. To this end, we report the selective binding properties of sulfated hyaluronic acid (s-HA). Selective biopolymers offer several advantages over antibodies or aptamers including cost effective and simple synthesis, and the ability to make nanoparticles or hydrogels for drug delivery applications or VEGF165a sequestration. Limiting sulfation to the C-6 hydroxyl (C-6 OH) in the N-acetyl-glucosamine repeat unit of hyaluronic acid (HA) resulted in a polymer with strong affinity for VEGF165a but not VEGF165b. Increased sulfation beyond the C-6 OH (i.e. greater than 1 sulfate group per HA repeat unit) resulted in s-HA polymers that bound both VEGF165a and VEGF165b. The C-6 OH sulfated HA (Mw 150 kDa) showed strong binding properties to VEGF165a with a fast association rate constant (Ka; 2.8 × 10(6) M(-1) s(-1)), slow dissociation rate constant (Kd; 2.8 × 10(-3) s(-1)) and strong equilibrium binding constant (KD; ∼1.0 nM)), which is comparable to the non-selective VEGF165 binding properties of the commercialized therapeutic anti-VEGF antibody (Avastin(®)). The C-6 OH sulfated HA also inhibited human umbilical vein endothelial cell (HUVEC) survival and proliferation and human dermal microvascular endothelial cell (HMVEC) tube formation. These results demonstrate that the semi-synthetic natural polymer, C-6 OH sulfated HA, may be a promising biomaterial for the treatment of angiogenesis

  12. 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

  13. Unfolding pathway in red kidney bean acid phosphatase is dependent on ligand binding.

    PubMed

    Cashikar, A G; Rao, N M

    1996-03-01

    Structural basis for ligand-induced protein stabilization was investigated in the case of an acid phosphatase (red kidney bean purple acid phosphatase (KBPAP)) from red kidney bean. Phosphate, a physiological ligand, increases the stability against solvent denaturation by 3.5 kcal/mol. Generality of phosphate stabilization was shown by similar effects with other KBPAP ligands viz. adenosine 5'-O-(thiotriphosphate), a nonhydrolyzable ligand, and arsenate, an inhibitor. The dissociation constant of phosphate obtained from denaturation curves matches with the dissociation constant estimated by conventional methods. The guanidinium chloride-mediated denaturation of KBPAP was monitored by several structural and functional parameters viz. activity, tryptophan fluorescence, 8-anilinonaphthalene 1-sulfonic acid binding, circular dichroism, and size exclusion chromatography, in the presence and absence of 10 mm phosphate. In the presence of phosphate, profiles of all the parameters shift to a higher guanidinium chloride concentration. Noncoincidence of these profiles in the absence of phosphate indicates multistate unfolding pathway for KBPAP; however, in the presence of phosphate, KBPAP unfolds with a single intermediate. Based on the crystal structure, we propose that the Arg258 may have an important role to play in stabilization mediated by phosphate.

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

    PubMed

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

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

  15. Structure of malonic acid-based inhibitors bound to human neutrophil collagenase. A new binding mode explains apparently anomalous data.

    PubMed Central

    Brandstetter, H.; Engh, R. A.; Von Roedern, E. G.; Moroder, L.; Huber, R.; Bode, W.; Grams, F.

    1998-01-01

    Matrix metalloproteinases (MMPs) are a family of zinc endopeptidases, which have been implicated in various disease processes. Various classes of MMP inhibitors, including hydroxamic acids, phosphinic acids, and thiols, have been previously described. Most of these mimic peptides, and most likely bind analogous to the corresponding peptide substrates. Among the hydroxamic acids, malonic acid derivatives have been used as MMP inhibitors, although optimization of their inhibition potency was not successful. Here we report the design of malonic acid-based inhibitors using the X-ray structure of a collagenase/inhibitor complex, which revealed a nonsubstrate-like binding mode. The proposed beta-type turn-like conformation for the improved inhibitors was confirmed by X-ray crystallography. The observation of nonsubstrate-like binding confirms the original strategy for structure-based modeling of improved malonic acid inhibitors, and explains kinetic data that are inconsistent with substrate-like binding. Detailed interactions for the improved inhibitors seen in the crystal structure also suggest possibilities for further modifications in cycles of structure based drug design. Indeed, we have designed nonpeptidic inhibitors with approximately 500-fold improved inhibition based on these structures. PMID:9655333

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

    PubMed Central

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

    2015-01-01

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

  17. 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.

  18. The Human CCHC-type Zinc Finger Nucleic Acid-Binding Protein Binds G-Rich Elements in Target mRNA Coding Sequences and Promotes Translation.

    PubMed

    Benhalevy, Daniel; Gupta, Sanjay K; Danan, Charles H; Ghosal, Suman; Sun, Hong-Wei; Kazemier, Hinke G; Paeschke, Katrin; Hafner, Markus; Juranek, Stefan A

    2017-03-21

    The CCHC-type zinc finger nucleic acid-binding protein (CNBP/ZNF9) is conserved in eukaryotes and is essential for embryonic development in mammals. It has been implicated in transcriptional, as well as post-transcriptional, gene regulation; however, its nucleic acid ligands and molecular function remain elusive. Here, we use multiple systems-wide approaches to identify CNBP targets and function. We used photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) to identify 8,420 CNBP binding sites on 4,178 mRNAs. CNBP preferentially bound G-rich elements in the target mRNA coding sequences, most of which were previously found to form G-quadruplex and other stable structures in vitro. Functional analyses, including RNA sequencing, ribosome profiling, and quantitative mass spectrometry, revealed that CNBP binding did not influence target mRNA abundance but rather increased their translational efficiency. Considering that CNBP binding prevented G-quadruplex structure formation in vitro, we hypothesize that CNBP is supporting translation by resolving stable structures on mRNAs.

  19. 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.

  20. 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

  1. 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.

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

    PubMed

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

    2011-02-01

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

  3. Lysophosphatidic acid directly activates TRPV1 through a C-terminal binding site.

    PubMed

    Nieto-Posadas, Andrés; Picazo-Juárez, Giovanni; Llorente, Itzel; Jara-Oseguera, Andrés; Morales-Lázaro, Sara; Escalante-Alcalde, Diana; Islas, León D; Rosenbaum, Tamara

    2011-11-20

    Since 1992, there has been growing evidence that the bioactive phospholipid lysophosphatidic acid (LPA), whose amounts are increased upon tissue injury, activates primary nociceptors resulting in neuropathic pain. The TRPV1 ion channel is expressed in primary afferent nociceptors and is activated by physical and chemical stimuli. Here we show that in control mice LPA produces acute pain-like behaviors, which are substantially reduced in Trpv1-null animals. Our data also demonstrate that LPA activates TRPV1 through a unique mechanism that is independent of G protein-coupled receptors, contrary to what has been widely shown for other ion channels, by directly interacting with the C terminus of the channel. We conclude that TRPV1 is a direct molecular target of the pain-producing molecule LPA and that this constitutes, to our knowledge, the first example of LPA binding directly to an ion channel to acutely regulate its function.

  4. 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.

  5. A first principles study of the binding of formic acid in catalase complementing high resolution X-ray structures

    NASA Astrophysics Data System (ADS)

    Rovira, Carme; Alfonso-Prieto, Mercedes; Biarnés, Xevi; Carpena, Xavi; Fita, Ignacio; Loewen, Peter C.

    2006-03-01

    Density functional molecular dynamics simulations using a QM/MM approach are used to get insight into the binding modes of formic acid in catalase. Two ligand binding sites are found, named A and B, in agreement with recent high resolution structures of catalase with bound formic acid. In addition, the calculations show that the His56 residue is protonated and the ligand is present as a formate anion. The lowest energy minimum structure ( A) corresponds to the ligand interacting with both the heme iron and the catalytic residues (His56 and Asn129). The second minimum energy structure ( B) corresponds to the situation in which the ligand interacts solely with the catalytic residues. A mechanism for the process of formic acid binding in catalase is suggested.

  6. 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

    2014-04-04

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

  7. 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

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

    PubMed

    Aki, H; Yamamoto, M

    1993-02-01

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

  9. 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

  10. 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

  11. Transformation of triclosan by laccase catalyzed oxidation: The influence of humic acid-metal binding process.

    PubMed

    Lu, Junhe; Shi, Yuanyuan; Ji, Yuefei; Kong, Deyang; Huang, Qingguo

    2017-01-01

    Laccase is a widely present extracellular phenoloxidase excreted by fungi, bacteria, and high plants. It is able to catalyze one-electron oxidation of phenolic compounds into radical intermediates that can subsequently couple to each other via covalent bonds. These reactions are believed to play an important role in humification process and the transformation of contaminants containing phenolic functionalities in the environment. In this study, we investigated the kinetics of triclosan transformation catalyzed by laccase. It was found that the rate of triclosan oxidation was first order to the concentrations of both substrate and enzyme. Humic acid (HA) could inhibit the reaction by quenching the radical intermediate of triclosan generated by laccase oxidation. Such inhibition was more significant in the presence of divalent metal cations. This is because that binding to metal ions neutralized the negative charge of HA molecules, thus making them more accessible to laccase molecule that is also negatively charged. Therefore, it has greater chance to quench the radical intermediate that is very unstable and can only diffuse a limited distance after being released from the enzyme catalytic center. Based on these understandings, a reaction model was developed by integration of metal-HA binding equilibriums and kinetic equations. This model precisely predicted the transformation rate of triclosan in the presence of HA and divalent metal ions including Ca(2+), Mg(2+), Cd(2+), Co(2+), Mn(2+), Ba(2+), and Zn(2+). Overall, this work reveals important insights into laccase catalyzed oxidative coupling process.

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

    PubMed

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

    2013-01-01

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

  13. Hepatic Induction of Fatty Acid Binding Protein 4 Plays a Pathogenic Role in Sepsis in Mice.

    PubMed

    Hu, Bingfang; Li, Yujin; Gao, Li; Guo, Yan; Zhang, Yiwen; Chai, Xiaojuan; Xu, Meishu; Yan, Jiong; Lu, Peipei; Ren, Songrong; Zeng, Su; Liu, Yulan; Xie, Wen; Huang, Min

    2017-03-06

    Sepsis is defined as the host's deleterious systemic inflammatory response to microbial infections. Herein, we report an essential role of the fatty acid binding protein 4 (FABP4; alias adipocyte protein 2 or aP2), a lipid-binding chaperone, in sepsis response. Bioinformatic analysis of the Gene Expression Omnibus data sets showed the level of FABP4 was higher in the nonsurvival sepsis patients' whole blood compared to the survival cohorts. The expression of Fabp4 was induced in a liver-specific manner in cecal ligation and puncture (CLP) and lipopolysaccharide treatment models of sepsis. The induction of Fabp4 may have played a pathogenic role, because ectopic expression of Fabp4 in the liver sensitized mice to CLP-induced inflammatory response and worsened the animal's survival. In contrast, pharmacological inhibition of Fabp4 markedly alleviated the CLP responsive inflammation and tissue damage and improved survival. We conclude that FABP4 is an important mediator of the sepsis response. Early intervention by pharmacological inhibition of FABP4 may help to manage sepsis in the clinic.

  14. 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

  15. 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

  16. Identification of the putative binding pocket of valerenic acid on GABAA receptors using docking studies and site‐directed mutagenesis

    PubMed Central

    Luger, D; Poli, G; Wieder, M; Stadler, M; Ke, S; Ernst, M; Hohaus, A; Linder, T; Seidel, T; Langer, T; Hering, S

    2015-01-01

    Background and Purpose β2/3‐subunit‐selective modulation of GABAA receptors by valerenic acid (VA) is determined by the presence of transmembrane residue β2/3N265. Currently, it is not known whether β2/3N265 is part of VA's binding pocket or is involved in the transduction pathway of VA's action. The aim of this study was to clarify the localization of VA's binding pocket on GABAA receptors. Experimental Approach Docking and a structure‐based three‐dimensional pharmacophore were employed to identify candidate amino acid residues that are likely to interact with VA. Selected amino acid residues were mutated, and VA‐induced modulation of the resulting GABAA receptors expressed in Xenopus oocytes was analysed. Key Results A binding pocket for VA at the β+/α− interface encompassing amino acid β3N265 was predicted. Mutational analysis of suggested amino acid residues revealed a complete loss of VA's activity on β3M286W channels as well as significantly decreased efficacy and potency of VA on β3N265S and β3F289S receptors. In addition, reduced efficacy of VA‐induced I GABA enhancement was also observed for α1M235W, β3R269A and β3M286A constructs. Conclusions and Implications Our data suggest that amino acid residues β3N265, β3F289, β3M286, β3R269 in the β3 subunit, at or near the etomidate/propofol binding site(s), form part of a VA binding pocket. The identification of the binding pocket for VA is essential for elucidating its pharmacological effects and might also help to develop new selective GABAA receptor ligands. PMID:26375408

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. Proton and aluminum binding properties of organic acids in surface waters of the northeastern U.S.

    PubMed

    Fakhraei, Habibollah; Driscoll, Charles T

    2015-03-03

    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).

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

    PubMed

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

    2007-01-01

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

  3. Synthesis and iron(III) binding properties of 3-hydroxypyrid-4-ones derived from kojic acid.

    PubMed

    Molenda, J J; Basinger, M A; Hanusa, T P; Jones, M M

    1994-08-01

    In an attempt to reduce the toxicity of the 3-hydroxypyrid-4-ones, the more hydrophilic derivatives of kojic acid were explored and compared to the standard, 1,2-dimethyl-3-hydroxypyrid-4-one, L1. The synthesis and iron(III) binding properties of these chelators are described. Neither these compounds nor the clinically effective 1,2-dimethyl-3-hydroxypyrid-4 one is able to completely remove all of the iron(III) from the Fe(III)EDTA complex in sodium acetate buffered solutions, when the 3-hydroxypyrid-4-one: Fe(III) ratio is 6:1. The ability of these compounds to enhance the urinary excretion of iron in rats indicates that the behavior of the 3-hydroxypyrid-4-ones derived from kojic acid is comparable to the analogous derivatives of maltol and ethyl maltol. The structure of the iron(III) complex of 3-hydroxy-6-hydroxymethyl-1-methylpyrid-4-one was determined by x-ray diffraction and found to be similar to the previously reported structure of the iron(III) complex of L1.

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

    Masuzaki, Hiroaki; Hosono, Masahiro; Nitta, Kazuo

    2017-01-01

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

  6. Rat androgen-binding protein: evidence for identical subunits and amino acid sequence homology with human sex hormone-binding globulin.

    PubMed

    Joseph, D R; Hall, S H; French, F S

    1987-01-01

    The cDNA for rat androgen-binding protein (ABP) was previously isolated from a bacteriophage lambda gt11 rat testis cDNA library and its identity was confirmed by epitope selection. Hybrid-arrested translation studies have now demonstrated the identity of the isolates. The nucleotide sequence of a near full-length cDNA encodes a 403-amino acid precursor (Mr = 44,539), which agrees in size with the cell-free translation product (Mr = 45,000) of ABP mRNA. Putative sites of N-glycosylation and signal peptide cleavage were identified. Comparison of the predicted amino acid sequence of rat ABP with the amino-terminal amino acid sequence of human sex hormone-binding globulin revealed that 17 of 25 residues are identical. On the basis of the predicted amino acid sequence the molecular weight of the primary translation product, lacking the signal peptide, was 41,183. Hybridization analyses indicated that the two subunits of ABP are coded for by a single gene and a single mRNA species. Our results suggest that ABP consists of two subunits with identical primary sequences and that differences in post-translational processing result in the production of 47,000 and 41,000 molecular weight monomers.

  7. 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.

  8. 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.

  9. Photoaffinity labeling of high affinity nicotinic acid adenine dinucleotide phosphate (NAADP)-binding proteins in sea urchin egg.

    PubMed

    Walseth, Timothy F; Lin-Moshier, Yaping; Jain, Pooja; Ruas, Margarida; Parrington, John; Galione, Antony; Marchant, Jonathan S; Slama, James T

    2012-01-20

    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 [(32)P-5-azido]nicotinic acid adenine dinucleotide phosphate ([(32)P-5N(3)]NAADP) as a photoaffinity probe. Photolysis of sea urchin egg homogenates preincubated with [(32)P-5N(3)]NAADP resulted in specific labeling of 45-, 40-, and 30-kDa proteins, which was prevented by inclusion of nanomolar concentrations of unlabeled NAADP or 5N(3)-NAADP, but not by micromolar concentrations of structurally related nucleotides such as NAD, nicotinic acid adenine dinucleotide, nicotinamide mononucleotide, nicotinic acid, or nicotinamide. [(32)P-5N(3)]NAADP binding was saturable and displayed high affinity (K(d) ∼10 nM) in both binding and photolabeling experiments. [(32)P-5N(3)]NAADP photolabeling was irreversible in a high K(+) buffer, a hallmark feature of NAADP binding in the egg system. The proteins photolabeled by [(32)P-5N(3)]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.

  10. Increased expression of fatty acid binding protein 4 in preeclamptic Placenta and its relevance to preeclampsia.

    PubMed

    Yan, Yuying; Peng, Huilian; Wang, Peng; Wang, Hanzhi; Dong, Minyue

    2016-03-01

    The aim of this investigation was to determine the expression of fatty acid binding protein 4 (FABP4) in the placenta from women with preeclampsia and normal pregnancy, and to delineate the regulatory effects on thophoblast cell by FABP4. We determined the expression of FABP4 by real-time polymerase chain reaction (PCR) for messenger ribonucleic acid (mRNA) or enzyme-linked immunesorbent assay (ELISA) and Western blotting for protein. Small interference of ribonucleic acid (siRNA) and specific FABP4 inhibitor were used to inhibit FABP4. The proliferation, migration and invasion of trophoblastic cells (Swan-71 and Jar) were evaluated with cell counting kit-8, wound-healing test and transwell analysis respectively. We found the expression of FABP4 was significantly higher in the placenta of preeclamptic women than that of women with normal pregnancy (t = 4.244, P < 0.001 for mRNA; t = 4.536, P < 0.001 for protein). FABP4 siRNA significantly reduced the proliferation of trophoblasts (P < 0.001). The specific inhibition of FABP4 inhibited the proliferation of trophoblasts in a dose-dependent manner (P < 0.001) and the inhibitory effect increased as the concentration of inhibitor increased. FABP4 siRNA and specific inhibitor significantly decreased the migration (P < 0.001) and invasion (P < 0.001) of trophoblasts. We concluded the increase in placental FABP4 expression in preeclampsia may affect the function of trophoblast, and this increase may have a role in the pathogenesis of preeclampsia.

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

    NASA Astrophysics Data System (ADS)

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

    2015-02-01

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

  12. Comparative studies on the interaction of caffeic acid, chlorogenic acid and ferulic acid with bovine serum albumin

    NASA Astrophysics Data System (ADS)

    Li, Shuang; Huang, Kelong; Zhong, Ming; Guo, Jun; Wang, Wei-zheng; Zhu, Ronghua

    2010-10-01

    The substitution of the hydrogen on aromatic and esterification of carboxyl group of the phenol compounds plays an important role in their bio-activities. In this paper, caffeic acid (CaA), chlorogenic acid (ChA) and ferulic acid (FA) were selected to investigate the binding to bovine serum albumin (BSA) using UV absorption spectroscopy, fluorescence spectroscopy and synchronous fluorescence spectroscopy. It was found that the methoxyl group substituting for the 3-hydroxyl group of CaA decreased the affinity for BSA and the esterification of carboxyl group of CaA with quinic acid increased the affinities. The affinities of ChA and FA with BSA were more sensitive to the temperature than that of CaA with BSA. Synchronous fluorescence spectroscopy and time-resolved fluorescence indicated that the Stern-Volmer plots largely deviated from linearity at high concentrations and were caused by complete quenching of the tyrosine fluorescence of BSA.

  13. Identification and characterization of nuclear retinoic acid-binding activity in human myeloblastic leukemia HL-60 cells.

    PubMed Central

    Nervi, C; Grippo, J F; Sherman, M I; George, M D; Jetten, A M

    1989-01-01

    Specific [3H]retinoic acid (RA)-binding sites in nuclear and cytosolic extracts prepared from human myeloblastic leukemia HL-60 cells have been detected by sucrose density gradient sedimentation and size-exclusion high-performance liquid chromatography (HPLC) analyses. This RA-binding activity migrated as a single peak with an apparent molecular weight of 50,000 and greater than 95% of the total binding activity was associated with the nuclear extract. Nuclear extracts prepared from COS-1 cells transfected with an expression vector for the nuclear RA receptors RAR alpha or RAR beta were enriched (20- to 100-fold) with a RA-binding activity that coeluted by size-exclusion HPLC with the putative RAR from HL-60 cells. The HL-60 nuclear receptor exhibited high-affinity binding of RA and its benzoic acid analogs Ch55, Ch30, Ro 13-7410, and SRI 6409-40 and low-affinity binding of retinol, Ro 8-8717, and SRI 5442-60, correlating well with the biological activity of these compounds in HL-60 cells. Saturation binding and Scatchard plot analyses of the binding of RA to the nuclear HL-60 receptor yielded an apparent dissociation constant of approximately 0.46 nM and 1400 +/- 100 receptor sites per cell. Northern blot analyses of poly(A)+ RNA with cDNA probes specific for RAR alpha and RAR beta indicated that HL-60 cells contain predominantly transcripts encoded by the RAR alpha gene. Our results suggest that the observed nuclear RA-binding activity in HL-60 cells might mediate the action of RA in these cells. Images PMID:2548190

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

    NASA Astrophysics Data System (ADS)

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

    2011-10-01

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

  15. 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

  16. 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

  17. Dephosphorylation of microtubule-binding sites at the neurofilament-H tail domain by alkaline, acid, and protein phosphatases.

    PubMed

    Hisanaga, S; Yasugawa, S; Yamakawa, T; Miyamoto, E; Ikebe, M; Uchiyama, M; Kishimoto, T

    1993-06-01

    The dephosphorylation-induced interaction of neurofilaments (NFs) with microtubules (MTs) was investigated by using several phosphatases. Escherichia coli alkaline and wheat germ acid phosphatases increased the electrophoretic mobility of NF-H and NF-M by dephosphorylation, and induced the binding of NF-H to MTs. The binding of NFs to MTs was observed only after the electrophoretic mobility of NF-H approached the exhaustively dephosphorylated level when alkaline phosphatase was used. The number of phosphate remaining when NF-H began to bind to MTs was estimated by measuring phosphate bound to NF-H. NF-H did not bind to MTs even when about 40 phosphates from the total of 51 had been removed by alkaline phosphatase. The removal of 6 further phosphates finally resulted in the association of NF-H with MTs. A similar finding, that the restricted phosphorylation sites in the NF-H tail domain, but not the total amount of phosphates, were important for binding to MTs, was also obtained with acid phosphatases. In contrast to alkaline and acid phosphatases, four classes of protein phosphatases (protein phosphatases 1, 2A, 2B, and 2C) were ineffective for shifting the electrophoretic mobility of NF proteins and for inducing the association of NFs to MTs.

  18. Identification and binding mechanism of phage displayed peptides with specific affinity to acid-alkali treated titanium.

    PubMed

    Sun, Yuhua; Tan, Jing; Wu, Baohua; Wang, Jianxin; Qu, Shuxin; Weng, Jie; Feng, Bo

    2016-10-01

    Acid-alkali treatment is one of means widely used for preparing bioactive titanium surfaces. Peptides with specific affinity to titanium surface modified by acid-alkali two-steps treatment were obtained via phage display technology. Out of the eight new unique peptides, titanium-binding peptide 54 displayed by monoclonal M13 phage at its pIII coat protein (TBP54-M13 phage) was proved to have higher binding affinity to the substrate. The binding interaction occurred at the domain from phenylalanine at position 1 to arginine at position 6 in the sequences of TBP54 (FAETHRGFHFSF) mainly via the reaction of these residues with the Ti surface. Together the coordination and electrostatic interactions controlled the specific binding of the phage to the substrate. The binding affinity was dependent on the surface basic hydroxyl group content. In addition, the phage showed a different interaction way with the Ti surface without acid-alkali treatment along with an impaired affinity. This study could provide more understanding of the interaction mechanism between the selected peptide and its specific substrate, and develop a promising method for the biofunctionalization of titanium.

  19. Hybridoma anti-DNA autoantibodies from patients with rheumatoid arthritis and systemic lupus erythematosus demonstrate similar nucleic acid binding characteristics.

    PubMed

    Rauch, J; Massicotte, H; Tannenbaum, H

    1985-01-01

    Hybridoma anti-DNA antibodies have been generated from the fusion of the GM 4672 lymphoblastoid line with peripheral blood lymphocytes from four normal subjects, nine patients with rheumatoid arthritis (RA), and 13 patients with systemic lupus erythematosus (SLE). A total of 441 hybridoma clones were obtained, of which 37 secreted anti-DNA autoantibodies. The nucleic acid binding characteristics of the anti-DNA antibodies produced by two hybridomas from normal subjects, nine hybridomas from RA patients, and 18 hybridomas from SLE patients are reported. The hybridoma anti-DNA antibodies from all three groups showed similar antigen-binding characteristics for denatured DNA (dDNA), native DNA (nDNA), poly(I), poly(dT), and cardiolipin, by both direct binding and competitive binding analyses. One difference noted between normal-derived anti-DNA antibodies and autoimmune-derived antibodies was the inability of the former to react with z-DNA. However, this requires further substantiation with larger numbers of normal-derived clones. The broad overlap of reactivity to nucleic acid antigens among individual anti-DNA autoantibodies found in two clinically different autoimmune diseases, namely RA and SLE, suggests that the pathogenicity of anti-DNA autoantibodies may bear no relationship to their nucleic acid antigen-binding characteristics.

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

    PubMed

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

    2013-11-01

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

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

    PubMed

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

    2017-01-01

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

  2. 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.

  3. 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

  4. Chicoric acid binds to two sites and decreases the activity of the YopH bacterial virulence factor.

    PubMed

    Kuban-Jankowska, Alicja; Sahu, Kamlesh K; Gorska, Magdalena; Tuszynski, Jack A; Wozniak, Michal

    2016-01-19

    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.

  5. 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.

  6. Envelope-binding domain in the cationic amino acid transporter determines the host range of ecotropic murine retroviruses.

    PubMed Central

    Albritton, L M; Kim, J W; Tseng, L; Cunningham, J M

    1993-01-01

    Infection of rodent cells by ecotropic type C retroviruses requires the expression of a cationic amino acid transporter composed of multiple membrane-spanning domains. By exchanging portions of cDNAs encoding the permissive mouse and nonpermissive human transporters and examining their abilities to specify virus infection upon expression in human 293 cells, we have identified the amino acid residues in the extracellular loop connecting the fifth and sixth membrane-spanning segments of the mouse transporter that are required for both envelope gp70 binding and infection. These findings strongly suggest that the role of the mouse transporter in determining infection is to provide an envelope-binding site. This role is analogous to those of host membrane proteins composed of a single membrane-spanning domain that serve as binding proteins or receptors for other enveloped viruses such as human immunodeficiency virus, Epstein-Barr virus, and murine and human coronaviruses. PMID:8445722

  7. Characterisation of the nucleic acid binding features of the PRRSV 7ap and its ability to induce antinuclear antibodies.

    PubMed

    Olasz, Ferenc; Dénes, Béla; Bálint, Ádám; Magyar, Tibor; Belák, Sándor; Zádori, Zoltán

    2017-03-01

    A short alternative open reading frame named ORF7a has recently been discovered within the nucleocapsid gene of the porcine reproductive and respiratory syndrome virus (PRRSV) genome. Proteins (7ap) translated from the ORF7a of two divergent strains - a type I and a type II - are able to completely reduce the motility of nucleic acids at relatively high molar charge ratios in gel retardation assays indicating strong dsDNA- and ssRNA-binding capability. Conserved RNA- and DNA-binding properties suggest that nucleic acid binding is a functional property of the divergent 7aps, and not an arbitrary consequence of their net positive charge. Sera from Hu7ap-immunised pigs and mice did not react with Hu7ap or Hu7ap-GFP; however, antinuclear antibodies were detected in the sera of the immunised animals, suggesting an ability of Hu7ap to interact with or mimic autoantigenic macromolecules.

  8. [Zinc-induced interactions of the metal-binding domain of beta-amyloid with nucleic acids and glycosaminoglycans].

    PubMed

    Khmeleva, S A; Kozin, S A; Kiseleva, Y Y; Mitkevich, V A; Makarov, A A; Radko, S P

    2016-01-01

    Zinc ions form complexes with β-amyloid peptides and play an important role in Alzheimer's disease pathogenesis. It has been demonstrated by turbidimetry and correlation spectroscopy that synthetic peptide Aβ16 representing the metal-binding domain of β-amyloid is able to interact with nucleic acids, chondroitin polysulfate, and dextran sulfates in the presence of zinc ions. The amino acid D7H substitution enhanced the peptide binding to polyanions, whereas the H6R and H6A-H13A substitutions abolished this interaction. It is suggested that the metal-binding domain may serve as a zinc-dependent site of β-amyloid interaction with biological polyanions including DNA, RNA, and glycosaminoglycans.

  9. 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.

  10. Up-regulation of the expression of the gene for liver fatty acid-binding protein by long-chain fatty acids.

    PubMed Central

    Meunier-Durmort, C; Poirier, H; Niot, I; Forest, C; Besnard, P

    1996-01-01

    The role of fatty acids in the expression of the gene for liver fatty acid-binding protein (L-FABP) was investigated in the well-differentiated FAO rat hepatoma cell line. Cells were maintained in serum-free medium containing 40 microM BSA/320 microM oleate. Western blot analysis showed that oleate triggered an approx. 4-fold increase in the cytosolic L-FABP level in 16 h. Oleate specifically stimulated L-FABP mRNA in time-dependent and dose-dependent manners with a maximum 7-fold increase at 16 h in FAO cells. Preincubation of FAO cells with cycloheximide prevented the oleate-mediated induction of L-FABP mRNA, showing that protein synthesis was required for the action of fatty acids. Run-on transcription assays demonstrated that the control of L-FABP gene expression by oleate was, at least in part, transcriptional. Palmitic acid, oleic acid, linoleic acid, linolenic acid and arachidonic acid were similarly potent whereas octanoic acid was inefficient. This regulation was also found in normal hepatocytes. Therefore long-chain fatty acids are strong inducers of L-FABP gene expression. FAO cells constitute a useful tool for studying the underlying mechanism of fatty acid action. PMID:8912685

  11. The adsorption of substrate-binding domain of PHB depolymerases to the surface of poly(3-hydroxybutyric acid).

    PubMed

    Shinomiya, M; Iwata, T; Doi, Y

    1998-04-01

    The binding characteristic of PHB depolymerase has been studied by using glutathione S-transferase (GST) fusion proteins with substrate-binding domain of three bacterial PHB depolymerases, Alcaligenes faecalis, Comamonas acidovorans and Comamonas testosteroni. Analysis using immuno-gold labeling technique and transmission electron microscopy indicated that a novel GST fusion protein derived from A. Faecalis enzyme adsorbed to the surface of poly(3-hydroxybutyric acid) (P(3HB)) single crystals like other fusion proteins. Comparison of inhibiting degree of P(3HB) powder hydrolysis activity of PHB depolymerase by fusion proteins indicated that three fusion proteins bind to P(3HB) powder in the same degree. The measurement of the surface hydrophobicity of proteins suggests that the interaction of the substrate-binding domain with insoluble P(3HB) may include not only a hydrophobic effect but also molecule-specific contacts.

  12. Structural analysis of site-directed mutants of cellular retinoic acid-binding protein II addresses the relationship between structural integrity and ligand binding

    SciTech Connect

    Vaezeslami, Soheila; Jia, Xiaofei; Vasileiou, Chrysoula; Borhan, Babak; Geiger, James H.

    2009-09-02

    The structural integrity of cellular retinoic acid-binding protein II (CRABPII) has been investigated using the crystal structures of CRABPII mutants. The overall fold was well maintained by these CRABPII mutants, each of which carried multiple different mutations. A water-mediated network is found to be present across the large binding cavity, extending from Arg111 deep inside the cavity to the {alpha} 2 helix at its entrance. This chain of interactions acts as a 'pillar' that maintains the integrity of the protein. The disruption of the water network upon loss of Arg111 leads to decreased structural integrity of the protein. A water-mediated network can be re-established by introducing the hydrophilic Glu121 inside the cavity, which results in a rigid protein with the {alpha}2 helix adopting an altered conformation compared with wild-type CRABPII.

  13. 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 Central

    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

  14. 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.

  15. 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

  16. Pharmacologically relevant receptor binding characteristics and 5alpha-reductase inhibitory activity of free Fatty acids contained in saw palmetto extract.

    PubMed

    Abe, Masayuki; Ito, Yoshihiko; Oyunzul, Luvsandorj; Oki-Fujino, Tomomi; Yamada, Shizuo

    2009-04-01

    Saw palmetto extract (SPE), used widely for the treatment of benign prostatic hyperplasia (BPH) has been shown to bind alpha(1)-adrenergic, muscarinic and 1,4-dihydropyridine (1,4-DHP) calcium channel antagonist receptors. Major constituents of SPE are lauric acid, oleic acid, myristic acid, palmitic acid and linoleic acid. The aim of this study was to investigate binding affinities of these fatty acids for pharmacologically relevant (alpha(1)-adrenergic, muscarinic and 1,4-DHP) receptors. The fatty acids inhibited specific [(3)H]prazosin binding in rat brain in a concentration-dependent manner with IC(50) values of 23.8 to 136 microg/ml, and specific (+)-[(3)H]PN 200-110 binding with IC(50) values of 24.5 to 79.5 microg/ml. Also, lauric acid, oleic acid, myristic acid and linoleic acid inhibited specific [(3)H]N-methylscopolamine ([(3)H]NMS) binding in rat brain with IC(50) values of 56.4 to 169 microg/ml. Palmitic acid had no effect on specific [(3)H]NMS binding. The affinity of oleic acid, myristic acid and linoleic acid for each receptor was greater than the affinity of SPE. Scatchard analysis revealed that oleic acid and lauric acid caused a significant decrease in the maximal number of binding sites (B(max)) for [(3)H]prazosin, [(3)H]NMS and (+)-[(3)H]PN 200-110. The results suggest that lauric acid and oleic acid bind noncompetitively to alpha(1)-adrenergic, muscarinic and 1,4-DHP calcium channel antagonist receptors. We developed a novel and convenient method of determining 5alpha-reductase activity using LC/MS. With this method, SPE was shown to inhibit 5alpha-reductase activity in rat liver with an IC(50) of 101 microg/ml. Similarly, all the fatty acids except palmitic acid inhibited 5alpha-reductase activity, with IC(50) values of 42.1 to 67.6 microg/ml. In conclusion, lauric acid, oleic acid, myristic acid, and linoleic acid, major constituents of SPE, exerted binding activities of alpha(1)-adrenergic, muscarinic and 1,4-DHP receptors and inhibited 5

  17. Spectroscopic studies on the binding interaction of novel 13-phenylalkyl analogs of the natural alkaloid berberine to nucleic acid triplexes.

    PubMed

    Bhowmik, Debipreeta; Buzzetti, Franco; Fiorillo, Gaetano; Lombardi, Paolo; Suresh Kumar, Gopinatha

    2014-01-01

    In this study we have characterized the capability of six 13-phenylalkyl analogs of berberine to stabilize nucleic acid triplex structures, poly(rA)⋅2poly(rU) and poly(dA)⋅2poly(dT). Berberine analogs bind to the RNA and DNA triplexes non-cooperatively. As the chain length of the substitution increased beyond CH2, the affinity enhanced up to critical length of (CH2)4, there after which the binding affinity decreased for both the triplexes. A remarkably stronger intercalative binding of the analogs compared to berberine to the triplexes was confirmed from ferrocyanide fluorescence quenching, fluorescence polarization and viscosity results. Circular dichroism results had indicated strong conformational changes in the triplexes on binding of the analogs. The analogs enhanced the stability of the Hoogsteen base paired third strand of both the triplexes while no significant change in the high-temperature duplex-to-single strand transitions was observed. Energetics of the interaction revealed that as the alkyl chain length increased, the binding was more entropy driven. This study demonstrates that phenylalkyl substitution at the 13-position of berberine increased the triplex binding affinity of berberine but a threshold length of the side chain is critical for the strong intercalative binding to occur.

  18. Aminosulfhydryl and Aminodisulfide Compounds Enhance Binding of the Glucocorticoid Receptor Complex to Deoxyribonucleic Acid-Coated Cellulose and to Chromatin

    DTIC Science & Technology

    1993-01-01

    glucocorticoid receptor [21]. Diaminosulfhydryl chloroacetic acid was obtained from the Fisher compounds are more active at enhancing GRC Scientific...phase consisting of 0. I M BASE containing 25mM KCI and 3 mM chloroacetic acid and 5mM d/-10-camphorsul- MgCI2, pH 7.6 at 0 0C) was added to each tube...Enhance Binding of the Glucocorticoid Receptor Complex to Deoxy- ribonucleic Acid -Coated Cellulose and to Chromatin 4. AUThOR(S)’ J.M. Karle, R. Olmeda and

  19. 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

  20. 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.

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

    PubMed

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

    2004-02-01

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

  2. 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.

  3. 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

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

    PubMed

    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.

  5. Calcite growth-rate inhibition by fulvic acid and magnesium ion—Possible influence on biogenic calcite formation

    NASA Astrophysics Data System (ADS)

    Reddy, Michael M.

    2012-08-01

    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.

  6. 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.

  7. 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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  9. Intestinal Fatty-Acid Binding Protein and Metronidazole Response in Premature Infants

    PubMed Central

    Sampson, Mario R.; Bloom, Barry T.; Arrieta, Antonio; Capparelli, Edmund; Benjamin, Daniel K.; Smith, P. Brian; Kearns, Gregory L.; van den Anker, John; Cohen-Wolkowiez, Michael

    2014-01-01

    Objectives In premature infants with suspected intra-abdominal infection, biomarkers for treatment response to antimicrobial therapy are lacking. Intestinal fatty acid-binding protein (I-FABP) is specific to the enterocyte and is released in response to intestinal mucosal injury. I-FABP has not been evaluated as a surrogate marker of disease response to antimicrobial therapy. We examined the relationship between metronidazole exposure and urinary I-FABP concentrations in premature infants with suspected intra-abdominal infection. Study design We conducted an intravenous metronidazole pharmacokinetic study, collecting ≤3 urine samples per infant for I-FABP concentration measurements. We analyzed the relationship between I-FABP concentrations and measures of metronidazole exposure and pharmacokinetics, maturational factors, and other covariates. Results Twenty-six samples from 19 premature infants were obtained during metronidazole treatment. When analyzed without regard to presence of necrotic gastrointestinal disease, there were no significant associations between predictor variables and I-FABP concentrations. However, when the sample was limited to premature infants with necrotic gastrointestinal disease, an association was found between average predicted metronidazole concentration and I-FABP concentration (p=0.006). Conclusion While a predictive association between urinary I-FABP and metronidazole systemic exposure was not observed, the data suggest the potential of this endogenous biomarker to serve as a pharmacodynamic surrogate for antimicrobial treatment of serious abdominal infections in neonates and infants. PMID:25318626

  10. 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

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

    PubMed

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

    2016-01-01

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

  12. Expression of liver fatty acid binding protein alters growth and differentiation of embryonic stem cells.

    PubMed

    Schroeder, F; Atshaves, B P; Starodub, O; Boedeker, A L; Smith, R R; Roths, J B; Foxworth, W B; Kier, A B

    2001-03-01

    Although expression of liver fatty acid binding protein (L-FABP) modulates cell growth, it is not known if L-FABP also alters cell morphology and differentiation. Therefore, pluripotent embryonic stem cells were transfected with cDNA encoding L-FABP and a series of clones expressing increasing levels of L-FABP were isolated. Untransfected ES cells, as well as ES cells transfected only with empty vector, spontaneously differentiated from rounded adipocyte-like to fibroblast-like morphology, concomitant with marked reduction in expression of stage-specific embryonic antigen (SSEA-1). These changes in morphology and expression of SSEA-1 were greatest in ES cell clones expressing L-FABP above a threshold level. Immunofluorescence confocal microscopy revealed that L-FABP was primarily localized in a diffuse-cytosolic pattern along with a lesser degree of punctate L-FABP expression in the nucleus. Nuclear localization of L-FABP was preferentially increased in clones expressing higher levels of L-FABP. In summary, L-FABP expression altered ES cell morphology and expression of SSEA-1. Taken together with the fact that L-FABP was detected in the nucleus, these data suggested that L-FABP may play a more direct, heretofore unknown, role in regulating ES cell differentiation by acting in the nucleus as well as cytoplasm.

  13. Fatty acid binding protein 4 in circulating leucocytes reflects atherosclerotic lesion progression in Apoe(-/-) mice.

    PubMed

    Agardh, Hanna E; Gertow, Karl; Salvado, Dolores M; Hermansson, Andreas; van Puijvelde, Gijs H; Hansson, Göran K; n-Berne, Gabrielle Paulsso; Gabrielsen, Anders

    2013-02-01

    Discovery of novel biomarkers for atherosclerosis is important to aid in early diagnosis of pre-symptomatic patients at high risk of cardiovascular events. The aim of the present study was therefore to identify potential biomarkers in circulating cells reflecting atherosclerotic lesion progression in the vessel wall. We performed gene arrays on circulating leucocytes from atherosclerosis prone Apoe(-/-) mice with increasing ages, using C57BL/6 mice as healthy controls. We identified fatty acid binding protein 4 (FABP4) mRNA to be augmented in mice with established disease compared with young Apoe(-/-) or controls. Interestingly, the transcript FABP4 correlated significantly with lesion size, further supporting a disease associated increase. In addition, validation of our finding on protein level showed augmented FABP4 in circulating leucocytes whereas, importantly, no change could be observed in plasma. Immunofluorescence analysis demonstrated FABP4 to be present mainly in circulating neutrophils and to some extent in monocytes. Moreover, FABP4-positive neutrophils and macrophages could be identified in the subintimal space in the plaque. Using human circulating leucocytes, we confirmed the presence of FABP4 protein in neutrophils and monocytes. In conclusion, we have showed that cellular levels of FABP4 in circulating leucocytes associate with lesion development in the experimental Apoe(-/-) model. The increased expression is primarily localized to neutrophils, but also in monocytes. We have identified FABP4 in leucocytes as a potential and easy accessible biomarker of atherosclerosis which could be of future clinical relevance.

  14. 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.

  15. Genetic ablation of the fatty acid binding protein FABP5 suppresses HER2-induced mammary tumorigenesis

    PubMed Central

    Levi, Liraz; Lobo, Glenn; Doud, Mary Kathryn; von Lintig, Johannes; Seachrist, Darcie; Tochtrop, Gregory P.; Noy, Noa

    2014-01-01

    The fatty acid binding protein FABP5 shuttles ligands from the cytosol to the nuclear receptor PPARβ/δ (encoded for by Pparδ), thereby enhancing the transcriptional activity of the receptor. This FABP5/PPARδ pathway is critical for induction of proliferation of breast carcinoma cells by activated EGFR. In this study, we show that FABP5 is highly upregulated in human breast cancers and we provide genetic evidence of the pathophysiological significance of FABP5 in mammary tumorigenesis. Ectopic expression of FABP5 was found to be oncogenic in 3T3 fibroblasts where it augmented the ability of PPARδ to enhance cell proliferation, migration and invasion. To determine whether FABP5 was essential for EGFR-induced mammary tumor growth, we interbred FABP5-null mice with MMTV-ErbB2/HER2 oncomice which spontaneously develop mammary tumors. FABP5 ablation relieved activation of EGFR downstream effector signals, decreased expression of PPARδ target genes that drive cell proliferation, and suppressed mammary tumor development. Our findings establish that FABP5 is critical for mammary tumor development, rationalizing the development of FABP5 inhibitors as novel anticarcinogenic drugs. PMID:23722546

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

    PubMed Central

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

    2016-01-01

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

  17. Heart fatty acid binding protein in the diagnosis of myocardial infarction: where do we stand today?

    PubMed

    Colli, Andrea; Josa, Miguel; Pomar, Jose Luis; Mestres, Carlos Alberto; Gherli, Tiziano

    2007-01-01

    Heart fatty acid binding protein (hFABP) is a novel small cytosolic protein that is abundant in the heart. It is highly cardiac-specific (i.e. expressed primarily in cardiac tissue), but is also expressed at low concentrations in tissues outside the heart. After myocardial ischemic damage, hFABP can be detected in the blood as early as 1-3 h after onset of chest pain, with peak values reached at 6-8 h and plasma levels returning to normal within 24-30 h. hFABP's clinical diagnostic value is very limited in the presence of renal failure and skeletal muscle diseases as it is completely renally eliminated. In these conditions, the diagnosis of acute myocardial infarction (AMI) may be overestimated. The combination of initial hFABP release after symptom onset, rapid kidney clearance from the circulation and high cardiac specificity suggests great potential for clinical use. Serial measurements of hFABP in the first 24 h after onset of symptoms in AMI patients can: (a) identify patients who are susceptible to reperfusion strategies, (b) detect perioperative AMIs, (c) distinguish patients who reperfuse their infarct-related artery from those who do not, as early as 30 min after starting thrombolytic treatment, (d) detect re-infarction if it occurs within 10 h after symptom onset, and (e) permit an accurate estimation of myocardial infarct size providing important prognosis information.

  18. 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

  19. 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.

  20. Dual mechanism of activation of plant plasma membrane Ca2+-ATPase by acidic phospholipids: evidence for a phospholipid binding site which overlaps the calmodulin-binding site.

    PubMed

    Meneghelli, Silvia; Fusca, Tiziana; Luoni, Laura; De Michelis, Maria Ida

    2008-09-01

    The effect of phospholipids on the activity of isoform ACA8 of Arabidopsis thaliana plasma membrane (PM) Ca2+-ATPase was evaluated in membranes isolated from Saccharomyces cerevisiae strain K616 expressing wild type or mutated ACA8 cDNA. Acidic phospholipids stimulated the basal Ca2+-ATPase activity in the following order of efficiency: phosphatidylinositol 4-monophosphate > phosphatidylserine > phosphatidylcholine approximately = phosphatidylethanolamine approximately = 0. Acidic phospholipids increased V(max-Ca2+) and lowered the value of K(0.5-Ca2+) below the value measured in the presence of calmodulin (CaM). In the presence of CaM acidic phospholipids activated ACA8 by further decreasing its K(0.5-Ca2+) value. Phosphatidylinositol 4-monophosphate and, with lower efficiency, phosphatidylserine bound peptides reproducing ACA8 N-terminus (aa 1-116). Single point mutation of three residues (A56, R59 and Y62) within the sequence A56-T63 lowered the apparent affinity of ACA8 for phosphatidylinositol 4-monophosphate by two to three fold, indicating that this region contains a binding site for acidic phospholipids. However, the N-deleted mutant Delta74-ACA8 was also activated by acidic phospholipids, indicating that acidic phospholipids activate ACA8 through a complex mechanism, involving interaction with different sites. The striking similarity between the response to acidic phospholipids of ACA8 and animal plasma membrane Ca2+-ATPase provides new evidence that type 2B Ca2+-ATPases share common regulatory properties independently of structural differences such as the localization of the terminal regulatory region at the N- or C-terminal end of the protein.

  1. Putative binding sites for arachidonic acid on the human cardiac Kv1.5 channel

    PubMed Central

    Bai, Jia‐Yu; Ding, Wei‐Guang; Kojima, Akiko; Seto, Tomoyoshi

    2015-01-01

    Background and Purpose In human heart, the Kv1.5 channel contributes to repolarization of atrial action potentials. This study examined the electrophysiological and molecular mechanisms underlying arachidonic acid (AA)‐induced inhibition of the human Kv1.5 (hKv1.5) channel. Experimental Approach Site‐directed mutagenesis was conducted to mutate amino acids that reside within the pore domain of the hKv1.5 channel. Whole‐cell patch‐clamp method was used to record membrane currents through wild type and mutant hKv1.5 channels heterologously expressed in CHO cells. Computer docking simulation was conducted to predict the putative binding site(s) of AA in an open‐state model of the Kv1.5 channel. Key Results The hKv1.5 current was minimally affected at the onset of depolarization but was progressively reduced during depolarization by the presence of AA, suggesting that AA acts as an open‐channel blocker. AA itself affected the channel at extracellular sites independently of its metabolites and signalling pathways. The blocking effect of AA was attenuated at pH 8.0 but not at pH 6.4. The blocking action of AA developed rather rapidly by co‐expression of Kvβ1.3. The AA‐induced block was significantly attenuated in H463C, T480A, R487V, I502A, I508A, V512A and V516A, but not in T462C, A501V and L510A mutants of the hKv1.5 channel. Docking simulation predicted that H463, T480, R487, I508, V512 and V516 are potentially accessible for interaction with AA. Conclusions and Implications AA itself interacts with multiple amino acids located in the pore domain of the hKv1.5 channel. These findings may provide useful information for future development of selective blockers of hKv1.5 channels. PMID:26292661

  2. Identification of amino acids important for binding of Clostridium perfringens epsilon toxin to host cells and to HAVCR1

    PubMed Central

    Ivie, Susan E.; McClain, Mark S.

    2012-01-01

    Clostridium perfringens epsilon toxin belongs to the aerolysin-like family of pore-forming toxins and is one of the most potent bacterial toxins known. The epsilon toxin causes fatal enterotoxemia in sheep, goats, and possibly humans. Evidence indicates that the toxin binds to protein receptors including hepatitis A virus cellular receptor 1 (HAVCR1), but the region of the toxin responsible for cell binding has not been identified. In the present study, we identify amino acids within the epsilon toxin important for this cell interaction. Site-specific mutagenesis was used to investigate the role of a surface-accessible cluster of aromatic amino acids, and purified mutant proteins were tested in a series of cell-culture assays to assess cytotoxic activity and cell binding. When added to cells, four mutant proteins (Etx-Y29E, Etx-Y30E, Etx-Y36E and Etx-Y196E) were severely impaired in their ability to not only kill host cells, but also in their ability to permeabilize the plasma membrane. Circular dichroism spectroscopy and thermal stability studies revealed that the wild-type and mutant proteins were similarly folded. Additional experiments revealed that these mutant proteins were defective in binding to host cells and to HAVCR1. These data indicate that an amino acid motif including Y29, Y30, Y36, and Y196 is important for the ability of epsilon toxin to interact with cells and HAVCR1. PMID:22938730

  3. 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.

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

    PubMed Central

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

    1995-01-01

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

  5. Characterization of epoxyeicosatrienoic acid binding site in U937 membranes using a novel radiolabeled agonist, 20-125i-14,15-epoxyeicosa-8(Z)-enoic acid.

    PubMed

    Yang, Wenqi; Tuniki, Venugopal Raju; Anjaiah, Siddam; Falck, J R; Hillard, Cecilia J; Campbell, William B

    2008-03-01

    Epoxyeicosatrienoic acids (EETs) are important regulators of vascular tone and homeostasis. Whether they initiate signaling through membrane receptors is unclear. We developed 20-iodo-14,15-epoxyeicosa-8(Z)-enoic acid (20-I-14,15-EE8ZE), a radiolabeled EET agonist, to characterize EET binding to membranes of U937 cells. 20-I-14,15-EE8ZE stimulated cAMP production in U937 cells with similar potency, but it decreased efficacy compared with 11,12-EET. Maximum cAMP production increased 4.2-fold, with an EC(50) value of 9 muM. Like 14,15-EET, 20-I-14,15-EE8ZE relaxed bovine coronary arteries, with a similar EC(50) value. Both 20-I-14,15-EE8ZE agonist activities were blocked by the EET antagonist 14,15-epoxyeicosa-5(Z)enoic acid (14,15-EE5ZE). Specific 20-(125)I-14,15-EE8ZE binding to U937 membranes reached equilibrium within 10 min and remained unchanged for 30 min at 4 degrees C. The binding was saturable, reversible, and exhibited K(D) and B(max) values of 11.8 +/- 1.1 nM and 5.8 +/- 0.2 pmol/mg protein, respectively. Pretreatment of the membranes with guanosine 5'-O-(3-thio)triphosphate reduced the B(max) in a concentration-related manner. 20-(125)I-14,15-EE8ZE binding was inhibited by eicosanoids with potency order of 11,12-EET >14,15-EE5ZE approximately 14,15-EET > 15-hydroxyeicosatetraenoic acid > 14,15-EET-thiirane >14,15-dihydroxyeicosatrienoic acid. This order is in agreement with the efficacy and potency of cAMP production. In summary, 20-(125)I-14,15-EE8ZE is a radiolabeled EET agonist that is useful to study binding and metabolism. Using this radioligand, we have identified a specific high-affinity and high-abundance EET binding site in U937 cell membranes. This binding site could represent a specific EET receptor, which is probably a G protein-coupled receptor.

  6. Inhibitory effect of fulvic acid extracted from Canadian sphagnum peat on chemical mediator release by RBL-2H3 and KU812 cells.

    PubMed

    Yamada, Parida; Isoda, Hiroko; Han, Jun Kyu; Talorete, Terence P N; Abe, Yukuo

    2007-05-01

    Fulvic acid (FA) was extracted and purified from Canadian Sphagnum peat (CP-FA) and characterized by using an element analysis meter, Fourier transform infrared (FT-IR) spectroscopy, electron spin resonance (ESR) spectroscopy, and (13)C-nuclear magnetic resonance ((13)C-NMR) spectroscopy. To investigate the antiallergic effect of CP-FA, we incubated rat basophilic leukemia (RBL-2H3) cells with 0.001-10.0 microg/ml of CP-FA and determined the beta-hexosaminidase release inhibition at different response stages. The intracellular calcium [Ca(2+)](i) level was also determined by using Fluo 3-AM, a calcium-specific fluorescent probe, and the cytotoxicity of CP-FA was determined by the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. The results revealed that RBL-2H3 cells incubated for 48 h with 0.001-10.0 microg/ml of CP-FA did not show any decreased viability. CP-FA inhibited the beta-hexosaminidase release by IgE-sensitized, antigen-stimulated RBL-2H3 cells at the antigen-antibody binding stage and the antibody-receptor binding stage. CP-FA also inhibited histamine release from A23187 plus PMA- or compound 48/80-stimulated KU812 cells. Furthermore, there was a decrease in the intracellular [Ca(2+)](i) level in IgE-sensitized cells incubated with CP-FA and stimulated with antigen. Our results show that CP-FA may be useful for the treatment or prevention of allergic diseases.

  7. 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.

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

    PubMed Central

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

    2011-01-01

    Brown adipose tissue has a central role in thermogenesis to maintain body temperature through energy dissipation in small mammals and has recently been verified to function in adult humans as well. Here, we demonstrate that the heart-type fatty acid-binding protein, FABP3, is essential for cold tolerance and efficient fatty acid oxidation in mouse brown adipose tissue, despite the abundant expression of adipose-type fatty acid-binding protein, FABP4 (also known as aP2). Fabp3−/− mice exhibit extreme cold sensitivity despite induction of uncoupling and oxidative genes and hydrolysis of brown adipose tissue lipid stores. However, using FABP3 gain- and loss-of-function approaches in brown adipocytes, we detected a correlation between FABP3 levels and the utilization of exogenous fatty acids. Thus, Fabp3−/− brown adipocytes fail to oxidize exogenously supplied fatty acids, whereas enhanced Fabp3 expression promotes more efficient oxidation. These results suggest that FABP3 levels are a determinant of fatty acid oxidation efficiency by brown adipose tissue and that FABP3 represents a potential target for modulation of energy dissipation. PMID:21044951

  9. Interaction of brain fatty acid-binding protein with the polyunsaturated fatty acid environment as a potential determinant of poor prognosis in malignant glioma

    PubMed Central

    Elsherbiny, Marwa E.; Emara, Marwan; Godbout, Roseline

    2015-01-01

    Malignant gliomas are the most common adult brain cancers. In spite of aggressive treatment, recurrence occurs in the great majority of patients and is invariably fatal. Polyunsaturated fatty acids are abundant in brain, particularly ω-6 arachidonic acid (AA) and ω-3 docosahexaenoic acid (DHA). Although the levels of ω-6 and ω-3 polyunsaturated fatty acids are tightly regulated in brain, the ω-6:ω-3 ratio is dramatically increased in malignant glioma, suggesting deregulation of fundamental lipid homeostasis in brain tumor tissue. The migratory properties of malignant glioma cells can be modified by altering the ratio of AA:DHA in growth medium, with increased migration observed in AA-rich medium. This fatty acid-dependent effect on cell migration is dependent on expression of the brain fatty acid binding protein (FABP7) previously shown to bind DHA and AA. Increased levels of enzymes involved in eicosanoid production in FABP7-positive malignant glioma cells suggest that FABP7 is an important modulator of AA metabolism. We provide evidence that increased production of eicosanoids in FABP7-positive malignant glioma growing in an AA-rich environment contributes to tumor infiltration in the brain. We discuss pathways and molecules that may underlie FABP7/AA-mediated promotion of cell migration and FABP7/DHA-mediated inhibition of cell migration in malignant glioma. PMID:23981365

  10. Alkali metal ion binding to amino acids versus their methyl esters: affinity trends and structural changes in the gas phase.

    PubMed

    Talley, Jody M; Cerda, Blas A; Ohanessian, Gilles; Wesdemiotis, Chrys

    2002-03-15

    The relative alkali metal ion (M(+)) affinities (binding energies) between seventeen different amino acids (AA) and the corresponding methyl esters (AAOMe) were determined in the gas phase by the kinetic method based on the dissociation of AA-M(+)-AAOMe heterodimers (M=Li, Na, K, Cs). With the exception of proline, the Li(+), Na(+), and K(+) affinities of the other aliphatic amino acids increase in the order AAacids, which are particularly basic molecules, the order AA>AAOMe is already observed for K(+). Proline binds more strongly than its methyl ester to all M(+) except Li(+). Ab initio calculations on the M(+) complexes of alanine, beta-aminoisobutyric acid, proline, glycine methyl ester, alanine methyl ester, and proline methyl ester show that their energetically most favorable complexes result from charge solvation, except for proline which forms salt bridges. The most stable mode of charge solvation depends on the ligand (AA or AAOMe) and, for AA, it gradually changes with metal ion size. Esters chelate all M(+) ions through the amine and carbonyl groups. Amino acids coordinate Li(+) and Na(+) ions through the amine and carbonyl groups as well, but K(+) and Cs(+) ions are coordinated by the O atoms of the carboxyl group. Upon consideration of these differences in favored binding geometries, the theoretically derived relative M(+) affinities between aliphatic AA and AAOMe are in good overall agreement with the above given experimental trends. The majority of side chain functionalized amino acids studied show experimentally the affinity order AAacids lysine and arginine, whose K(+) (for arginine) and Cs(+) complexes (for both) follow the affinity order AA>AAOMe. The latter ranking is attributed to salt bridge formation.

  11. Amino acid polymorphisms in the fibronectin-binding repeats of fibronectin-binding protein A affect bond strength and fibronectin conformation.

    PubMed

    Casillas-Ituarte, Nadia N; Cruz, Carlos H B; Lins, Roberto D; DiBartola, Alex C; Howard, Jessica; Liang, Xiaowen; Höök, Magnus; Viana, Isabelle F T; Sierra-Hernández, M Roxana; Lower, Steven K

    2017-04-11

    The Staphylococcus aureus cell surface contains cell wall-anchored proteins such as fibronectin-binding protein A (FnBPA) that bind to host ligands (e.g. fibronectin; Fn) present in the extracellular matrix of tissue or coatings on cardiac implants. Recent clinical studies have found a correlation between cardiovascular infections caused by S. aureus and nonsynonymous single nucleotide polymorphisms (SNPs) in FnBPA. Atomic force microscopy (AFM), surface plasmon resonance (SPR), and molecular simulations were used to investigate interactions between Fn and each of eight, 20-mer peptide variants containing amino acids A, H, I, K, N, and Q at positions equivalent to 782 and/or 786 in Fn-binding repeat-9 (FnBR-9) of FnBPA. Experimentally measured bond lifetimes (1/koff ) and dissociation constants (Kd = koff / kon ), determined by mechanically dissociating the Fn-peptide complex at loading rates relevant to the cardiovascular system varied from the lowest-affinity H782A+K786A peptide (0.011 sec, 747 µM) to the highest-affinity H782Q+K786N peptide (0.192 sec, 15.7 µM). These AFM results tracked remarkably well to metadynamics simulations in which peptide detachment was defined solely by the free-energy landscape. Simulations and SPR experiments suggested that an Fn conformational change may enhance the stability of the binding complex for peptides with K786I or H782Q+K786I (Kd(app) = 0.2 to 0.5 µM. as determined by SPR) compared with the lowest-affinity double alanine peptide (Kd(app) = 3.8 µM). Together, these findings demonstrate that amino acid substitutions in FnBR-9 can significantly affect bond strength and influence the conformation of Fn upon binding. They provide a mechanistic explanation for the observation of nonsynonymous SNPs in fnbA) among clinical isolates of S. aureus that cause endovascular infections.

  12. 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.

  13. 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.

  14. Characterization of an intracellular hyaluronic acid binding site in isolated rat hepatocytes

    SciTech Connect

    Frost, S.J.; Raja, R.H.; Weigel, P.H. )

    1990-11-13

    125I-HA, prepared by chemical modification at the reducing sugar, specifically binds to rat hepatocytes in suspension or culture. Intact hepatocytes have relatively few surface 125I-HA binding sites and show low specific binding. However, permeabilization of hepatocytes with the nonionic detergent digitonin results in increased specific 125I-HA binding (45-65%) and a very large increase in the number of specific 125I-HA binding sites. Scatchard analysis of equilibrium 125I-HA binding to permeabilized hepatocytes in suspension at 4 degrees C indicates a Kd = 1.8 x 10(-7) M and 1.3 x 10(6) molecules of HA (Mr approximately 30,000) bound per cell at saturation. Hepatocytes in primary culture for 24 h show the same affinity but the total number of HA molecules bound per cell at saturation decreases to approximately 6.2 x 10(5). Increasing the ionic strength above physiologic concentrations decreases 125I-HA binding to permeable cells, whereas decreasing the ionic strength above causes an approximately 4-fold increase. The divalent cation chelator EGTA does not prevent binding nor does it release 125I-HA bound in the presence of 2 mM CaCl2, although higher divalent cation concentrations stimulate 125I-HA binding. Ten millimolar CaCl2 or MnCl2 increases HA binding 3-6-fold compared to EGTA-treated cells. Ten millimolar MgCl2, SrCl2, or BaCl2 increased HA binding by 2-fold. The specific binding of 125I-HA to digitonin-treated hepatocytes at 4{degrees}C increased greater than 10-fold at pH 5.0 as compared to pH 7.

  15. 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.

  16. CSF levels of heart fatty acid binding protein are altered during early phases of Alzheimer's disease.

    PubMed

    Chiasserini, Davide; Parnetti, Lucilla; Andreasson, Ulf; Zetterberg, Henrik; Giannandrea, David; Calabresi, Paolo; Blennow, Kaj

    2010-01-01

    Heart fatty acid binding protein (HFABP) has been proposed as a putative marker for dementia disorders. To evaluate the value of this protein as an early marker of Alzheimer's disease (AD), we analyzed HFABP level and the classical biomarkers amyloid-β (Aβ)1-42, total tau (t-tau), and phosphorylated tau (p-tau) in cerebrospinal fluid (CSF) of patients with mild cognitive impairment (MCI) followed up for four years (n=41), AD (n=32), and subjects with other neurological diseases without dementia (OND, n=25). HFABP levels were higher in AD patients and in MCI converting to AD (MCI-AD) with respect to OND and to cognitively stable MCI patients (MCI-MCI). The receiver operator characteristics analysis for HFABP alone showed a sensitivity of 87% and a specificity of 81% for AD versus OND (area under the curve, AUC=0.83); sensitivity and specificity were 46% and 94%, respectively, when comparing MCI-MCI versus MCI-AD. CSF HFABP levels showed a strong positive correlation with both t-tau and p-tau. Interestingly, the ratio between HFABP and Aβ1-42 improved the performance in distinguishing AD from OND (sensitivity: 90%; specificity 82%, AUC=0.89), and gave the best accuracy in discriminating MCI-AD from MCI-MCI (sensitivity: 80%; specificity 100%, AUC=0.90). Survival analysis by means of Kaplan-Meier curve showed a significantly higher proportion of MCI patients converting to AD in the group with higher values of HFABP/Aβ1-42 ratio (cut-off=0.7). A significant correlation between HFABP/Aβ1-42 ratio and MMSE annual decrease rate was also documented (p<0.0001). HFABP /Aβ1-42 ratio might be a useful predictor of conversion in MCI patients.

  17. The binding of okadaic acid analogs to recombinant OABP2.1 originally isolated from the marine sponge Halichondria okadai.

    PubMed

    Konoki, Keiichi; Onoda, Tatsuya; Furumochi, Sachie; Cho, Yuko; Yotsu-Yamashita, Mari; Yasumoto, Takeshi

    2013-11-01

    The binding between [24-(3)H]okadaic acid (OA) and a recombinant OA binding protein OABP2.1 was examined using various OA analog, including methyl okadaate, norokadanone, 7-deoxy OA, and 14,15-dihydro OA, 7-O-palmitoyl DTX1, to investigate the structure activity relationship. Among them, 7-O-palmitoyl DTX1, which is one of the diarrhetic shellfish poisoning (DSP) toxins identified in shellfish, displayed an IC50 for [24-(3)H]OA binding at 51±6.3nM (Mean±SD). In addition, a synthetic compound, N-pyrenylmethyl okadamide, exhibited its IC50 at 10±2.9nM (Mean±SD). These results suggested that the recombinant OABP2.1 and the N-pyrenylmethyl okadamide might be core substances in a novel assay for the DSP toxins.

  18. In situ fluorescence labelling of jasmonic acid binding sites in plant tissues with cadmium-free quantum dots.

    PubMed

    Liao, Qiumei; Yu, Ying; Cao, Yujuan; Lin, Bixia; Wei, Jingjing

    2015-02-01

    The fluorescence labelling of plant hormone binding sites is an important analytical technique in research on the molecular mechanisms of plant hormone activities. The authors synthesised a jasmonic acid (JA)-conjugated ZnS:Mn quantum dot (QD) probe, with a cubic structure and average hydrodynamic sizes of about 17.0 nm. The maximum fluorescence emission of the probe was recorded at about 585 nm. The probe was used for fluorescence labelling of JA binding sites in mung bean seedling tissues. Analysis revealed that the probe exhibited high selectivity to JA binding sites and good performance in eliminating interference from background fluorescence in plant tissues. In addition, the probe did not exhibit any apparent biotoxicity, and is much more suitable than probes constructed from CdTe QDs for the analysis of biological samples.

  19. 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.

  20. Liver fatty acid binding protein is the mitosis-associated polypeptide target of a carcinogen in rat hepatocytes.

    PubMed Central

    Bassuk, J A; Tsichlis, P N; Sorof, S

    1987-01-01

    Hepatocytes in normal rat liver were found previously to contain a cytoplasmic 14,000-dalton polypeptide (p14) that is associated with mitosis and is the principal early covalent target of activated metabolites of the carcinogen N-2-fluorenylacetamide (2-acetylaminofluorene). The level of immunohistochemically detected p14 was low when growth activity of hepatocytes was low, was markedly elevated during mitosis in normal and regenerating livers, but was very high throughout interphase during proliferation of hyperplastic and malignant hepatocytes induced in rat liver by a carcinogen (N-2-fluorenylacetamide or 3'-methyl-4-dimethylaminoazobenzene). We report here that p14 is the liver fatty acid binding protein. The nucleotide sequence of p14 cDNA clones, isolated by screening a rat liver cDNA library in bacteriophage lambda gt11 using p14 antiserum, was completely identical to part of the sequence reported for liver fatty acid binding protein. Furthermore, the two proteins shared the following properties: size of mRNA, amino acid composition, molecular size according to NaDodSO4 gel electrophoresis, and electrophoretic mobilities in a Triton X-100/acetic acid/urea gel. Their pI values overlapped in 2-dimensional isoelectric focusing/NaDodSO4 gel electrophoresis and showed the same response to delipidation. Either polypeptide reacted with and blocked the antiserum raised against the other polypeptide. The two polypeptides bound oleic acid similarly. Finally, identical elevations of cytoplasmic immunostain were detected specifically in mitotic hepatocytes with either antiserum. The collected findings are suggestive that liver fatty acid binding protein may carry ligands that promote hepatocyte division and may transport certain activated chemical carcinogens. Images PMID:3478711

  1. 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.

  2. Using spin labels to study molecular processes in soils: Covalent binding of aromatic amines to humic acids of soils

    NASA Astrophysics Data System (ADS)

    Aleksandrova, O. N.; Kholodov, V. A.; Perminova, I. V.

    2015-08-01

    Interactions of aliphatic and aromatic amines with soil and humic acids isolated from it are studied by means of spin labels and electron paramagnetic resonance (EPR) spectroscopy. Nitroxyl radicals containing amino groups are used as spin labels. It is found experimentally that aromatic amines are instantaneously converted to the bound state. It is shown that the microareas of their incorporation are characterized by a significant delay in the reduction of the nitroxyl fragment of spin-label molecules, indicating the formation of condensed structures typical of an oxidative binding mechanism. It is concluded that aliphatic amines do not bind to humic acids. It is noted that the studied process allows elucidating the formation of bound xenobiotic residues in soils.

  3. 3-(4-Hydroxyphenyl)propionic acid: the forgotten detection substrate for ligand-binding assay-based bioanalysis.

    PubMed

    Jordan, Gregor; Stubenrauch, Kay-Gunnar; Heinrich, Julia; Staack, Roland F

    2017-02-01

    Ligand-binding assays are ideal for routine bioanalysis, but we reason that the straightforward replacement of the conventional chromogenic horseradish peroxidase substrate, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid, of a routinely used preclinical immunoassay to detect hIgG, with the fluorogenic 3-(4-hydroxyphenyl)propionic acid would broaden the narrow dynamic range. The replacement leads to a sensitivity of 0.47 (minimum required dilution [MRD] 10) and 1.02 (MRD 50) ng/ml, and dynamic ranges of 3.3 (MRD 10) and 3.6 (MRD 50) orders of magnitude, and thereby had improved sensitivity and dynamic range compared with other conventional colorimetric ELISAs, other ligand-binding assay technologies or LC-MS assays. Improvements in sensitivity and dynamic range were achieved for the sera of horse, mice and monkeys without assay optimization.

  4. In vitro binding of acetic acid and its chlorinated derivatives by the soluble glutathione S-transferases from rat liver.

    PubMed

    Dierickx, P J

    1984-05-01

    The in vitro interaction of acetic acid and its chlorinated derivatives with rat liver glutathione S-transferases (GST) was studied, using glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB) as substrates. The investigated compounds inhibited the GST activity in crude extracts in a dose dependent manner. Each of the different GST isoenzymes was inhibited by each of the compounds under study, albeit at very different degrees. Kinetic studies never revealed competitive inhibition kinetics, with GSH nor CDNB as the variable substrate. Titration of remaining GSH in appropriate incubation mixtures revealed no GST catalyzed conjugation with GSH. It is concluded that acetic acid and its chlorinated derivatives interact with GST by direct binding to these proteins. This binding could have a protective function against these compounds.

  5. The Magnesium-Chelatase H Subunit Binds Abscisic Acid and Functions in Abscisic Acid Signaling: New Evidence in Arabidopsis1[W][OA

    PubMed Central

    Wu, Fu-Qing; Xin, Qi; Cao, Zheng; Liu, Zhi-Qiang; Du, Shu-Yuan; Mei, Chao; Zhao, Chen-Xi; Wang, Xiao-Fang; Shang, Yi; Jiang, Tao; Zhang, Xiao-Feng; Yan, Lu; Zhao, Rui; Cui, Zi-Ning; Liu, Rui; Sun, Hai-Li; Yang, Xin-Ling; Su, Zhen; Zhang, Da-Peng

    2009-01-01

    Using a newly developed abscisic acid (ABA)-affinity chromatography technique, we showed that the magnesium-chelatase H subunit ABAR/CHLH (for putative abscisic acid receptor/chelatase H subunit) specifically binds ABA through the C-terminal half but not the N-terminal half. A set of potential agonists/antagonists to ABA, including 2-trans,4-trans-ABA, gibberellin, cytokinin-like regulator 6-benzylaminopurine, auxin indole-3-acetic acid, auxin-like substance naphthalene acetic acid, and jasmonic acid methyl ester, did not bind ABAR/CHLH. A C-terminal C370 truncated ABAR with 369 amino acid residues (631–999) was shown to bind ABA, which may be a core of the ABA-binding domain in the C-terminal half. Consistently, expression of the ABAR/CHLH C-terminal half truncated proteins fused with green fluorescent protein (GFP) in wild-type plants conferred ABA hypersensitivity in all major ABA responses, including seed germination, postgermination growth, and stomatal movement, and the expression of the same truncated proteins fused with GFP in an ABA-insensitive cch mutant of the ABAR/CHLH gene restored the ABA sensitivity of the mutant in all of the ABA responses. However, the effect of expression of the ABAR N-terminal half fused with GFP in the wild-type plants was limited to seedling growth, and the restoring effect of the ABA sensitivity of the cch mutant was limited to seed germination. In addition, we identified two new mutant alleles of ABAR/CHLH from the mutant pool in the Arabidopsis Biological Resource Center via Arabidopsis (Arabidopsis thaliana) Targeting-Induced Local Lesions in Genomes. The abar-2 mutant has a point mutation resulting in the N-terminal Leu-348→Phe, and the abar-3 mutant has a point mutation resulting in the N-terminal Ser-183→Phe. The two mutants show altered ABA-related phenotypes in seed germination and postgermination growth but not in stomatal movement. These findings support the idea that ABAR/CHLH is an ABA receptor and reveal

  6. Helicobacter pylori acidic stress response factor HP1286 is a YceI homolog with new binding specificity.

    PubMed

    Sisinni, Lorenza; Cendron, Laura; Favaro, Gabriella; Zanotti, Giuseppe

    2010-04-01

    HP1286 from Helicobacter pylori is among the proteins that play a relevant role in bacterial colonization and persistence in the stomach. Indeed, it was demonstrated to be overexpressed under acidic stress conditions, together with other essential virulence factors. Here we describe its crystal structure, determined at 2.1 A resolution. The molecular model, a dimer characterized by two-fold symmetry, shows that HP1286 structurally belongs to the YceI-like protein family, which in turn is characterized by the lipocalin fold. The latter characterizes proteins possessing an internal cavity with the function of binding and/or transport of amphiphilic molecules. Surprisingly, a molecule of erucamide was found bound in the internal cavity of each monomer of recombinant HP1286, cloned and expressed in an Escherichia coli heterologous system. The shape and length of the cavity indicate that, at variance with other members of the family, HP-YceI has a binding specificity for amphiphilic compounds with a linear chain of about 22 carbon atoms. These features, along with the fact that the protein is secreted by the bacterium and is involved in adaptation to an acidic environment, suggest that its function could be that of sequestering specific fatty acids or amides from the environment, either to supply the bacterium with the fatty acids necessary for its metabolism, or to protect and detoxify it from the detergent-like antimicrobial activity of fatty acids that are eventually present in the external milieu.

  7. 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

  8. Ferulic acid improves lipid and glucose homeostasis in high-fat diet-induced obese mice.

    PubMed

    Naowaboot, Jarinyaporn; Piyabhan, Pritsana; Munkong, Narongsuk; Parklak, Wason; Pannangpetch, Patchareewan

    2016-02-01

    Ferulic acid (FA) is a plant phenolic acid that has several pharmacological effects including antihyperglycaemic activity. Thus, the objective of this study is to investigate the effect of FA on glucose and lipid metabolism in high-fat diet (HFD)-induced obese mice. Institute for Cancer Research (ICR) mice were fed a HFD (45 kcal% fat) for 16 weeks. At the ninth week of induction, the obese mice were orally administered with daily FA doses of 25 and 50 mg/kg for the next eight weeks. The results show that FA significantly reduced the elevated blood glucose and serum leptin levels, lowered the insulin resistance, and increased the serum adiponectin level. Moreover, serum lipid level, and liver cholesterol and triglyceride accumulations were also reduced. The histological examination showed clear evidence of a decrease in the lipid droplets in liver tissues and smaller size of fat cells in the adipose tissue in the obese mice treated with FA. Interestingly, FA reduced the expression of hepatic lipogenic genes such as sterol regulatory element-binding protein 1c (SREBP1c), fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC). It could also up-regulate hepatic carnitine palmitoyltransferase 1a (CPT1a) gene and peroxisome proliferator-activated receptor alpha (PPARα) proteins. The FA treatment was also found to suppress the protein expressions of hepatic gluconeogenic enzymes, phosphoenolpyruvate carboxylase (PEPCK) and glucose-6-phosphatase (G6Pase). In conclusion, the findings of this study demonstrate that FA improves the glucose and lipid homeostasis in HFD-induced obese mice probably via modulating the expression of lipogenic and gluconeogenic genes in liver tissues.

  9. Importance of the proline-rich multimerization domain on the oligomerization and nucleic acid binding properties of HIV-1 Vif.

    PubMed

    Bernacchi, Serena; Mercenne, Gaëlle; Tournaire, Clémence; Marquet, Roland; Paillart, Jean-Christophe

    2011-03-01

    The HIV-1 viral infectivity factor (Vif) is required for productive infection of non-permissive cells, including most natural HIV-1 targets, where it counteracts the antiviral activities of the cellular cytosine deaminases APOBEC-3G (A3G) and A3F. Vif is a multimeric protein and the conserved proline-rich domain (161)PPLP(164) regulating Vif oligomerization is crucial for its function and viral infectivity. Here, we expressed and purified wild-type Vif and a mutant protein in which alanines were substituted for the proline residues of the (161)PPLP(164) domain. Using dynamic light scattering, circular dichroism and fluorescence spectroscopy, we established the impact of these mutations on Vif oligomerization, secondary structure content and nucleic acids binding properties. In vitro, wild-type Vif formed oligomers of five to nine proteins, while Vif AALA formed dimers and/or trimers. Up to 40% of the unbound wild-type Vif protein appeared to be unfolded, but binding to the HIV-1 TAR apical loop promoted formation of β-sheets. Interestingly, alanine substitutions did not significantly affect the secondary structure of Vif, but they diminished its binding affinity and specificity for nucleic acids. Dynamic light scattering showed that Vif oligomerization, and interaction with folding-promoting nucleic acids, favor formation of high molecular mass complexes. These properties could be important for Vif functions involving RNAs.

  10. Binding of α,α-Disubstituted Amino Acids to Arginase Suggests New Avenues for Inhibitor Design1

    PubMed Central

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

    2011-01-01

    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-(di