和厚朴酚对幽门螺杆菌生长和空泡毒素A表达及活性的影响

【目的】幽门螺杆菌(Helicobacter pylori, H. pylori)是胃炎和消化性溃疡的病原体, 但其具有潜在的正常菌群的特性。本研究通过评价临床常用中药厚朴的活性成分和厚朴酚对H. pylori的抑制作用及对其空泡毒素A表达和活性的影响, 以反映其对H. pylori具有的潜在去除毒性的作用。【方法】使用平皿稀释固体法和脑心浸液液体法测定和厚朴酚对H. pylori的最低抑菌浓度, 进一步通过中性红摄入法评价经无抑菌效果的低浓度和厚朴酚干预后, H. pylori培养上清中空泡毒素A (Vacuolating cytotoxin A, VacA)的毒性作用; 并通过RT-PCR和Western blot方法检测经低浓度和厚朴酚处理后, H. pylori菌体及分泌上清中VacA mRNA和蛋白的表达水平。【结果】发现高浓度(0.75 g/L)和厚朴酚对H. pylori 具有抑制作用; 而在远低于最低抑菌浓度时, 和厚朴酚可有效抑制H. pylori VacA的形成和分泌。【结论】和厚朴酚具有下调H. pylori毒性的潜在作用, 这为基于“致病性H. pylori的非致病性转变”这一机理的干预性治疗提供了有希望的研究前景。     

Helicobacter pylori VacA cytotoxin interacts with fibronectin and alters HeLa cell adhesion and cytoskeletal organization in vitro

Helicobacter pylori vacuolating cytotoxin VacA causes multiple effects on epithelial cell function and morphology, but the effects of VacA on signal transduction pathways and the cytoskeleton have not been investigated in detail. In this study, we analyzed the effects of native VacA on HeLa and AGS cell adhesion to fibronectin and laminin under serum-free conditions. Confocal microscopic examination revealed increased number of cells with rounded morphology and inhibition of actin fiber formation, in the presence of VacA. VacA binds to fibronectin in vitro in a dose-dependent manner. This interaction was partly inhibited by a peptide containing an arginine-glycine-aspartic acid motif. The adhesion of HeLa cells to fibronectin, but not to laminin, was decreased in the presence of VacA. Thus, VacA may interact with fibronectin and influence integrin receptor-induced cell signaling and cytoskeleton-dependent cell functions.     

Chiral recognition of dextran sulfate with D- and L-cystine studied by multiwavelength surface plasmon resonance

A multiwavelength surface plasmon resonance (mwSPR) approach has been developed to study the chiral discrimination between D- and L-cystine (Cys). A monolayer of the two enantiomers was separately assembled on a pair of gold films of about 50 nm in thickness and their resonance wavelength shifts, Deltalambda, were measured under a continuous flow of an identical chiral probe solution. Dextran sulfate (DS) was found to be an excellent chiral probe because it has rich chiral centers and is large enough to produce sensitive mwSPR response. The chiral discrimination was investigated either by Deltalambda(max), the maximum resonance wavelength shift in recognition equilibrium, or by recognition kinetics (Deltalambda vs time). The equilibrium data showed that D-Cys yielded always the smaller Deltalambda(max) as compared to L-Cys at pH 5.0 or above. This differentiation was enlarged by raising the probe content and became naught at pH <4.5. The kinetic results showed that, as pH increased from 5.0 to 7.5, the non-equilibrium Deltalambda for D-Cys rose above the level for L-Cys at the first 30s of recognition but came back gradually to its equilibrium position after about 150 s, with crossing at 50--150 s depending on DS concentration. This phenomenon was thought to be the result of molecular orientation adjustment after DS binding to D-Cys. Both kinetic and thermodynamic mechanisms were thus considered to be deeply involved in the investigated chiral recognition system.     

Inhibition of heparan sulphate and other glycosaminoglycans binding to Helicobacter pylori by various polysulphated carbohydrates

Abstract Heparan sulphate binding to Helicobacter pylori at pH 4 to 5 was inhibited with various sulphated polysaccharides (heparin and chondroitin sulphates, fucoidan, carrageenans and some others), but not by carboxylated or nonsulphated compounds. Heparin binding proteins are exposed on the cell surface.     

Complementary DNA display selection of high‐affinity peptides binding the vacuolating toxin (VacA) of Helicobacter pylori

Artificial peptides designed for molecular recognition of a bacterial toxin have been developed. Vacuolating cytotoxin A protein (VacA) is a major virulence factor of Helicobacter pylori, a gram‐negative microaerophilic bacterium inhabiting the upper gastrointestinal tract, particularly the stomach. This study attempted to identify specific peptide sequences with high affinity for VacA using systematic directed evolution in vitro, a cDNA display method. A surface plasmon resonance‐based biosensor and fluorescence correlation spectroscopy to examine binding of peptides with VacA identified a peptide (GRVNQRL) with high affinity. Cyclization of the peptide by attaching cysteine residues to both termini improved its binding affinity to VacA, with a dissociation constant (K_d) of 58 nm . This study describes a new strategy for the development of artificial functional peptides, which are promising materials in biochemical analyses and medical applications. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

High molecular weight factor in FCS inhibits Helicobacter pylori VacA-binding to its receptor,RPTPbeta, on AZ-521

VacA, a secretory product of Helicobacter pylori, binds to its cell surface receptor, receptor tyrosine phosphatase (RPTP) beta, leading to cytoplasmic vacuolization of gastric epithelial AZ-521 cells. VacA binding to the cell surface and VacA-dependent vacuolization were inhibited by cell culture media containing fetal calf serum (FCS). The high molecular weight fraction of FCS isolated by Superose 12 gel filtration chromatography inhibited VacA binding, whereas only weak effects were observed with other fractions. These data show that the high molecular weight fraction of FCS inhibits VacA action though its ability to block toxin binding to its receptor, RPTPbeta, on AZ-521 cells.     

Using surface plasmon resonance to directly measure slow binding of low-molecular mass inhibitors to a VanX chip

VanX, a d,d-dipeptidase, is one of five gene products responsible for vancomycin resistance in pathogenic bacteria and is an attractive drug target in circumventing clinical drug resistance. Our previous combinatorial search of VanX substrates in a dipeptide library of d-X(1)-d-X(2) (19(2)=361) forms has led to the discovery of three new compounds (d-Ala-d-Phe, d-Ala-d-Tyr, and d-Ala-d-Trp) having higher k(cat)/K(M) values than those of its natural substrate, d-Ala-d-Ala. Based on structures of newly identified substrates, two representative transition state analogs of substrates, d-Ala(P,O)d-Phe (6a) and d-Ala(P,O)d-Ala (6b) dipeptide phosphonates, used as VanX inhibitor were rationally designed and chemically synthesized. In the synthesis, eight synthetic steps in total were employed for preparing each VanX inhibitor, and their overall isolated yields were 21 and 11% for 6a and 6b, respectively. Binding interactions of d-Ala(P,O)d-Phe (6a) and d-Ala(P,O)d-Ala (6b) with VanX were confirmed unambiguously and measured quantitatively by surface plasmon resonance. The result reveals that both dipeptide phosphonates are slow-binding inhibitors of VanX (for 6a, k(on)=1.18 x 10(3)M(-1)s(-1), k(off)=2.31 x 10(-3) s(-1), K(D)=1.96 microM, chi(2)=0.0737; for 6b, k(on)=1.09 x 10(3)M(-1)s(-1), k(off)=1.80 x 10(-2)s(-1), K(D)=16.5 microM, chi(2)=0.0599). This suggests that only a fraction of the conformers of the inhibitors in solution adopts a conformation best suited for binding interaction with VanX and that the VanX-inhibitor complex may concomitantly undergo a conformational isomerization from an initial but fast weak-binding adduct to slowly convert to a tight-binding complex with a more stable bound geometry. Moreover, in comparison with 6b, an additional aromatic interaction of 6a with the Phe79 residue in the active site of the enzyme, through an energetically favorable face-to-face offset stacked orientation, may account for its higher affinity than 6b to VanX.     

Grating-coupled surface plasmon resonance: a cell and protein microarray platform

Grating-coupled surface plasmon resonance (GCSPR) is a method for the accurate assessment of analyte in a multiplexed format using small amounts of sample. In GCSPR, the analyte is flowed across specific receptors (e.g. antibodies or other proteins) that have been immobilized on a sensor chip. The chip surface is illuminated with p-polarized light that couples to the gold surface's electrons to form a surface plasmon. At a specific angle of incidence, the GCSPR angle, the maximum amount of coupling occurs, thus reducing the intensity of reflected light. Shifts in the GCSPR angle can be correlated with refractive index increases following analyte capture by chip-bound receptors. Because regions of the chip can be independently analyzed, this system can assess 400 interactions between analyte and receptor on a single chip. We have used this label-free system to assess a number of molecules of immunological interest. GCSPR can simultaneously detect an array of cytokines and other proteins using the same chip. Moreover, GCSPR is also compatible with assessments of antigen expression by intact cells, detecting cellular apoptosis and identifying T cells and B cells. This technology represents a powerful new approach to the analysis of cells and molecular constituents of biological samples.     

Direct analysis of a GPCR-agonist interaction by surface plasmon resonance

Despite their clinical importance, detailed analysis of ligand binding at G-protein coupled receptors (GPCRs) has proved difficult. Here we successfully measure the binding of a GPCR, neurotensin receptor-1 (NTS-1), to its ligand, neurotensin (NT), using surface plasmon resonance (SPR). Specific responses were observed between NT and purified, detergent-solublised, recombinant NTS-1, using a novel configuration where the biotinylated NT ligand was immobilised on the biosensor surface. This SPR approach shows promise as a generic approach for the study of ligand interactions with other suitable GPCRs.     

Single-cycle kinetic analysis of ternary DNA complexes by surface plasmon resonance on a decaying surface

Complexes involving three DNA strands were used to demonstrate that the single-cycle kinetics (SCK) method, which consists in injecting sequentially samples at increasing concentrations and until now used exclusively to investigate bimolecular complexes by surface plasmon resonance, can be extended to the kinetic analysis of ternary complexes. DNA targets, B, were designed with sequences of variable lengths on their 3' sides that recognise a surface-immobilized biotinylated DNA anchor, A. These targets displayed on their 5' sides sequences that recognise DNA oligonucleotides of variable lengths, C, namely the analytes. Combinations of B and C DNA oligonucleotides on A generated ternary complexes each composed of two Watson-Crick helices displaying different kinetic properties. The target-analyte B-C duplexes were formed by sequentially injecting three increasing concentrations of the analytes C during the dissociation phase of the target B from the anchor A. The sensorgrams for the target-analyte complexes dissociating from the functionalized surface were successfully fitted by the SCK method while the target dissociated from the anchor, i.e. on a decaying surface. Within the range of applicability of the method which is driven by the rate of dissociation of the target from the anchor, the rate and equilibrium constants characteristic of these target-analyte duplexes of the ternary complexes did not depend on how fast the targets dissociated from the immobilized DNA anchor. In addition the results agreed very well with those obtained when such duplexes were analysed directly as bimolecular complexes, i.e. when the target, modified with a biotin, was directly immobilized onto a streptavidin sensor chip surface rather than captured by an anchor. Therefore the method we named SCKODS (Single-Cycle Kinetics On a Decaying Surface) can also be used to investigate complexes formed during a dissociation phase, in a ternary complex context. The SCKODS method can be combined with the SCK one to fully characterize the two bimolecular complexes of a ternary complex.     

Stimulation of human olfactory receptor 17-40 with odorants probed by surface plasmon resonance

We report here the results of human olfactory receptor (OR) 17-40 stimulation with some odorants probed by means of the double-channel surface plasmon resonance platform NanoSPR-6. OR 17-40 tagged with N-terminal cmyc sequence was heterologously co-expressed with Galpha(olf) protein in yeast, and receptor-carrying nanosomes were prepared from yeast membrane fraction. Then, receptors were specifically captured via anti-cmyc antibody attached to the gold-coated substrate in orientated or random way. Measurement of odorants effects were carried out in the presence of GTP-gamma-S in differential mode in order to compensate bulk changes of refractive index. For the first time, biosensing efficiency of olfactory films was discussed in terms of their thickness and Galpha(olf) accessibility to GTP-gamma-S. Bell-shaped response profile with two maxima (near 1 nM and near 1 muM) was established for helional, which is documented as highly specific agonist of OR 17-40. Unrelated odorant heptanal used as control, did not evoke significant variations of differential signal.     

Probing the binding of tetraplatinum(pyridyl)porphyrin complexes to DNA by means of surface plasmon resonance

Tetrapyridylporphyrins containing four chloro(2,2′-bipyridine)platinum(II) complexes attached at the meta (3-H₂TPtPyP) and para (4-H₂TPtPyP) positions of the peripheral pyridine ligands were synthesized and their interaction with DNA investigated. The compounds were isolated in the solid state and characterized by means of spectroscopic and analytical techniques. According to molecular simulations, the two isomers exhibit contrasting structural characteristics, consistent with a saddle shape configuration for 3-H₂TPtPyP and a planar geometry for 4-H₂TPtPyP. Surface plasmon resonance studies were carried out on the interaction of the complexes with calf thymus DNA, revealing a preferential binding of 3-H₂TPtPyP, presumably at the DNA major grooves.     

The binding of cytochrome c to neuroglobin: a docking and surface plasmon resonance study

It has recently been proposed that the role of neuroglobin in the protection of neurons from ischaemia induced cell death requires the formation of a transient complex with cytochrome c. No such complex has yet been isolated. Here, we present the results of soft docking calculations, which indicate one major binding site for cytochrome c to neuroglobin. The results yield a plausible structure for the most likely complex structure in which the hemes of each protein are in close contact. NMR analysis identifies the formation of a weak complex in which the heme group of cytochrome c is involved. surface plasmon resonance studies provide a value of 45muM for the equilibrium constant for cytochrome c binding to neuroglobin, which increases significantly as the ionic strength of the solution increases. The temperature dependence of the binding constant indicates that the complex formation is associated with a small unfavourable enthalpy change (1.9kcalmol(-1)) and a moderately large, favourable entropy change (14.8calmol(-1)deg(-1)). The sensitivity of the binding constant to the presence of salt suggests that the complex formation involves electrostatic interactions.     

Whole cell-based surface plasmon resonance measurement to assess binding of anti-TNF agents to transmembrane target

We developed a technique for the measurement of surface plasmon resonance (SPR) to detect interactions of anti-tumor necrosis factor (TNF) agents with transmembrane TNF-α (mTNF-α) on living whole cells. The injection of a suspension of mTNF-α expressing Jurkat cells, used as an analyte, gave a clear binding response to anti-TNF agents, such as etanercept, infliximab and adalimumab, immobilized on sensorchip. The binding response of the analyte cells increased in a concentration-dependent manner and was competitively reduced by adding soluble TNF receptors to the analyte cell suspension. Treatment of analyte cells with free anti-TNF agent before injection reduced the binding response between the analyte cells and immobilized-etanercept on sensorchip, and the inhibitory effect of free anti-TNF agent was concordant with the affinity of anti-TNF agent for soluble TNF-α. These findings indicate that the SPR response arises from specific binding between anti-TNF agent and its target on cell membrane.     

Serum antibody screening by surface plasmon resonance using a natural glycan microarray

A surface plasmon resonance (SPR) based natural glycan microarray was developed for screening of interactions between glycans and carbohydrate-binding proteins (CBPs). The microarray contained 144 glycan samples and allowed the real-time and simultaneous screening for recognition by CBPs without the need of fluorescent labeling. Glycans were released from their natural source and coupled by reductive amination with the fluorescent labels 2-aminobenzamide (2AB) or anthranilic acid (AA) followed by high-performance liquid chromatography (HPLC) fractionation making use of the fluorescent tag. The released and labeled glycans, in addition to fluorescently labeled synthetic glycans and (neo)glycoproteins, were printed on an epoxide-activated chip at fmol amounts. This resulted in covalent immobilization, with the epoxide groups forming covalent bonds to the secondary amine groups present on the fluorescent glycoconjugates. The generated SPR glycan array presented a subset of the glycan repertoire of the human parasite Schistosoma mansoni. In order to demonstrate the usefulness of the array in the simultaneous detection of glycan-specific serum antibodies, the anti-glycan antibody profiles from sera of S. mansoni-infected individuals as well as from non-endemic uninfected controls were recorded. The SPR screening was sensitive for differences between infection sera and control sera, and revealed antibody titers and antibody classes (IgG or IgM). All SPR analyses were performed with a single SPR array chip, which required regeneration and blocking of the chip before the application of a serum sample. Our results indicate that SPR-based arrays constructed from glycans of natural or synthetic origin, pure or as mixture, can be used for determining serum antibody profiles as possible markers for the infection status of an individual.     

Using surface plasmon resonance to directly identify molecules in a tripeptide library that bind tightly to a vancomycin chip

This paper describes a procedure, based on direct binding, for identifying tight-binding ligands for a receptor immobilized on a sensor chip from an array of equimolar tripeptides using surface plasmon resonance. Vancomycin and a library of 96 tripeptides, with molecular weight ranging from 316 to 560 Da, were used as a model system to illustrate the procedure. A consensus structure of the strongest interacting peptides consisted of D-Ala at the C terminus and aromatic amino acid in the penultimate position. Ligands having this structure bound more tightly to vancomycin than the known D-Ala-D-Ala peptide. The throughput of our continuous assay is 96 compounds in 3.3 h, and the sample consumption is less than 2 microg per peptide and 1 ng for vancomycin. This procedure should be applicable to peptide libraries of greater complexity than that used here and to mixtures of small organic compounds.     

Analysis of the carbohydrate binding specificity of the mushroom Pleurotus ostreatus lectin by surface plasmon resonance

The sugar binding specificity of the mushroom Pleurotus ostreatus lectin (POL) was analyzed by surface plasmon resonance. The lectin was immobilized to a sensor chip, and asialo-bovine submaxillary mucin (asialo-BSM), one of the most potent inhibitors in the hemagglutination inhibition assay, tightly bound to the lectin. The binding specificity of various mono- or oligosaccharides to the lectin was evaluated by the coinjection method. The dissociation of asialo-BSM was promoted by injection of some haptenic saccharides. For the most part, the order of acceleration ability of the sugars to the dissociation in the coinjection experiment agreed with that of the inhibitory potency of each sugar evaluated by the hemagglutination inhibition assay. In conclusion, POL recognized a galactosyl residue, and the specificity was increased by substitution at the C-2 position of the galactosyl residue with a fucosyl or acetylamino group. This method using the coinjection method proved useful in analysis of carbohydrate-lectin binding specificity.     

IgG subclass specificity to C1q determined by surface plasmon resonance using Protein L capture technique

Recombinant monoclonal antibodies (mAbs) have become an important category of biological therapeutics. mAbs share the same structures and biological functions as endogenous IgG molecules. One function is complement-dependent cytotoxicity (CDC) initiation by binding of C1q. Traditionally, ELISA methods have been utilized to measure C1q binding. A new robust capture method was established in this study to measure the binding affinity of C1q to antibodies by surface plasmon resonance (SPR). The utility of this method was demonstrated by determination of the difference in IgG subclass specificity of C1q binding.     

Characterization of osteoprotegerin binding to glycosaminoglycans by surface plasmon resonance: role in the interactions with receptor activator of nuclear factor kappaB ligand (RANKL) and RANK

Osteoprotegerin (OPG) is a decoy receptor for receptor activator of nuclear factor kappaB ligand (RANKL), a key inducer of osteoclastogenesis via its receptor RANK. We previously showed that RANK, RANKL, and OPG are able to form a tertiary complex and that OPG must be also considered as a direct effector of osteoclast functions. As OPG contains a heparin-binding domain, the present study investigated the interactions between OPG and glycosaminoglycans (GAGs) by surface plasmon resonance and their involvement in the OPG functions. Kinetic data demonstrated that OPG binds to heparin with a high-affinity (KD: 0.28 nM) and that the pre-incubation of OPG with heparin inhibits in a dose-dependent manner the OPG binding to the complex RANK-RANKL. GAGs from different structure/origin (heparan sulfate, dermatan sulfate, and chondroitin sulfate) exert similar activity on OPG binding. The contribution of the sulfation pattern and the size of the oligosaccharide were determined in this inhibitory mechanism. The results demonstrated that sulfation is essential in the OPG-blocking function of GAGs since a totally desulfated heparin loses its capacity to bind and to block OPG binding to RANKL. Moreover, a decasaccharide is the minimal structure that totally inhibits the OPG binding to the complex RANK-RANKL. Western blot analysis performed in 293 cells surexpressing RANKL revealed that the pre-incubation of OPG with these GAGs strongly inhibits the OPG-induced decrease of membrane RANKL half-life. These data support an essential function of the related glycosaminoglycans heparin and heparan sulfate in the activity of the triad RANK-RANKL-OPG.     

Determining OMP topology by computation, surface plasmon resonance and cysteine labelling: the test case of OMPG

Bacterial outer-membrane proteins (OMP) are important in pathogenicity and the recently solved structure of OmpG provides an excellent test case for topological predictions since it is monomeric. Here we compare the results of applying several computerised structure prediction algorithms to the sequence of OmpG. Furthermore, we probe the OmpG topology by both an established chemical labelling approach and a new method which combines epitope insertion and surface plasmon resonance. The computational approaches are broadly accurate but the exact choice of the number of beta strands remains difficult. The algorithms also tend to predict the entire beta strand rather than just the transmembrane region. Epitope insertion clearly pinpoints exposed loops but its utility in defining buried or periplasmic sites is less clear cut. Cysteine-mutant labelling is largely confined to exposed residues but one periplasmic cysteine may be labelled by reagents entering via the OmpG pore.