Differences In Binding Properties Research Articles

Contribution of different adherent properties of Granulicatella adiacens and Abiotrophia defectiva to their associations with oral colonization and the risk of infective endocarditis.

Granulicatella adiacens (G. adiacens) and Abiotrophia defectiva (A. defectiva) colonize the oral cavity and form part of the normal flora in the intestinal and genitourinary tracts. As reported previously, the frequency of isolation of G. adiacens from the oral cavity was much higher than that of A. defectiva. However, it has been reported that compared with G. adiacens, A. defectiva was isolated at considerably higher frequencies from the blood of patients with infective endocarditis (IE). Hence, in this study, the in vitro interaction of G. adiacens and A. defectiva strains with host surfaces and biofilm formation was examined to assess whether their different adhesive properties contribute to their associations with oral colonization and IE, respectively. G. adiacens exhibited an increased binding ability to saliva-coated hydroxyapatite beads than A. defectiva following the addition of CaCl2. Furthermore, biofilm formation was observed only for G. adiacens with the use of a polystyrene tube and scanning electron microscopy analysis. Conversely, A. defectiva displayed significantly greater adherence to human umbilical vein endothelial cells and immobilized fibronectin than G. adiacens. These findings suggest that differences in binding properties to host components imply specific binding mechanisms in G. adiacens and A. defectiva, which might mediate selective colonization in the oral cavity or are associated with the pathogenicity of endocarditis.

Surface complexation modeling of interactions between freshwater and marine diatom species and trace elements (Mo, W, Cr, Ge, Ga, Al)

Towards a better understanding of photosynthetic algae interaction with less common trace elements, we performed adsorption experiments of Al, Ga, Ge, Cr (VI), Mo, and W onto four diatom species typical for marine, estuarine, and freshwater aquatic systems. Using a surface complexation approach, we tentatively identified the nature of functional groups involved in the complexation of trace elements with diatom surface moieties and we quantified the stability of surface complexes formed at the diatom – solution interface. The differences in binding properties between freshwater and marine species are linked to variable proportion of three main binding groups at the diatom surfaces, namely amine, carboxylate and silanol. Under acidic conditions, the oxyanion (MoO42−, WO42−, CrO42−) sorption occurred electrostatically with a 1:1 ratio to the protonated amino groups. Trivalent cations (Ga3+ and Al3+) were complexed by deprotonated carboxyl groups, while the anionic hydroxylated forms of these metals (Ga(OH)4− and Al(OH)4−) formed mononuclear monodentate complex with positively charged amino groups at higher pH. Finally, Ge(IV) sorption was controlled by interaction with neutral carboxylic, silanol and amino groups. Taken together, the surface complexation model developed in this study allows rigorous description of anions and trivalent hydrolysates adsorption onto diatom surfaces and demonstrates potential importance of diatoms for the control of trace element transfer in aquatic settings.

COMPARISON OF BINDING PROPERTIES OF THK5117, THK5351, PBB3 AND T807 IN AUTOPSIES OF ALZHEIMER DISEASE CASES

Several families of tau PET tracers recently developed have been studied in vivo in patients after having shown good in vitro binding-properties. Some differences in binding properties have been observed between the tau PET tracers in Alzheimer disease and non-AD tauopathies. Recent observations report presence of off-target binding. Here we aimed at characterizing the binding properties as well as possible off-target binding of the tau tracers by comparing two THK compounds, THK5117 and THK5351 with T807 and PBB3, head-to-head in the same human brain tissue, using autoradiography on large frozen section from AD cases and binding assay in brain homogenates. Autoradiographies on large frozen section were done using 3H-THK5351, 3H-THK5117, 11C-THK5351, 11C-PBB3 and 18F-T807. Competition autoradiography using unlabeled T807 were performed for the 11C compound. In vitro binding assay competition using 3H-THK5351 with unlabeled THK5351, THK5117 and T807 were performed in hippocampus homogenates. Competitions binding assay and autoradiography were performed using 3H-THK5117, 3H-L-deprenyl with unlabeled THK5117, R-deprenyl, rasagiline and chlorgyline to characterize the MAO off-target component. 11C-PPB3, 11C-THK5351 and 18F-T807 showed similar regional distribution in autoradiography but with different intensity. Unlabeled THK5351 displaced 50% of 11C-THK5351 binding and less than 10% of 11C-PBB3 binding in all regions. Unlabeled PBB3 displaced 50 % of 11C-PBB3 binding. Unlabeled T807 displaced up to 70 % of 18F-T807 binding in frontal and temporal cortex, 30% of 11C-THK5351 in frontal and temporal cortex and up to 40% in the basal ganglia, and displaced less than 20 %of 11C-PBB3 in all regions. In vitro binding assay using 3H-THK5351 in competition with unlabeled THK5351, THK5117 and T807 showed at least two binding sites. While a 4 times differences in affinity for the binding site in nanomolar range was observed between THK5351 and T807, they shared one binding site in the picomolar range. Tau tracers from three different families showed all different binding properties in AD cases. Greater similarity in binding properties was observed between THK5351 and T807 than with PBB3. These results may have an influence on the outcome of the clinical use in vivo PET study targeting different form of tauopathies.

Label-Free Detection of Sequence-Specific DNA Based on Fluorescent Silver Nanoclusters-Assisted Surface Plasmon-Enhanced Energy Transfer.

We have developed a label-free method for sequence-specific DNA detection based on surface plasmon enhanced energy transfer (SPEET) process between fluorescent DNA/AgNC string and gold nanoparticles (AuNPs). DNA/AgNC string, prepared by a single-stranded DNA template encoded two emitter-nucleation sequences at its termini and an oligo spacer in the middle, was rationally designed to produce bright fluorescence emission. The proposed method takes advantage of two strategies. The first one is the difference in binding properties of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) toward AuNPs. The second one is SPEET process between fluorescent DNA/AgNC string and AuNPs, in which fluorescent DNA/AgNC string can be spontaneously adsorbed onto the surface of AuNPs and correspondingly AuNPs serve as "nanoquencher" to quench the fluorescence of DNA/AgNC string. In the presence of target DNA, the sensing probe hybridized with target DNA to form duplex DNA, leading to a salt-induced AuNP aggregation and subsequently weakened SPEET process between fluorescent DNA/AgNC string and AuNPs. A red-to-blue color change of AuNPs and a concomitant fluorescence increase were clearly observed in the sensing system, which had a concentration dependent manner with specific DNA. The proposed method achieved a detection limit of ∼2.5 nM, offering the following merits of simple design, convenient operation, and low experimental cost because of no chemical modification, organic dye, enzymatic reaction, or separation procedure involved.

The molecular mechanism of betacellulin induced corneal epithelial wound healing

The epidermal growth factor receptor (EGFR) has been shown to be necessary and sufficient for mediating corneal epithelial homeostasis and wound healing. Experimentally, activation of EGFR by epidermal growth factor (EGF), increases healing in a corneal wound; however, therapeutic use of EGF does not improve corneal healing in patients. This study looks at wound healing elicited by other EGFR ligands, specifically betacellulin (BTC). Using human telomerase‐immortalized corneal epithelial cells (hTCEpi) in an in vitro wound healing assay, BTC was shown to be more efficacious than EGF during wound closure. Since BTC can also stimulate ErbB4, an EGFR family member, the efficacy of BTC may be due to activation of this receptor population. However, mRNA and protein levels were assessed in both hTCEpi and primary human corneal epithelial cells and revealed the absence of ErbB4. Differences in ligand binding properties could also explain BTC efficacy. Using radiolabeled ligands to assess binding sites, ligand affinity, pH sensitivity and competition revealed differences between BTC and EGF in these areas. We conclude that the increased efficacy seen with BTC over EGF is not from stimulation of ErbB4 but is, at least in part, from differences in binding properties. Funding: NIH 1R01GM092874 and P20 RR 017703.

Study of Receptor-Chaperone Interactions Using the Optical Technique of Spectroscopic Ellipsometry

This work describes a detailed quantitative interaction study between the novel plastidial chaperone receptor OEP61 and isoforms of the chaperone types Hsp70 and Hsp90 using the optical method of total internal reflection ellipsometry (TIRE). The receptor OEP61 was electrostatically immobilized on a gold surface via an intermediate layer of polycations. The TIRE measurements allowed the evaluation of thickness changes in the adsorbed molecular layers as a result of chaperone binding to receptor proteins. Hsp70 chaperone isoforms but not Hsp90 were shown to be capable of binding OEP61. Dynamic TIRE measurements were carried out to evaluate the affinity constants of the above reactions and resulted in clear discrimination between specific and nonspecific binding of chaperones as well as differences in binding properties between the highly similar Hsp70 isoforms.

Evaluation of an approach for the characterization of reactive and available pools of 20 potentially toxic elements in soils: Part II – Solid-solution partition relationships and ion activity in soil solutions

To assess environmental risks related to contaminants in soil it is essential to predict the available pool of inorganic contaminants at regional scales, accounting for differences between soils from variable geologic and climatic origins. An approach composed of a well-accepted soil extraction procedure (0.01 M CaCl 2) and empirical Freundlich-type models in combination with mechanistically based models which to date have been used only in temperate regions was applied to 136 soils from a South European area and evaluated for its possible general use in risk assessment. Empirical models based on reactive element pools and soil properties (pH, organic carbon, clay, total Al, Fe and Mn) provided good estimations of available concentrations for a broad range of contaminants including As, Ba, Cd, Co, Cu, Hg, Mo, Ni, Pb, Sb, Se and Zn ( r 2: 0.46–0.89). The variation of the pools of total Al in soils expressed the sorptive capacity of aluminosilicates and Al oxides at the surfaces and edges of clay minerals better than the actual variability of clay contents. The approach has led to recommendations for further research with particular emphasis on the impact of clay on the solubility of As and Sb, on the mechanisms controlling Cr and U availability and on differences in binding properties of soil organic matter from different climatic regions. This study showed that such approach may be included with a good degree of certainty for first step risk assessment procedures to identify potential risk areas for leaching and uptake of inorganic contaminants in different environmental settings.

Species Differences in the Pharmacology and Toxicology of PEGylated Helper-Dependent Adenovirus

Clinically relevant doses of helper-dependent adenoviruses (HDAds) provoke the host response against capsid proteins in primates and rodents. To determine if PEGylation truly affects this, baboons and mice were given either HDAd or PEG-HDAd expressing beta-galactosidase at 5 × 10¹¹ or 3 × 10¹² virus particles per kilogram (vp/kg) by iv infusion. Serum cytokines and blood chemistries were assessed for 96 h. PEG-HDAd reduced IL-6 6-fold in mice and 3-fold in the primate. This vector reduced IL-12 by 50% in both animal models. PEGylation reduced serum transaminases by approximately 50% at each dose in the primate and the mouse. PEGylation did not alter hepatic transduction efficiency in the mouse but did reduce transduction efficiency in the liver and the spleen of primates. Unmodified and PEGylated virus suppressed hepatic CYP3A activity in both animal models. PEGylation doubled the half-life (t(½)) of the virus in the mouse and cut plasma clearance (CL) in half without affecting the half-life in primates. These results suggest that there are notable species-specific differences in the biodistribution of and response to PEG-modified vectors which may be linked to differences in binding properties to coagulation factors, receptor density and tissue architecture in the liver.

Replacement of a Nitrogen by a Phosphorus Donor in Biomimetic Copper Complexes: a Surprising and Informative Case Study with Calix[6]arene-Based Cryptands

The aim of the paper is to characterize Cu complexes in the P(Ar)N(3) environment provided by ligands derived from triphenylphosphine P(C(6)H(4)CH(2)NHR)(3) and compare their coordination behavior and reactivity with those obtained with all-nitrogen ligands such as tren. It is shown that coordination of the PN(3) ligand (R = iPr) to Cu(I) and Cu(II) leads to complexes whose coordination sphere is hardly controlled as they readily undergo decoordination of either one N or the P donor together with oxidation of the latter. In strong contrast, when grafted on the small rim of a calix[6]arene, the P(Ar)N(3) is geometrically constrained into a tripod that enforces the metal center to remain in the same environment with a P-Cu bond for both oxidation states. These calix[6]PN(3)-based Cu(I) and Cu(II) complexes react readily with exogenous ligands, making a comparison with calix[6]tren-based copper complexes possible. Indeed, reactivity studies in solution highlight very different behaviors. The complex [Cu(calix[6]PN(3))](2+) shows an unusual affinity for weak sigma-donors (e.g., MeCN > EtOH), while the analogous cuprous complex, [Cu(calix[6]PN(3))](+), displays a surprising affinity for hard O-donor ligands (EtOH, DMF), which has never been observed for the tren analogues. Even more surprising is the lack of reactivity of [Cu(calix[6]PN(3))](+) toward dioxygen, which contrasts strongly with the high reactivity of the [Cu(calix[6]tren)](+) complex. In an attempt to explain the observed differences in binding properties and reactivity, Density Functional Theory calculations and electronic spectra simulations were undertaken. They suggest that coordination of the soft P(Ar)(3) center allows to tune the metal ion properties, either by absorbing excess electron density from Cu(I), or by increasing the electronic density of Cu(II). This is due to the simultaneous presence of the phosphorus atom (sigma-donor) in apical position and the aromatic groups (pi-acceptors) bound to the P-atom.

Alanine Scanning of a Putative Receptor Binding Surface of Insulin-like Growth Factor-I

Current evidence supports a binding model in which the insulin molecule contains two binding surfaces, site 1 and site 2, which contact the two halves of the insulin receptor. The interaction of these two surfaces with the insulin receptor results in a high affinity cross-linking of the two receptor alpha subunits and leads to receptor activation. Evidence suggests that insulin-like growth factor-I (IGF-I) may activate the IGF-I receptor in a similar mode. So far IGF-I residues structurally corresponding to the residues of the insulin site 1 together with residues in the C-domain of IGF-I have been found to be important for binding of IGF-I to the IGF-I receptor (e.g. Phe(23), Tyr(24), Tyr(31), Arg(36), Arg(37), Val(44), Tyr(60), and Ala(62)). However, an IGF-I second binding surface similar to site 2 of insulin has not been identified yet. In this study, we have analyzed whether IGF-I residues corresponding to the six residues of the insulin site 2 have a role in high affinity binding of IGF-I to the IGF-I receptor. Six single-substituted IGF-I analogues were produced, each containing an alanine substitution in one of the following positions (corresponding insulin residues in parentheses): Glu(9) (His(B10)), Asp(12) (Glu(B13)), Phe(16) (Leu(B17)), Asp(53) (Ser(A12)), Leu(54) (Leu(A13)), and Glu(58) (Glu(A17)). In addition, two analogues with 2 and 3 combined alanine substitutions were also produced (E9A,D12A IGF-I and E9A,D12A,E58A IGF-I). The results show that introducing alanine in positions Glu(9), Asp(12), Phe(16), Leu(54), and Glu(58) results in a significant reduction in IGF-I receptor binding affinity, whereas alanine substitution at position 53 had no effect on IGF-I receptor binding. The multiple substitutions resulted in a 33-100-fold reduction in IGF-I receptor binding affinity. These data suggest that IGF-I, in addition to the C-domain, uses surfaces similar to those of insulin in contacting its cognate receptor, although the relative contribution of the side chains of homologous residues varies.

MECHANISM AND DYNAMICS OF CADHERIN ADHESION

Cadherins are essential cell adhesion molecules involved in tissue morphogenesis and the maintenance of tissue architecture in adults. The adhesion and selectivity functions of cadherins are located in their extracellular regions. Biophysical studies show that the adhesive activity is not confined to a single interface. Instead, multiple cadherin domains contribute to binding. By contrast, the specificity-determining site maps to the N-terminal domains, which adhere by the reciprocal binding of Trp2 residues from opposing proteins. Structural cooperativity can transmit the effects of subtle structural changes or ligand binding over large distances in the protein. Increasingly, studies show that differential cadherin-mediated adhesion, rather than exclusive homophilic binding between identical cadherins, direct cell segregation and the organization of tissue interfaces during morphogenesis. Force measurements quantified both kinetic and strength differences between different classical cadherins that may underlie cell sorting behavior. Despite the complex adhesion mechanisms and differences in binding properties, cadherin-mediated cell adhesion is also regulated by many other biochemical processes. Elucidating the mechanisms by which cadherins organize cell junctions and tissue architecture requires not only quantitative, mechanistic investigations of cadherin function but also investigations of the biochemical and cellular processes that can modulate those functions.

Anion control of isomerism, crystal packing and binding properties in a mononuclear zinc complex

The coordination chemistry of the tetradentate pyridyl N-donor ligand cis-3,5- bis-[2-pyridinyleneamin]- trans-hydroxycyclohexane (DDOP) has been investigated with zinc(II) nitrate and triflate. The resulting complexes, [Zn(DDOP)(H 2O)(NO 3)](NO 3) ( 1), and [Zn(DDOP)(H 2O)(OTf)](OTf) ( 2) differ not only in their counterions, but also the arrangement of the axial ligands and their solid state hydrogen bonded networks. Isothermal titration calorimetry was used to assess the difference in binding properties exhibited by the two zinc complexes at physiological pH in an aqueous environment. A series of coordinating amino acids were found to preferentially bind to the mononuclear zinc triflate ( 1) complex over the corresponding nitrate ( 2) assembly, with histidine exhibiting a two centre binding mode.

High density lipoproteins bind Aβ and apolipoprotein C-II amyloid fibrils

Disease-associated amyloid deposits contain both fibrillar and nonfibrillar components. The majority of these amyloid components originate or coexist in the bloodstream. To understand the nature of the interaction between the nonfibrillar and fibrillar components, we have developed a centrifugation method to isolate fibril binding proteins from human serum. Amyloid fibrils composed of either Abeta peptide or apolipoprotein C-II (apoC-II) cosedimented with specific serum proteins. Gel electrophoresis, mass spectrometry peptide fingerprinting, and Western analysis identified the major binding species as proteins found in HDL particles, including apoA-I, apoA-II, apoE, clusterin, and serum amyloid A. Sedimentation analysis showed that purified human HDL and recombinant apoA-I lipid particles bound directly to Abeta and apoC-II amyloid fibrils. These studies reveal a novel function of HDL that may contribute to the well-established protective effect of this lipoprotein class in heart disease.

Protein Interaction Analysis of ST14 Domains and Their Point and Deletion Mutants

ST14 (suppression of tumorigenicity 14) is a transmembrane serine protease that contains a serine protease catalytic (SP) domain, an SEA domain, two complement subcomponent C1r/s (CUB) domains, and four low density lipoprotein receptor class A domains. Glutathione S-transferase fusion proteins with SP, CUB, and low density lipoprotein receptor domains and their corresponding mutants were generated to analyze protein interactions with these domains. Modified glutathione S-transferase pull-down assays demonstrated the interaction between the SP domain and hepatocyte growth factor activator inhibitor-1. With the same method, a CUB domain-interacting protein was isolated and turned out to be the transmembrane protein with epidermal growth factor-like and two follistatin-like domains 1 (TMEFF1). Quantitative real time PCR revealed that the expression of the TMEFF1 gene was dependent on the transfection of the ST14 gene in the RKO cell line. Our results also suggested that ST14 and TMEFF1 were co-expressed in the human breast cancer cell line MCF7, human placenta, kidney, and liver tissues. Interestingly, these two genes were co-up-regulated in kidney tumors versus normal tissues, consistent with our results that showed the dependence of TMEFF1 expression on ST14 in RKO cells. Finally, homology modeling studies suggested that TMEFF1 might form a complex with ST14 by an interaction between epidermal growth factor and CUB domains.

Interaction of chromium(III) complex of chiral binaphthyl tetradentate ligand with DNA

Since conformation of the molecule plays a vital role in the activity of drug, we have investigated the DNA interaction of a chromium(III) complex with ligands in two conformations. Chromium(III) complexes derived from chiral binaphthyl Schiff base ligands, viz. R- and S-2,2′-bis(salicylideneamino) 1,1′-binaphthyl, have been synthesized and characterized by mass, IR, and electronic spectra. The interaction of these R- and S-binaphthyl Schiff base chromium(III) complexes with CT–DNA was investigated with the goal of examining whether the chirality has an influence on the chromium(III)–DNA binding properties. The difference in chirality of the ligand did not show any striking difference in binding properties. The binding constants for R and S conformers were estimated to be 18 (±0.4) × 10 3 and 9.4 (±0.3) × 10 3 M −1, respectively, through spectroscopic titrations. All the experimental results are suggestive that both the isomers are DNA groove binders. The results of steady-state as well as time-resolved fluorescence experiments, however, suggest that the R conformer has restricted mobility when bound to DNA because it is more deeply buried in the groove of DNA compared to the S isomer.

Multiple Extracellular Loops Contribute to Substrate Binding and Transport by the Escherichia coli Cobalamin Transporter BtuB

The Escherichia coli outer membrane TonB-dependent transporters for iron complexes and cobalamins recognize their multiple and diverse substrates with high specificity and affinity. The X-ray crystallographic structures of several transporters show that the substrate-binding surfaces are comprised of residues from the internal globular domain and multiple extracellular loops. The extracellular loops on the N-terminal half of the transmembrane beta-barrel of the cobalamin transporter BtuB participate in binding of the cofactor calcium atoms and undergo substantial conformation changes upon substrate binding. The functional relevance of the five C-terminal loops was examined by examining the effects of short in-frame deletions. Each loop contributed in different ways to the binding of BtuB substrates. Deletions in loops 7, 8, 9, and 11 strongly decreased cobalamin binding and transport, whereas deletions in loops 8, 9, and 10 affected binding and entry of phage BF23. None of the loops were essential for the action of colicin E1 or E3, which is consistent with the crystallographic observation that the colicin E3 receptor-binding domain can contact almost all of the loops. A deletion in loop 9 or 11 eliminated the ability of cobalamin to inhibit the action of colicin E1. These phenotypes show that there are multiple independent binding elements and point out similarities and differences in binding properties among the TonB-dependent transporters.

Differences in binding properties of two proton pump inhibitors on the gastric H +,K +-ATPase in vivo

Restoration of acid secretion after treatment with covalently-bound proton pump inhibitors may depend on protein turnover and on reversal of inhibition by reducing agents such as glutathione. Glutathione incubation of the H +,K +-ATPase isolated from omeprazole or pantoprazole-treated rats reversed 88% of the omeprazole inhibition but none of the pantoprazole inhibition. The present study was designed to measure binding properties of omeprazole or pantoprazole in vivo. Rats were injected with 14C-omeprazole or 14C-pantoprazole after acid stimulation. The specific binding to the gastric H +,K +-ATPase was measured at timed intervals as well as reversal of binding by glutathione reduction. The stoichiometry of omeprazole and pantoprazole binding to the catalytic subunit of the H +,K +-ATPase was 2 moles of inhibitor per mole of the H +,K +-ATPase phosphoenzyme. Omeprazole bound to one cysteine between transmembrane segments 5/6 and one between 7/8, pantoprazole only to the two cysteines in the TM5/6 domain. Loss of drug from the pump was biphasic, the fast component accounted for 84% of omeprazole binding and 51% of pantoprazole binding. Similarly, only 16% of omeprazole binding but 40% of pantoprazole binding was not reversed by glutathione. The residence time of omeprazole and pantoprazole on the ATPase in vivo depends on the reversibility of binding. Binding of pantoprazole at cysteine 822 is irreversible whereas that of omeprazole at cysteine 813 and 892 is reversible both in vivo and in vitro. This is consistent with the luminal exposure of cysteine 813 and 892 and the intra-membranal location of cysteine 822 in the 3D structure of the H +,K +-ATPase.

Molecular Basis of the Differences in Binding Properties of the Highly Related C-type Lectins DC-SIGN and L-SIGN to Lewis X Trisaccharide and Schistosoma mansoni Egg Antigens

The dendritic cell-specific C-type lectin DC-SIGN functions as a pathogen receptor that recognizes Schistosoma mansoni egg antigens through its major glycan epitope Galbeta1,4(Fucalpha1,3)GlcNAc (Lex). Here we report that L-SIGN, a highly related homologue of DC-SIGN found on liver sinusoidal endothelial cells, binds to S. mansoni egg antigens but not to the Lex epitope. L-SIGN does bind the Lewis antigens Lea, Leb, and Ley, similar as DC-SIGN. A specific mutation in the carbohydrate recognition domain of DC-SIGN (V351G) abrogates binding to all Lewis antigens. In L-SIGN Ser363 is present at the corresponding position of Val351 in DC-SIGN. Replacement of this Ser into Val resulted in a "gain of function" L-SIGN mutant that binds to Lex, and shows increased binding to the other Lewis antigens. These data indicate that Val351 is important for the fucose specificity of DC-SIGN. Molecular modeling and docking of the different Lewis antigens in the carbohydrate recognition domains of L-SIGN, DC-SIGN, and their mutant forms, demonstrate that Val351 in DC-SIGN creates a hydrophobic pocket that strongly interacts with the Fucalpha1,3/4-GlcNAc moiety of the Lewis antigens. The equivalent amino acid residue Ser363 in L-SIGN creates a hydrophilic pocket that prevents interaction with Fucalpha1,3-GlcNAc in Lex but supports interactions with the Fucalpha1,4-GlcNAc moiety in Lea and Leb antigens. These data demonstrate for the first time that DC-SIGN and L-SIGN differ in their carbohydrate binding profiles and will contribute to our understanding of the functional roles of these C-type lectin receptors, both in recognition of pathogen and self-glycan antigens.

Photocontrolled release and uptake of a porphyrin guest by dithienylethene-tethered beta-cyclodextrin host dimers.

Two photoswitchable dithienylethene-tethered beta-cyclodextrin dimers were synthesized to function as host molecules with an externally controllable binding affinity. The cyclodextrin cavities of these dimers are linked through their secondary sides by a photochromic dithienylethene unit that is connected to the secondary rim either directly (4) or through propyl spacers (9). Irradiation with light switches these dimers between a relatively flexible (open) and a rigid (closed) form. The binding properties of the dimers depend on the configuration of the dithienylethene spacer, as is shown by microcalorimetry performed with tetrakis-sulfonatophenyl porphyrin (TSPP) as a guest molecule. The differences in binding properties are most pronounced for the more rigid dimer 4, which binds TSPP 35 times more strongly in the open form (4 a) than in the closed form (4 b). The values found for the enthalpy of binding (deltaH degrees ) indicate that this difference in binding is due to the loss of cooperativity between the two beta-cyclodextrin cavities in the closed form. Molecular modeling shows that 4 b is not able to bind TSPP effectively in both cyclodextrin cavities. The open and closed forms of the more flexible dimer 9 show no substantial difference in their binding of TSPP. Thermodynamic values indicative of strong binding of TSPP by two beta-cyclodextrin cavities were measured for both forms of the dimer, and molecular modeling confirms that both are flexible enough to tightly bind TSPP. The binding differences between the forms of dimer 4 allow the photocontrolled release and uptake of TSPP, which renders control of the ratio of complexed to free TSPP in solution possible.

Relationship between haemolytic and sphingomyelinase activities in a partially purified β-like toxin from Staphylococcus schleiferi

β-Toxins of staphylococcal species possess dual activity in that they can both lyse erythrocytes (by ‘hot-cold’ lysis) and catalyse hydrolysis of membrane-associated sphingomyelin. However, the precise relationship between these two activities has not been extensively studied. We have partially purified a β-like toxin from culture supernatants of Staphylococcus schleiferi N860375 which exhibits both ‘hot-cold’ lysis of erythrocytes and neutral sphingomyelinase activities. This toxin has a strong preference for sheep erythrocytes, the membranes of which are rich in sphingomyelin. Kinetic analysis suggests that haemolysis and sphingomyelinase activities are very closely associated obeying identical Michaelis–Menten kinetics. However, pre-treatment with antibodies to Staphylococcus aureus β-toxin, Ca 2+, dithiothreitol and phenylmethylsulfonyl fluoride appear to inhibit sphingomyelinase activity significantly more strongly than haemolysis while Mg 2+ activates sphingomyelinase activity more strongly than haemolysis. We attribute these effects to differences in binding properties in the two assays. Micropurification by both sphingosylphosphocholine-agarose affinity chromatography and preparative electrophoresis revealed that the 34-kDa toxin associates non-covalently with individual proteins.