Littérature scientifique sur le sujet « Sulfate recognition »

ABSTRACT Toxoplasma gondii is an obligate intracellular parasite that actively invades a wide variety of vertebrate cells, although the basis of this pervasive cell recognition is not understood. We demonstrate here that binding to the substratum and to host cells is partially mediated by interaction with sulfated glycosaminoglycans (GAGs). Addition of excess soluble GAGs blocked parasite attachment to serum-coated glass, thereby preventing gliding motility of extracellular parasites. Similarly, excess soluble GAGs decreased the attachment of parasites to human host cells from a variety of lineages, including monocytic, fibroblast, endothelial, epithelial, and macrophage cells. The inhibition of parasite attachment by GAGs was observed with heparin and heparan sulfate and also with chondroitin sulfates, indicating that the ligands for attachment are capable of recognizing a broad range of GAGs. The importance of sulfated proteoglycan recognition was further supported by the demonstration that GAG-deficient mutant host cells, and wild-type cells treated enzymatically to remove GAGs, were partially resistant to parasite invasion. Collectively, these studies reveal that sulfated proteoglycans are one determinant used for substrate and cell recognition by Toxoplasma. The widespread distribution of these receptors may contribute to the broad host and tissue ranges of this highly successful intracellular parasite.

SUMMARYHeparin-binding proteins (HBPs) have been demonstrated in both infective forms of Trypanosoma cruzi and are involved in the recognition and invasion of mammalian cells. In this study, we evaluated the potential biological function of these proteins during the parasite-vector interaction. HBPs, with molecular masses of 65·8 kDa and 59 kDa, were isolated from epimastigotes by heparin affinity chromatography and identified by biotin-conjugated sulfated glycosaminoglycans (GAGs). Surface plasmon resonance biosensor analysis demonstrated stable receptor-ligand binding based on the association and dissociation values. Pre-incubation of epimastigotes with GAGs led to an inhibition of parasite binding to immobilized heparin. Competition assays were performed to evaluate the role of the HBP-GAG interaction in the recognition and adhesion of epimastigotes to midgut epithelial cells of Rhodnius prolixus. Epithelial cells pre-incubated with HBPs yielded a 3·8-fold inhibition in the adhesion of epimastigotes. The pre-treatment of epimastigotes with heparin, heparan sulfate and chondroitin sulfate significantly inhibited parasite adhesion to midgut epithelial cells, which was confirmed by scanning electron microscopy. We provide evidence that heparin-binding proteins are found on the surface of T. cruzi epimastigotes and demonstrate their key role in the recognition of sulfated GAGs on the surface of midgut epithelial cells of the insect vector.

Significant structural contraction and expansion of flexible Pd₆L₈ cages by encapsulation of alkyl sulfate were demonstrated. The contact angles on the fine-ground microcrystal layers shift according to the chain length of the alkyl sulfate.

The macrophage and epithelial cell mannose receptor (MR) binds carbohydrates on foreign and host molecules. Two portions of MR recognize carbohydrates: tandemly arranged C-type lectin domains facilitate carbohydrate-dependent macrophage uptake of infectious organisms, and the NH2-terminal cysteine-rich domain (Cys-MR) binds to sulfated glycoproteins including pituitary hormones. To elucidate the mechanism of sulfated carbohydrate recognition, we determined crystal structures of Cys-MR alone and complexed with 4-sulfated-N-acetylgalactosamine at 1.7 and 2.2 Å resolution, respectively. Cys-MR folds into an approximately three-fold symmetric β-trefoil shape resembling fibroblast growth factor. The sulfate portions of 4-sulfated-N-acetylgalactosamine and an unidentified ligand found in the native crystals bind in a neutral pocket in the third lobe. We use the structures to rationalize the carbohydrate binding specificities of Cys-MR and compare the recognition properties of Cys-MR with other β-trefoil proteins.

L'objectif de cette thèse est le développement d'une langue électronique avec une méthode simplifiée d'obtention de récepteurs à réactivité croisée. Ces récepteurs sont préparés par une approche combinatoire novatrice qui consiste au mélange et à l'auto-assemblage de deux disaccharides. Le couplage de ces récepteurs avec un système de détection d'imagerie par résonance des plasmons de surface nous a permis de réaliser une langue électronique capable de différencier des échantillons de différentes complexités, y compris des protéines pures et des mélanges complexes. Cela se fait grâce aux profils et images d'évolution continue, assimilés à des « empreintes digitales » des échantillons. D'un autre côté, ce système peut être utilisé en tant qu'outil pour la conception de surfaces biomimétiques 2D et 3D. Ce système est prometteur pour l'étude des interactions sucre-protéine et pour la préparation de nanovecteurs biomimétiques qui ciblent de façon spécifique des protéines d'intérêt
L'objectif de cette thèse est le développement d'une langue électronique avec une méthode simplifiée d'obtention de récepteurs à réactivité croisée. Ces récepteurs sont préparés par une approche combinatoire novatrice qui consiste au mélange et à l'auto-assemblage de deux disaccharides. Le couplage de ces récepteurs avec un système de détection d'imagerie par résonance des plasmons de surface nous a permis de réaliser une langue électronique capable de différencier des échantillons de différentes complexités, y compris des protéines pures et des mélanges complexes. Cela se fait grâce aux profils et images d'évolution continue, assimilés à des « empreintes digitales » des échantillons. D'un autre côté, ce système peut être utilisé en tant qu'outil pour la conception de surfaces biomimétiques 2D et 3D. Ce système est prometteur pour l'étude des interactions sucre-protéine et pour la préparation de nanovecteurs biomimétiques qui ciblent de façon spécifique des protéines d'intérêt

Disabled-2 (Dab2) is an adaptor protein that plays critical roles in various biological processes, including protein endocytosis, platelet activation and aggregation, tumor growth, and development. In platelets, Dab2 associates with membrane sulfatide at the platelet surface, modulating platelet inside-out and outside-in signaling pathways. A Dab2-derived peptide, named the sulfatide-binding peptide (SBP), is the minimal unit of Dab2 to exert its function as a negative regulator of platelet activation and aggregation. The work of this thesis refines the model of Dab2 SBP binding to sulfatide and provides structural and functional insights into the mechanism by which Dab2 SBP modulates platelet activation. Using molecular docking, lipid-protein overlay assay, nuclear magnetic resonance, and surface plasma resonance tools, this work identifies the critical residues within two major regions responsible for sulfatide interaction. First, docking a sulfatide to Dab2 SBP, a hydrophilic region, primarily mediated by Arg42, is thought to be responsible for the association with the sulfatide headgroup. We observed that Arg 42 could directly interact with sulfatide by forming hydrogen bonds with the OS atoms in the sulfatide head group. Further lipid-protein overlay assay and surface plasma resonance experiments confirmed that both the positive charge and stereochemistry of the side chain of Dab2 SBP Arg42 are required for the sulfatide binding. Moreover, Arg42 is found to be critical in the inhibition of P-selectin expression on activated platelets. The residues nearby Arg42 (i.e., Glu33, Ty38, and Lys 44) also contribute to sulfatide interaction. Second, the second polybasic motif located at the C-terminal -helix 2 is considered to interact with the acyl chain through hydrophobic interactions rather than direct binding to the charged sulfatide head group. Lysine residues in this region are suggested to exert a dual role in sulfatide association, that is, by favoring electrostatic interactions with the negatively-charged sulfatide and/or by employing their flexible hydrocarbon spacers for hydrophobic interactions with membrane lipids. Consistent with this suggestion, we found a hydrophobic patch in the wild type Dab2 SBP structure surrounded by Lys49, Lys51, and Lys53. Furthermore, the role of the second sulfatide binding motif in sulfatide binding is confirmed by mutagenesis analysis and lipid-protein overlay assays, highlighting the ability of molecular docking to accurately predict critical residues responsible for sulfatide binding. In summary, this work provides a detailed structural basis for Dab2 recognition by sulfatide through multiple biophysical methods. The corresponding biological implications in the inhibition of platelet activation are also evaluated by flow cytometry. By elucidating the underlying mechanisms of Dab2 mediating platelet activation through sulfatide binding, we provided structural and functional insights for designing a Dab2-derived peptide with altered sulfatide recognition features in platelets, which can be further employed in antiplatelet therapy.
Doctor of Philosophy
Platelets are blood cells that are fundamentally intended to help form clots to stop bleeding. They do so by being activated after getting signals from damaged blood vessels and reaching the injury site. Consequently, they form aggregates by attracting more platelets to clump on the clot. However, platelet activation induced by a tumor cell can, in turn, protect the tumor cell from immune system elimination and facilitates their growth and spread. This platelet-tumor complex formation suggests platelets as a therapeutic target for reducing tumor migration out of the bloodstream. Our study investigates the mechanism of a Disabled-2-derived peptide, named Dab2 SBP, which upon binding to a sulfatide lipid, can reduce the platelet activation extent, using molecular and cellular approaches. The results of this study may be instrumental in the generation of Dab2 SBP-derived peptides with altered sulfatide binding ability and selectivity, which may lead to a design of an antiplatelet drug that can limit the ability of tumor cells to invade other tissues.

Les glycosaminoglycannes (GAGs) sont des molécules polysaccharidiques polysulfatées intervenant dans des processus aussi variés que la prolifération, différenciation ou migration cellulaire, la coagulation sanguine ou l‟infection virale. Il est généralement admis qu‟une séquence particulière de GAG doit être associée à une fonction biologique spécifique. Les structures chimiques globales des GAGs sont connues. Cependant, contrairement au séquençage des gènes ou des protéines, la détermination de la séquence saccharidique exacte impliquée dans une fonction biologique particulière n‟est encore pas possible. Le séquençage « glycomique » constitue donc un enjeu majeur. L‟une des technologies les plus novatrices pour aborder ce problème de séquençage des GAGs semble être l‟impression moléculaire. En effet, elle permet d‟obtenir des polymères (MIPs pour Molecular Imprinted Polymer) spécifiquement imprimés par la forme structurale d‟une molécule cible.En nous appuyant sur des travaux antérieurs réalisés avec des modèles saccharidiques sulfatés simples, nous avons appliqué cette technologie à la reconnaissance de glycannes sulfatés complexes d‟intérêt biologique tels qu‟une héparine de bas poids moléculaire ou un mimétique ayant une activité anticoagulante. Il a été démontré une reconnaissance spécifique et sélective selon la molécule étudiée à l‟aide de MIPs spécialement conçus pour chaque GAG. De plus, nous avons obtenu des MIPs qui, en immobilisant temporairement un sucre, permettraient leur substitution de façon stéréospécifique. La détermination des conditions optimales de synthèse des MIPs s‟est avéré une étape nécessaire à l‟obtention d‟une bonne reconnaissance. Ces travaux ouvrent des perspectives d‟application de la technique d‟impression moléculaire à l‟analyse des séquences de GAGs d‟intérêt biologique
Glycosaminoglycans (GAGs) are polysulfated polysaccharide molecules involved in many biological processes such as cellular proliferation, differentiation or migration, blood clotting or viral infection. It is generally admitted that a particular GAG sequence is connected to a specific biological function. Depending on their composition in disaccharides, GAGs are classified into subfamilies whose overall chemical structures are known. Unlike gene or protein sequencing, determination of the exact saccharidic sequence involved in a particular biological function is not yet possible with the available technological tools. "Glycomics" is a real challenge nowadays. One of the most innovative technologies to achieve this goal seems to be the molecular imprinting. Indeed, it provides polymers (MIPs for Molecular Imprinted Polymer) imprinted by the structural form of a target molecule.Based on previous studies performed with simple sulfated saccharides, this technology has been applied to the recognition of complex sulfated glycans. MIPs were achieved demonstrating specific and selective recognition for a Low Molecular Weight Heparin or a synthetic anticoagulant mimetic. Other MIPs were able to temporally immobilize sugars which make them available for stereo-specific modifications. Screening of optimal synthesis conditions of MIPs appeared a necessary step to obtain a specific and selective recognition. These studies open further possibilities to analyze GAG sequences carrying biological functions by the molecular imprinting technology

El presente proyecto de investigación está enfocado al desarrollo de sensores químicos fluoro-cromogénicos, para la detección y determinación de especies químicas de interés biológico, industrial y medioambiental de forma selectiva y con alta sensibilidad. En forma general, se busca el diseñar nuevos sistemas sensores basados en compuestos (receptores) formados por dos unidades: una unidad coordinante que interacciona con el anión a determinar y una unidad generadora de señal que alerta del reconocimiento molecular efectuado. Durante este estudio se están preparando diversas moléculas receptoras funcionalizandas con grupos modificadores de estructura para evaluar su influencia sobre las capacidades de detección y selectividad como receptores de especies específicas en diferentes condiciones y medios. Las diferentes aproximaciones en prueba implican a su vez el diseño y síntesis molecular, así como el análisis de las diferentes señales ópticas producidas en el reconocimiento, con el fin de diseñar sistemas de alta eficacia y eficiencia, y con posibilidades reales de aplicación.
Santos Figueroa, LE. (2014). New approaches for the development of chromo-fluorogenic sensors for chemical species of biological, industrial and environmental interest [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/43216
TESIS
Premiado

[EN] Abstract The present PhD thesis entitled "Supramolecular Chemistry: New chemodosimeters and hybrid materials for the chromo-fluorogenic detection of anions and neutral molecules" is based on the application of supramolecular chemistry and material science principles for the development of optical chemosensors for anions and neutral molecules detection. The second chapter of this PhD thesis is devoted to the preparation of chemodosimeters for the chromo-fluorogenic detection of fluoride, diisopropyl fluorophosphates (DFP) and hydrogen sulfide. The optical detection of fluoride anion was achieved by using a pyridine derivative containing a t-butyldimethylsilyl ether group. Aqueous solutions of the chemodosimeter were colorless but turned yellow upon addition of fluoride anion. Also a remarkable enhancement in emission was observed only upon the addition of fluoride. The optical changes were ascribed to a fluoride-induced hydrolysis of the silyl ether moiety. Also a chemodosimeter for the optical recognition of DFP, a nerve agent simulant, was prepared. In this case, the chemodosimeter was based on a stilbene pyridinium derivative functionalized with hydroxyl and silyl ether moieties. Aqueous solutions of the chemodosimeter were colorless changing to yellow upon DFP addition. The optical changes were ascribed to a hydroxyl phosphorylation followed by a fluoride-induced hydrolysis of the silyl ether group. Besides, that probe was implemented in test strips and DFP detection in gas phase was accomplished. Finally, the fluorogenic recognition of hydrogen sulfide anion was explored. For this purpose different fluorophores were selected and fucntionalized with 2,4-dinitrophenyl ether groups. The prepared probes were neraly non-emissive but remarkable emission enhancements upon addition of hydrogen sulfide were observed. The emission enhancements observed were due to a selective sulfide-induced hydrolysis of the 2,4-dinitrophenyl ether moiety that yielded the free fluorophores. Another set of chemodosimeters equipped with azide and sulfonylazide moieties were prepared. Again these probes were non-fluorescent but upon addition of hydrogen sulfide an important enhancement in emission was found. The selective response was ascribed to a reduction of the azide and sulfonylazide moieties to amine and sulfonylamide induced by hydrogen sulfide anion. Besides, the viability assays showed that these dosimeters were essentially non-toxic and real-time fluorescence imaging measurements confirmed their ability to detect intracellular hydrogen sulfide at micromolar concentrations. The third chapter of this PhD thesis was devoted to the preparation of nanoscopic gated materials and their use in sensing protocols. In a first step a gated material for the optical detection of glutathione (GSH) was prepared. For this purpose MCM-41 mesoporous silca nanoparticles were selected as inorganic scaffold. The pores were loaded with safranine O and the external surface was functionalized with disulfide-containing oligo(ethylene glycol) moieties. Dye delivery from aqueous suspensions of the sensory material was only observed in the presence of GSH. The signalling paradigm was ascribed to the selective reduction of the disulfide bond by GSH which induced pore opening and dye release. Also capped organic-inorganic hybrid materials for the selective detection of hydrogen sulfide were prepared and characterized. In this case the same MCM-41 support was used and charged with [Ru(bipy)3]2+ dye. Then, the external surface was functionalized with Cu(II)-macorcyclic complexes and finally, the pores were capped by the addition of the bulky anion hexametaphosphate. Aqueous suspensions of this material showed negligible dye release whereas in the presence of hydrogen sulfide anion a remarkable colour change was observed. This optical response was ascribed to a demetallation process of the Cu(II) complex induced by hydrogen sulfide.
[ES] Resumen La presente tesis doctoral titulada "Química supramolecular: Nuevos dosímetros químicos y materiales híbridos para la detección cromo-fluorogénica de aniones y moléculas neutras." está basada en la aplicación de principios básicos de la química supramolecular y de la ciencia de materiales en el desarrollo de sensores ópticos para aniones y moléculas neutras. El segundo capítulo de esta tesis doctoral está dedicado a la preparación de dosímetros químicos para la detección cromo-fluorogénica de fluoruro, diisopropil fluorofosfato (DFP) y sulfuro de hidrógeno. Para la detección óptica del anión fluoruro se sintetizó un derivado de piridina funcionalizado con un t-butildimetilsilil éter. En este capítulo también se describe la preparación de un dosímetro químico para la detección de DFP, que es un simulante de agentes nerviosos. Este dosímetro está basado en un estilbeno funcionalizado con una sal de piridinio que contiene grupos hidroxilo y silil éter en su estructura. Finalmente se prepararon dos familias de sensores para la detección óptica de hidrógeno sulfuro. La primera familia de sensores consiste en fluoróforos comunes funcionalizados con 2,4-dinitrofenil éteres. Los sensores preparados no presentaron una emisión de fluorescencia importante mientras que, en presencia del anión hidrógeno sulfuro, se observó un aumento significativo. La segunda familia de dosímetros también estaba compuesta por ciertos fluorofóros pero, en este caso, funcionalizados con grupos azida y sulfonilazida. Los dosimétros preparados, siguiendo esta segunda aproximación, tampoco dieron una fluorescencia significativa observándose un aumento de la misma al añadir el anión hidrógeno sulfuro. El tercer capítulo de esta tesis doctoral está dedicado a la preparación de materiales híbridos nanoscópicos funcionalizados con puertas moleculares y su aplicación en protocolos de reconocimiento. En primer lugar se preparó un material para la detección óptica de glutatión (GSH). Para ello se emplearon nanopartículas de MCM-41 mesoporosas como soporte inorgánico. Los poros del soporte fueron cargados con el colorante safranina O y la superficie externa funcionalizada con oligo(etilenglicol) conteniendo enlaces disulfuro. También se prepararon y caracterizaron varios materiales híbridos para la detección selectiva del anión hidrógeno sulfuro. En este caso también se empleó, como soporte inorgánico, sílice mesoporosa MCM-41. Los poros del soporte inorgánico fueron cargados con [Ru(bipy)3]2+ y la superficie externa funcionalizada con varios complejos macrocíclicos de Cu(II). El material sensor final fue obtenido al añadir el anion hexametafosfato, que compleja con los complejos de Cu(II), produciendo un bloqueo de los poros.
[CAT] Resum La present tesi doctoral titulada "Química supramolecular: Nous dosímetres químics i materials híbrids per a la detecció cromo-fluorogènica d'anions i molècules neutres." està basada en l'aplicació dels principis bàsics de la química supramolecular i de la ciència dels materials en el desenvolupament de sensors òptics per a anions i molècules neutres. El segon capítol d'aquesta tesi doctoral està dedicat a la preparació de dosímetres químics per a la detecció cromo-fluorogènica de fluorur, diisopropil fluorofosfat (DFP) i sulfur d'hidrogen. Per a la detecció òptica de l'anió fluorur es va sintetitzar un derivat de piridina funcionalitzat amb un t-dibutildimetilsilil èter. En aquest capítol també es descriu la preparació d'un dosímetre químic per a la detecció de DFP, que és un simulant d'agents nerviosos. Aquest dosímetre està basat en un estilbè funcionalitzat amb una sal de piridina que conté grups hidroxil i silis èter en la seua estructura. Finalment varen ser preparades dues famílies de sensors per a la detecció òptica de sulfur d'hidrogen. La primera família consisteix en fluoròfors comuns funcionalitzats amb 2,4-dinitrofenil èters. Els sensors preparats no presentaren una emissió de fluorescència significativa mentre que, en presencia de l'anió hidrogen sulfur, es va observar un augment significatiu. La segona família de dosímetres també estava composada per certs fluròfors però, en aquest cas, funcionalitzats amb grups azida i sulfonilazida. Els dosímetres preparats, seguint aquesta segona aproximació, tampoc donaren una fluorescència significativa observant-se un augment de la mateixa al afegir l'anió hidrogen sulfur. El tercer capítol d'aquesta tesi doctoral està dedicat a la preparació de materials híbrids nanoscòpics funcionalitzats amb portes moleculars i la seua aplicació en protocols de reconeixement. En primer lloc es va preparar un material per a la detecció òptica de glutatió (GSH). Per a aquest propòsit es varen emprar nanopartícules MCM-41 mesoporoses com a suport inorgànic. Els porus del suport varen ser carregats amb el colorant safranina O i la superfície externa funcionalitzada amb oligo(etilenglicol) que contenia enllaços disulfurs. També varen ser preparats i caracteritzats diversos materials híbrids per a la detecció selectiva de l'anió hidrogen sulfur. En aquest cas també es va emprar, com a suport inorgànic, sílice mesoporosa MCM-41. Els porus del suport inorgànic varen ser carregats amb [Ru(bipy)3]2+ i la superfície externa funcionalitzada amb diversos complexos macrocíclics de Cu(II). El material sensor final es va obtindre al afegir l'anió hexametafosfat, que es complexa amb macrocicles de Cu(II), produint un bloqueig dels porus.
El Sayed Shehata Nasr, S. (2015). Supramolecular Chemistry: New chemodosimeters and hybrid materials for the chromo-fluorogenic detection of anions and neutral molecules [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/52598
TESIS

The present work describes new aspects of organic and supramolecular chemistry. The scientific contribution consists of two parts, which focus on the development of receptors for the sulfate anion and quantitative assessment of stacking interactions between an anthracene dye and nucleobases in an aqueous solution. In Chapter 1, basic concepts concerning supramolecular chemistry and recognition of cations and anions are discussed, as well as modern methods for the determination of binding constants. Particular attention is paid to fluorescence sensing of ions and underlying mechanisms of binding-induced fluorescence responses. Chapter 2 is dedicated to the design and synthesis of new fluorescent sulfate receptors functioning in aqueous solution. After a short review of the most effective sulfate receptors/probes created so far, a new design of PET probes for sulfate sensing is presented. The syntheses and anion binding properties of new compounds are described. The experimental data obtained for the receptors are discussed in detail to reveal the origin of high selectivity towards sulfate. Chapter 3 explores the importance of nucleobase–arene stacking interactions in recognition of nucleotides by synthetic receptors. Various experimental and theoretical approaches are presented to assess dispersion interactions between aromatic rings and nucleobases in the receptor–nucleotide complexes.

Mannose receptor (MR) is widely expressed on macrophages, immature dendritic cells, and a variety of epithelial and endothelial cells. It is a 180 kD type I transmembrane receptor whose extracellular region consists of three parts: the amino-terminal cysteine-rich domain (Cys-MR); a fibronectin type II-like domain; and a series of eight tandem C-type lectin carbohydrate recognition domains (CRDs). Two portions of MR have distinct carbohydrate recognition properties: Cys-MR recognizes sulfated carbohydrates and the tandem CRD region binds terminal mannose, fucose, and N-acetyl-glucosamine (GlcNAc). The dual carbohydrate binding specificity allows MR to interact with sulfated and nonsulfated polysaccharide chains, and thereby facilitating the involvement of MR in immunological and physiological processes. The immunological functions of MR include antigen capturing (through binding non-sulfated carbohydrates) and antigen targeting (through binding sulfated carbohydrates), and the physiological roles include rapid clearance of circulatory luteinizing hormone (LH), which bears polysaccharide chains terminating with sulfated and non-sulfated carbohydrates.

We have crystallized and determined the X-ray structures of unliganded Cys-MR (2.0 Å) and Cys-MR complexed with different ligands, including Hepes (1.7 Å), 4SO_4-N-Acetylgalactosamine (4SO_4-GalNAc; 2.2 Å), 3SO_4-Lewis^x (2.2 Å), 3S04-Lewis^a (1.9 Å), and 6SO_4-GalNAc (2.5 Å). The overall structure of Cys-MR consists of 12 anti-parallel β-strands arranged in three lobes with approximate three fold internal symmetry. The structure contains three disulfide bonds, formed by the six cysteines in the Cys-MR sequence. The ligand-binding site is located in a neutral pocket within the third lobe, in which the sulfate group of ligand is buried. Our results show that optimal binding is achieved by a carbohydrate ligand with a sulfate group that anchors the ligand by forming numerous hydrogen bonds and a sugar ring that makes ring-stacking interactions with Trpll7 of CysMR. Using a fluorescence-based assay, we characterized the binding affinities between CysMR and its ligands, and rationalized the derived affinities based upon the crystal structures. These studies reveal the mechanism of sulfated carbohydrate recognition by Cys-MR and facilitate our understanding of the role of Cys-MR in MR recognition of its ligands.

This article deals with the determination of technically important properties, the recognition of microstructure and pore structure, and the mortar resistance of a new cement kind NONRIVAL CEM I 52.5 N containing 7.94% wt. of C3A to 5% sodium sulfate solution. Both reference types of cement were industrially manufactured: 1) ordinary Portland cement CEM I 42.5 R and 2) Portland cement CEM I 42.5 R – SR 0, declared as sulfate resistant because of C3A = 0%. The research was carried out at standardized mortars. The used sodium sulfate solution, which contained 33802.8 mg of aggressive SO4 2− per liter, exceeded approximately 5 to 10 times the concentration of the third degree of aggressiveness of the XA chemical environment according to STN EN 206 + A1. The reference medium was drinking water. The 5-year results of non-destructive and destructive physical-mechanical tests as well as the formed microstructure and pore structure in both liquid media were evaluated. The cause of the NONRIVAL CEM I 52.5 N sulfate resistance was explained, despite the manufacturer’s declared C3A content of up to 8% by weight. Sulfate resistance of NONRIVAL CEM I 52.5 N is found comparable to that of sulfate resistant CEM I 42.5 R – SR 0.

In this, the last major chapter of the book, we turn our attention to the applications of modern electronic structure models and concepts to more general geochemical problems; namely, those described by Goldschmidt as being concerned with the “distribution of elements in the geochemical spheres and the laws governing the distribution of the elements” (see Preface). The majority of minerals and rocks originally formed by crystallization from melts, and so the first section of this chapter is devoted to considering the nature of melts (and glasses), structure and bonding in melts, and the partitioning of elements (particularly transition elements) between the melt and crystallizing solid phases. The classic work of Bowen (1928) led to the recognition of particular sequences of crystallization and crystal-melt reaction relationships in the silicate melts from which major rock types form, as enshrined in the “Bowen Reaction Series.” Attempts were also made to explain the incorporation of particular elements into particular mineral structures using simple crystal chemical arguments, notably as laid down in “Goldschmidt’s Rules” (Goldschmidt, 1937). Such concepts are reappraised in the light of modern electronic structure theories. The other major realm of formation of minerals and rocks, and the most important medium of transport and redistribution of the chemical elements at the Earth’s surface, is the aqueous solution. The molecular and electronic structures of aqueous solutions, their behavior at elevated temperatures, formation and stabilities of complexes in solution, and the mechanisms of reactions in solution are all considered in the second section of this chapter. The surfaces of minerals (or other crystalline solids) differ from the bulk material in terms of both crystal structure and electronic structure. A great variety of spectroscopic, diffraction, scanning, and other techniques are now available to study the nature of solid surfaces, and models are being developed to interpret and explain the experimental data. These approaches are discussed with reference to a few examples of oxide and sulfide minerals. Although relatively few studies have been undertaken specifically of the surfaces of minerals, many of the reaction phenomena that occur in natural systems take place at mineral surfaces, so that such surface studies represent an important area of future research.

This paper describes a case study involving chronic plugging of 5μm particulate filters in a gas turbine diesel fuel system in a Red Sea refinery. Rapid plugging of the filters was caused by water-in-diesel emulsions generated in diesel fuel supply tanks. Sludge with a wax-like appearance recovered from the 5 μm filters was, in fact, found to be composed of up to 50 percent water with no significant wax content. X-ray studies of the filter catch solids revealed a variety of iron oxide phases, sodium chloride, and high concentrations of sodium sulfate. Microbial cultures inoculated from storage tank ‘rag’ layers yielded moderate to high counts of general aerobic bacteria (GAB), moderate fungal cultures (yeast and molds) and low sulfate reducing bacteria (SRB). Elemental analysis of water in supply tanks where microbial activity was highest revealed ion concentrations similar to those found in Red Sea water. Sulfur isotope ratios in sulfate from filter catches suggest that much of the sulfate was derived from microbial metathesis of sulfur-bearing hydrocarbons. Frequent contamination of on-shore liquid hydrocarbon fuel tanks with sea water can cause corrosion and create a favorable environment for bacterial growth. Surfactant byproducts of microbial activity are capable of stabilizing emulsions, suspending water soluble salts such as sodium sulfate, and metals such as lead and copper. Copper is well known to promote gum formation, while all of these contaminants are potentially corrosive to gas turbine hot gas path components.

V1tronect1n/S-Prote1n (VN/SP) is a glycoprotein present at a concentration of 200-400 ug/ml 1n plasma and serum. It has been shown to promote cel 1-substratum adhesion and to act as an Inhibitor of the membrane attack complex of complement and of the inactivation of thrombin by antithrombin III in the presence of low levels of heparin. We have previously shown that VN/SP binds more avidly to heparln-agarose and to a monoclonal antibody (MaVN/SP)-Sepharose column when present 1n serum rather than 1n plasma. In order to examine the possibility of a serum-induced conformational change, we utilized, 1n this study, an Indirect enzyme-linked Immunosorbent system to test for the exposure of new antigenic determinants. When MaVN/SP was Incubated with plasma or serum, recognition of VN/SP 1n serum was approximately 50 fold greater than recognition of VN/SP in plasma. Since VN/SP has been shown to Interact strongly with the thromb1n-ant1thrombin complex, we examined the antigenicity of VN/SP when Incubated with thrombin and antithrombin 1n the presence and absence of heparin. Incubation of VN/SP with heparin promoted a 2.5-fold Increase 1n recognition by MaVN/SP. When MaVN/SP was Incubated with thromb1n-ant1thrombin but not thrombin or antithrombin alone, recognition was Increased by 7-fold 1n the absence of heparin and by 32-fold 1n the presence of heparin. This differential recognition of VN/SP was not observed with a second monoclonal antibody raised originally against S-Prote1n. Treatment of VN/SP with various glycosaminoglycans and polysaccharides demonstrated the following relative potencies for Induction of the partial antigenic change: dextran sulfate>fucoidan>heparin> dermatan suIfate>hyaluronic acid. No effect was detected upon Incubation of VN/SP with keratan sulfate, heparan sulfate or chondroltln sulfate. These data suggest a conformational change Induced by thrombin-antlthrombin which may allow VN/SP to Interact more avidly with other molecules such as heparin. The physiological role of this putative conformational change is under investigation.

In vivo treatment of experimental animals with vitamin K-antagonists induces the accumulation of non-carboxylated coagulation factor precursors in the liver, where they are tightly bound to vitamin K-dependent carboxylase. If hepatic carboxylase is isolated from warfarin-treated animals, it is obtained therefore almost exclusively in the form of an enzyme/substrate complex. If carboxylase is prepared from non-treated animals, on the other hand, the resulting enzyme is predominantly substrate-free. Small substrates like F L E E L or decarboxylated osteocalcinare carboxylated equally well by both types of carboxylase, but protein substrates(Mr > 30 000) are recognized exclusively by substrate-free carboxylase.Initial attempts to purify carboxylasewere performed with livers from warfarin-treated cows as a starting material. Antibodies against the normal blood coagulation factors crossreact with the hepatic precursor proteins so that the enzyme/substrate complexes could be specifically extracted from detergent-solubilized microsomes by the substrate/antibody interaction. This procedure resulted ina substantial purification of carboxylase, but because its endogenous substrate remained firmly bound, even after it had been carboxylated in vitro, the enzyme system was not suitable for the carboxylation of protein substrates.Therefore a second strategy was developed by which substrate-free carboxylase (from normal livers) was partly purified by sequential extraction of the microsomal membranes with detergents, followed by ammonium sulfate precipitation and size exclusion chromatography.This procedure resulted in a soluble carboxylase complex, still consisting of 7 proteins and phosphatidylcholine. Although further dissociation of the complex resulted in a complete loss of activity, it is not sure if all components play a role in the carboxylation reaction. Exogenous substrates which could be carboxylated by substrate-free carboxylase were: the penta-peptide F L E E L, descarboxyprothrombin from bovine plasma, thermally decarboxylated osteocalcin from bovine bone and non-car-boxy lated coagulaton factor precursors which had been produced by recombinant-DNA techniques in various laboratories. The . efficiency of CO^ incorporation was: 1 mole per 100 moles of F L E E L, 1 mole per 240 moles of descarboxy-prothrombin, 1 mole per mole of decarboxylated osteocalcin and 8 moles per mole of a recombinant factor IX precursor. We assume that the high efficiency with which the recombinant coagulation factor precursors were carboxylated is due to the presence of at least part of their leader sequence. The importance of the aminoacid chain preceding the first carboxylatable Glu residue is demonstrated by the fact that descarboxylated osteocalcin of bovine origin is carboxylated with a relatively high efficiency, whereas descarboxylated osteocalcin from monkey bone is not recognized atal.. Yet the only difference between the two substrates is found in their aminoacids 3 and 4, whereas the first carboxylatable Glu occurs at position 17. It seems, therefore, that the aminoacids 1-16 in bovine osteocalcin mimic to some extent part of the leader sequence in the coagulation factor precursors. Chemical or biochemical modification of decarboxylated osteocalcin might reveal which structural features contribute to its recognition by hepatic carboxylase.The optimal conditions for carboxylation include a high concentration of dithiols (e.g. DTT) and under these conditions disulfide bridges are reduced. Obviously this will lead to a complete destruction of the biological activity of various carboxylated products. Therefore we have searched for a more natural reducing system and it was found that the bacterial thioredoxin/thiore-doxin-reductase system in the presence of 40 uM NADFH was able to replace DTT in the reaction mixtures. Since a comparable system also occurs in calf liver it seems not unlikely that this is the physiological counterpart of the dithiols used in vitro.

In this paper, the cause and nondestructive test technology of backside cracks were studied. On the one hand, failure analysis revealed that, the backside cracks, which were found universally in the stainless steel inner linings of urea reactors in recent years, were caused by the interaction of wet hydrogen sulfide (H2S) and chloride ions (Cl−) stress corrosion cracking (SCC), and the source of the H2S and Cl− was the leak detection steam. On the other hand, the use of Lamb wave inspection was studied because of the inapplicability of other inspection methods. The dispersion curves of phase and group velocities of 316LMod stainless steel linings were derived out according to the Rayleigh-Lamb equation. The main Lamb wave mode of the backside defects was drawn out by 2D FFT to echo signals of the test plate including artificial defects. Then, the Lamb wave testing parameters of 8mm 316LMod stainless steel were defined. The defect equivalent evaluation diagram was given through the defect recognition of the test plate including artificial defects. And the quantitative evaluation of Lamb wave inspection to the backside defects was realized. After all the analysis done, the measures to avoid backside cracks were put forward, and the inspection technology was verified by applications on urea reactors.

Semiconductor nanocrystals have unique optical properties due to quantum confinement effects, and a variety of promising approaches have been devised to interface the nanomaterials with biomolecules for bioimaging and therapeutic applications. Such bio-interface can be facilitated via a DNA template for nanoparticles as oligonucleotides can mediate the aqueous-phase nucleation and capping of semiconductor nanocrystals.[1,2] Here, we report a novel scheme of synthesizing fluorescent nanocrystal quantum dots (NQDs) using DNA aptamers and the use of this biotic/abiotic nanoparticle system for growth inhibition of MCF-7 human breast cancer cells for the first time. Particularly, we used two DNA sequences for this purpose, which have been developed as anti-cancer agents: 5-GGT GGT GGT GGT TGT GGT GGT GGT GG-3 (also called, AGRO) and 5-(GT)15-3.[3–5] This study may ultimately form the basis of unique nanoparticle-based therapeutics with the additional ability to optically report molecular recognition. Figure 1a shows the photoluminescence (PL) spectra of GT- and AGRO-passivated PbS QD that fluoresce in the near IR, centered at approximately 980 nm. A typical synthesis procedure involves rapid addition of sodium sulfide in the mixture solution of DNA and Pb acetate at a molar ratio of 2:4:1. The resulting nanocrystals are washed to remove unreacted DNA and ions by adding mixture solution of NaCl and isopropanol, followed by centrifugation. The precipitated nanocrystals are collected and re-suspended in aqueous solution by mild sonication. Optical absorption measurements reveal that approximately 90 and 77% of GT and AGRO DNA is removed after the washing process. The particle size distribution in Figure 1b suggests that the GT sequence-capped PbS particles are primarily in 3–5 nm diameter range. These nanocrystals can be easily incorporated with mammalian cells and remain highly fluorescent in sub-cellular environments. Figure 1c serially presents an optical image of a MCF-7 cell and a PL image of the AGRO-capped QD incorporated with the cell. Figure 1. (a) Normalized fluorescence spectra of PbS QD synthesized with GT and AGRO sequences, which were previously developed as anti-cancer agents. The DNA-capped QD fluoresce in the near IR centered at ∼980 nm. (b) TEM image of GT-templated nanocrystals ranging 3–5 nm in diameter. (c) Optical image of an MCF-7 human breast cancer cell after a 12-hour exposure to aptamer-capped QD. (d) PL image of AGRO-QD incorporated with the cell, indicating that these nanocrystals remain highly fluorescent in sub-cellular environments. One immediate concern for interfacing inorganic nanocrystals with cells and tissue for labeling or therapeutics is their cytotoxicity. The nanoparticle cytotoxicity is primarily determined by material composition and surface chemistry, and QD are potentially toxic by generating reactive oxygen species or by leaching heavy metal ions when decomposed.[6] We examined the toxicity of aptamer-passivated nanocrystals with NIH-3T3 mouse fibroblast cells. The cells were exposed to PbS nanocrystals for 2 days before a standard MTT assay as shown in Figure 2, where there is no apparent cytotoxicity at these doses. In contrast, Pb acetate exerts statistically significant toxicity. This observation suggests a stable surface passivation by the DNA aptamers and the absence of appreciable Pb2+ leaching. Figure 2. Viability of 3T3 mouse fibroblast cells after a 2-day exposure to DNA aptamer-capped nanocrystals. There is no apparent dose-dependent toxicity, whereas a statistically significant reduction in cell viability is observed with Pb ions. Note that Pb acetate at 133 μM is equivalent to the Pb2+ amount that was used for PbS nanocrystal synthesis at maximum concentration. Error bars are standard deviations of independent experiments. *Statistically different from control (p<0.005). Finally, we examined if these cyto-compatible nanoparticle-aptamers remained therapeutically active for cancer cell growth inhibition. The MTT assay results in Figure 3a show significantly decreased growth of breast cancer cells incorporated with AGRO, GT, and the corresponding templated nanocrystals, as anticipated. In contrast, 5-(GC)15-3 and the QDs synthesized with the same sequence, which were used as negative controls along with zero-dose control cells, did not alter cell viability significantly. Here, we define the growth inhibition efficacy as (100 − cell viability) per DNA of a sample, because the DNA concentration is significantly decreased during the particle washing. The nanoparticle-aptamers demonstrate 3–4 times greater therapeutic activities compared to the corresponding aptamer drugs (Figure 3b). We speculate that when a nanoparticle-aptamer is internalized by the cancer cells, it forms an intracellular complex with nucleolin and nuclear factor-κB (NF-κB) essential modulator, thereby inhibiting NF-κB activation that would cause transcription of proliferation and anti-apoptotic genes.[7] The nanoparticle-aptamers may more effectively block the pathways for creating anti-apoptotic genes or facilitate the cellular delivery of aptamers via nanoparticle uptake. Our additional investigation indicates that the same DNA capping chemistry can be utilized to produce aptamer-mediated Fe3O4 nanocrystals, which may be potentially useful in MRI and therapeutics, considering their magnetic properties and biocompatibility. In summary, the nanoparticle-based therapeutic schemes developed here should be valuable in developing a multifunctional drug delivery and imaging agent for biological systems. Figure 3. Anti-proliferation of MCF-7 human breast cancer cells with aptamer-passivated nanocrystals. (a) Viability of MCF-7 cells exposed to AGRO and GT sequences, and AGRO-/GT-capped QD for 7 days. The DNA concentration was 10 uM, while the particles were incubated with cells at 75 nM. (b) Growth inhibition efficacy is defined as (100 − cell viability) per DNA to correct the DNA concentration after particle washing.