Molecular simulation of N-acetylneuraminic acid analogs and molecular dynamics studies of cholera toxin-Neu5Gc complex

Cholera toxin (CT) is an AB₅ protein complex secreted by the pathogen Vibrio cholera, which is responsible for cholera infection. N-acetylneuraminic acid (NeuNAc) is a derivative of neuraminic acid with nine-carbon backbone. NeuNAc is distributed on the cell surface mainly located in the terminal components of glycoconjugates, and also plays an important role in cell–cell interaction. In our current study, molecular docking and molecular dynamic (MD) simulations were implemented to identify the potent NeuNAc analogs with high-inhibitory activity against CT protein. Thirty-four NeuNAc analogs, modified in different positions C-1/C-2/C-4/C-5/C-7/C-8/C-9, were modeled and docked against the active site of CT protein. Among the 34 NeuNAc analogs, the analog Neu5Gc shows the least extra precision glide score of ?9.52 and glide energy of ?44.71?kcal/mol. NeuNAc analogs block the CT active site residues HIS:13, ASN:90, LYS:91, GLN:56, GLN:61, and TRP:88 through intermolecular hydrogen bonding. The MD simulation for CT-Neu5Gc docking complex was performed using Desmond. MD simulation of CT-Neu5Gc complex reveals the stable nature of docking interaction.     

Monosialogangliosides and Their Interaction with Cholera Toxin—Investigation by Molecular Modeling and Molecular Mechanics

Abstract

Molecular mechanics and molecular dynamics studies are performed to investigate the conformational preference of cell surface monosialogangliosides (GM3, GM2 and GM1) in aqueous environment. Water mediated hydrogen bonding network plays a significant role in the structural stabilization of GM3, GM2 and GM1. The spatial flexibility of NeuNAc of gangliosides at the binding site of cholera toxin reveals a limited allowed eulerian space of 2.4% with a much less allowed eulerian space (1.4%) for external galactose of GM1. The molecular mechanics of monosialoganglioside-cholera toxin complex reveals that cholera toxin can accommodate the monosialogangliosides in three different modes. Direct and water mediated hydrogen bonding interactions stabilize these binding modes and play an essential role in defining the order of specificity for different monosialogangliosides towards cholera toxin. This study identifies the NeuNAc binding site as a site for design of inhibitors that can restrict the pathogenic activity of cholera toxin.     

Conformations of higher gangliosides and their binding with cholera toxin - investigation by molecular modeling, molecular mechanics, and molecular dynamics

Molecular mechanics and molecular dynamics studies are performed to investigate the conformational preference of cell surface higher gangliosides (GT1A and GT1B) and their interaction with Cholera Toxin. The water mediated hydrogen bonding network exists between sugar residues in gangliosides. An integrated molecular modeling, molecular mechanics, and molecular dynamics calculation of cholera toxin complexed with GT1A and GT1B reveal that, the active site of cholera toxin can accommodate these higher gangliosides. Direct and water mediated hydrogen bonding interactions stabilize these binding modes and play an essential role in defining the order of specificity for different higher ganglioside towards cholera toxin. This study identifies that the binding site of cholera toxin is shallow and can accommodate a maximum of two NeuNAc residues. The NeuNAc binding site of cholera toxin may be crucial for the design of inhibitors that can prevent the infection of cholera.     

Conformations of Higher Gangliosides and Their Binding with Cholera Toxin—Investigation by Molecular Modeling,Molecular Mechanics,and Molecular Dynamics

Abstract

Molecular mechanics and molecular dynamics studies are performed to investigate the conformational preference of cell surface higher gangliosides (GT1A and GT1B) and their interaction with Cholera Toxin. The water mediated hydrogen bonding network exists between sugar residues in gangliosides. An integrated molecular modeling, molecular mechanics, and molecular dynamics calculation of cholera toxin complexed with GT1A and GT1B reveal that, the active site of cholera toxin can accommodate these higher gangliosides. Direct and water mediated hydrogen bonding interactions stabilize these binding modes and play an essential role in defining the order of specificity for different higher ganglioside towards cholera toxin. This study identifies that the binding site of cholera toxin is shallow and can accommodate a maximum of two NeuNAc residues. The NeuNAc binding site of cholera toxin may be crucial for the design of inhibitors that can prevent the infection of cholera.     

Disialogangliosides and their interaction with cholera toxin - investigation by molecular modeling, molecular mechanics and molecular dynamics

Molecular mechanics and molecular dynamics studies are performed to investigate the conformational preference of cell surface disialogangliosides (GD1A, GD1B and GD3) in aqueous environment. The molecular mechanics calculation reveals that water mediated hydrogen bonding network plays a significant role in the structural stabilization of GD1A, GD1B and GD3. These water mediated hydrogen bonds not only exist between neighboring residues but also exist between residues that are separated by 2 to 3 residues in between. The conformational energy difference between different conformational states of gangliosides correlates very well with the number of water mediated and direct hydrogen bonds. The spatial flexibility of NeuNAc of gangliosides at the binding site of cholera toxin is worked out. The NeuNAc has a limited allowed eulerian space at the binding site of Cholera Toxin (2.4%). The molecular modeling, molecular mechanics and molecular dynamics of disialoganglioside-cholera toxin complex reveal that cholera toxin can accommodate the disialoganglioside GD1A in three different modes. A single mode of binding is permissible for GD1B and GD3. Direct and water mediated hydrogen bonding interactions stabilizes these binding modes and play an essential role in defining the order of specificity for different disialogangliosides towards cholera toxin. This study not only provides models for the disialoganglioside-cholera toxin complexes but also identifies the NeuNAc binding site as a site for design of inhibitors that can restrict the pathogenic activity of cholera toxin.     

Regulation of phosphate-activated glutaminase (PAG) by glutamate analogues

The ability of structural analogues of glutamate (GLU) to modulate phosphate activated glutaminase (PAG) was assessed in the present series of studies. A number of GLU receptor agonists and antagonists were tested for their ability to inhibit synaptosomal PAG activity. PAG activity was determined by measuring GLU formation from 0.5mM glutamine (GLN) in the presence of 10 mM phosphate. GLU analogues at 5–10 mM were found to significantly inhibit PAG activity. It was determined that PAG inhibition occurred regardless of whether the GLU analogues were receptor agonists or antagonists, however, PAG inhibition was influenced by analogue chain length, isomeric form and substituent substitution. The glutamate uptake blockers, dihydrokainic acid and DL-threo--hydroxyaspartic acid were relatively weak inhibitors of PAG (<25% inhibition) as were the receptor agonists, ibotenic acid and (±)cis-2,3-piperidine-dicarboxylic acid. Other GLU analogues produced inhibition of PAG in the range of 40–70%. PAG inhibition by GLU analogues did not appear to differ substantially among the brain regions evaluated (cortex, striatum and hippocampus). The endogenous amino acids, glycine, taurine and N-acetylaspartic acid, also significantly inhibited PAG activity in the 5–10 mM range. The noncompetitive NMDA antagonists, (+)MK801 and ketamine, at a concentration of 5 mM, significantly stimulated PAG activity 1.5–2 fold over control values. The activation of PAG by (+)MK801 was dose-related, stereoselective and appeared to result from a synergistic interaction with phosphate to enhance substrate (GLN) binding to PAG. The results of these studies suggest that GLU analogues could potentially alter neurotransmitter GLU synthesis if sufficient concentrations of these drugs are used in in vitro or in vivo studies. Furthermore, preliminary evidence suggests that other endogenous amino acids (glycine, taurine, N-acetylaspartic acid) may modulate PAG activity. These studies have further characterized the structural requirements for the allosteric regulation of PAG by glutamate and its analogues.     

Disialogangliosides and Their Interaction with Cholera Toxin—Investigation by Molecular Modeling,Molecular Mechanics and Molecular Dynamics

Abstract

Molecular mechanics and molecular dynamics studies are performed to investigate the conformational preference of cell surface disialogangliosides (GD1A, GD1B and GD3) in aqueous environment. The molecular mechanics calculation reveals that water mediated hydrogen bonding network plays a significant role in the structural stabilization of GD1A, GD1B and GD3. These water mediated hydrogen bonds not only exist between neighboring residues but also exist between residues that are separated by 2 to 3 residues in between. The conformational energy difference between different conformational states of gangliosides correlates very well with the number of water mediated and direct hydrogen bonds. The spatial flexibility of NeuNAc of gangliosides at the binding site of cholera toxin is worked out. The NeuNAc has a limited allowed eulerian space at the binding site of Cholera Toxin (2.4%). The molecular modeling, molecular mechanics and molecular dynamics of disialo- ganglioside-cholera toxin complex reveal that cholera toxin can accommodate the disialo- ganglioside GD1A in three different modes. A single mode of binding is permissible for GD1B and GD3. Direct and water mediated hydrogen bonding interactions stabilizes these binding modes and play an essential role in defining the order of specificity for different disialogangliosides towards cholera toxin. This study not only provides models for the disialoganglioside-cholera toxin complexes but also identifies the NeuNAc binding site as a site for design of inhibitors that can restrict the pathogenic activity of cholera toxin.     

Monosialogangliosides and their interaction with cholera toxin - investigation by molecular modeling and molecular mechanics

Molecular mechanics and molecular dynamics studies are performed to investigate the conformational preference of cell surface monosialogangliosides (GM3, GM2 and GM1) in aqueous environment. Water mediated hydrogen bonding network plays a significant role in the structural stabilization of GM3, GM2 and GM1. The spatial flexibility of NeuNAc of gangliosides at the binding site of cholera toxin reveals a limited allowed eulerian space of 2.4% with a much less allowed eulerian space (1.4%) for external galactose of GM1. The molecular mechanics of monosialoganglioside-cholera toxin complex reveals that cholera toxin can accommodate the monosialogangliosides in three different modes. Direct and water mediated hydrogen bonding interactions stabilize these binding modes and play an essential role in defining the order of specificity for different monosialogangliosides towards cholera toxin. This study identifies the NeuNAc binding site as a site for design of inhibitors that can restrict the pathogenic activity of cholera toxin.     

Intrafollicular amino acid concentration and the effect of amino acids in a defined maturation medium on porcine oocyte maturation, fertilization, and preimplantation development

The objective of this study was to determine the intrafollicular concentrations of free amino acids in pigs and to examine the effect of amino acids in a chemically defined maturation medium on oocyte maturation, in vitro fertilization (IVF), and embryo development in vitro. Pooled follicular fluid aspirated separately from small (<3mm in diameter), medium (3-8mm), and large follicles (>8mm) in three pairs of ovaries was analyzed for amino acid concentration. In addition, oocyte maturation, fertilization, and embryo development were examined after in vitro maturation (IVM) of oocytes in a defined maturation medium supplemented individually with glutamate (GLU), glutamine (GLN), glycine (GLY), aspartate (ASP), asparagine (ASN), arginine (ARG), alanine (ALA), leucine (LEU), lysine (LYS), proline (PRO), and valine (VAL). Irrespective of follicle size, GLY, GLU, ALA, GLN, and PRO were the most abundant amino acids in pig follicular fluid (pFF). Sperm penetration was not altered by amino acid treatment during IVM, but monospermic fertilization was increased (P<0.05) by GLN, ASP, and VAL. All amino acids except ASP and ASN stimulated (P<0.05) male pronuclear formation after IVF. ARG and ALA treatment during IVM improved (P<0.05) blastocyst formation. In conclusion, GLY, GLU, ALA, GLN, and PRO were the most abundant amino acids in pFF and amino acids in a defined medium improved porcine monospermic fertilization, male pronuclear formation, and preimplantation development.     

Exploration of fluoroquinolone resistance in Streptococcus pyogenes: comparative structure analysis of wild‐type and mutant DNA gyrase

Quinolone resistance‐determining region is known to be the druggability site of the target protein that undergoes frequent mutation and thus renders quinolone resistance. In the present study, ligands were tested for their inhibitory activity against DNA gyrase of Streptococcus pyogenes involved in DNA replication. In silico mutational analysis on modelled gyrase A revealed that GLU85 had the most possible interactions with all the ligands used for the study. The amino acid residue GLU85 had also been predicted with an essential role of maintaining the three‐dimensional structure of the protein. When introduced with a mutation (GLU 85 LYS) on this particular residue, it had readily denatured the whole α‐helix (from 80 to 90 amino acids). This was confirmed through the molecular dynamics simulation and revealed that this single mutation can cause many functional and structural changes. Furthermore, LYS85 mutation has altered the original secondary structure of the protein, which in turn led to the steric hindrance during the ligand–receptor interaction. The results based on the G‐score revealed that ligands have reduced interaction with the mutant protein. The semisynthetic fluoroquinolone 6d, which is an exception, forms a strong interaction with the mutant protein and was experimentally verified using the antimicrobial test. Hence, the present study unravels the fact that mutation at the drug binding site is the major cause for different level of resistance by the S. pyogenes when exposed against the varying concentrations of the fluoroquinolones. Furthermore, a comparative assessment of quinolone derivative with the older generation fluoroquinolones will be of great impact for S. pyogenes–related infections. Copyright © 2013 John Wiley & Sons, Ltd.     

Solution dynamics of the oligosaccharide moiety of ganglioside GM1: Comparison of solution conformations with the bound state conformation in association with cholera toxin B-pentamer

The solution dynamics of the oligosaccharide moiety of ganglioside G_M1 have been determined by use of a combination of ¹H rotating frame Overhauser effect measurements and restrained molecular dynamics simulations, It is found that the Galβ1-3 and NeuNAc moieties which are primarily recognized by cholera toxin both exhibit considerable torsional flexibility about their respective glycosidic linkages. A comparison with the bound state conformation of the ganglioside in association with cholera toxin B-pentamer, shows that a low energy conformation of the oligosaccharide, which closely approximates the globel minimum, is selected upon binding.     

Cloning, expression, and characterization of sialic acid synthases

The most commonly occurring sialic acid, N-acetylneuraminic acid, is the repeating unit in polysialic acid chain of human neuronal cell adhesion molecule as well as in capsular polysialic acid of neuroinvasive bacteria, Escherichia coli K1 and Neisseria meningitidis. Sialic acid synthesis and polymerization occur in slightly different pathways in animals and bacteria. N-Acetylneuraminic acid (NeuNAc) is synthesized by the condensation of phosphoenolpyruvate and N-acetylmannosamine by NeuNAc synthase in bacteria. The mammalian homologue N-acetylneuraminic acid-9-phosphate (NeuNAc-9-P) synthase uses N-acetylmannosamine-6-phosphate in the condensation reaction to produce NeuNAc-9-P. Both subfamilies of sialic acid synthases possess N-terminal triosephosphate isomerase barrel domain and C-terminal antifreeze protein domain. We report cloning of the genes, expression, purification, and characterization of human NeuNAc-9-P synthase and N. meningitidis NeuNAc synthase. Stability of the purified enzymes and effects of pH and temperature on their activities were evaluated. Enzyme kinetics and preliminary mutagenesis experiments reveal the importance of C-terminal antifreeze protein domain and a conserved cysteine residue for the enzyme activities.     

Crystal structure of cholera toxin B-pentamer bound to receptor GM1 pentasaccharide.

Cholera toxin (CT) is an AB5 hexameric protein responsible for the symptoms produced by Vibrio cholerae infection. In the first step of cell intoxication, the B-pentamer of the toxin binds specifically to the branched pentasaccharide moiety of ganglioside GM1 on the surface of target human intestinal epithelial cells. We present here the crystal structure of the cholera toxin B-pentamer complexed with the GM1 pentasaccharide. Each receptor binding site on the toxin is found to lie primarily within a single B-subunit, with a single solvent-mediated hydrogen bond from residue Gly 33 of an adjacent subunit. The large majority of interactions between the receptor and the toxin involve the 2 terminal sugars of GM1, galactose and sialic acid, with a smaller contribution from the N-acetyl galactosamine residue. The binding of GM1 to cholera toxin thus resembles a 2-fingered grip: the Gal(beta 1-3)GalNAc moiety representing the "forefinger" and the sialic acid representing the "thumb." The residues forming the binding site are conserved between cholera toxin and the homologous heat-labile enterotoxin from Escherichia coli, with the sole exception of His 13. Some reported differences in the binding affinity of the 2 toxins for gangliosides other than GM1 may be rationalized by sequence differences at this residue. The CTB5:GM1 pentasaccharide complex described here provides a detailed view of a protein:ganglioside specific binding interaction, and as such is of interest not only for understanding cholera pathogenesis and for the design of drugs and development of vaccines but also for modeling other protein:ganglioside interactions such as those involved in GM1-mediated signal transduction.     

Binding of fluorescently labeled cholera toxin subunit B to glycolipids in the human submandibular gland and inhibition of binding by periodate oxidation and by galactose

FITC-labeled cholera toxin subunit B (CTB) stained the surfaces of cells of mucous acini in the submandibular gland. CTB, also called choleragenoid, binds to the GM1 glycolipid in the cell membrane. The binding in most acini was inhibited by periodic acid oxidation of the sections, while some acini remained unaffected even after increased oxidation. Staining with the subunit was also reduced significantly by adding galactose to the incubation medium. Binding of CTB to cell surfaces apparently requires intact sialic groups on most, but not all, cell surfaces. Oxidation of the sialic acid residues may influence the structure of the sialylated GM1 molecules on the cell surface in different ways. It is possible that both the sialic acid residue and the terminal galactose are oxidized. Alternatively, the sialic acid may be resistant to acid hydrolysis in gangliosides in which the sialic acid is attached to the internal galactose residue linked to GalNAc, as in the GM1 glycolipid. Inhibition of the GM1 receptor binding to cholera toxin has potential for protection of humans against cholera. Galactose and agents that modify sialic acid inhibit the accessibility of the toxin to the GM1 carbohydrate receptor. Human milk contains high levels of sialic acid glycoconjugates that may provide defense mechanisms.     

Elucidating the molecular interaction of Zebrafish (Danio rerio) peptidoglycan recognition protein 2 with diaminopimelic acid and lysine type peptidoglycans using in silico approaches

Abstract

Peptidoglycan recognition proteins (PGRPs) belong to the family of pattern recognition receptor, represent the major constituent of innate immunity. Although PGRPs are structurally conserved through evolution, their involvement in innate immunity is different in vertebrates and invertebrates. They are highly specific towards recognition of ligands and can hydrolyze bacterial peptidoglycans (PGNs). Zebrafish PGRPs (zPGRPs) have both peptidoglycans lytic amidase activity and broad-spectrum bactericidal activity, but far less is known about how these receptors recognize these microbial ligands. Such studies are hindered due to lack of structural and functional configuration of zPGRPs. Therefore, in this study, we predicted the three-dimensional structure of zPGRP2 through theoretical modeling, investigated the conformational and dynamic properties through molecular dynamics simulations. Molecular docking study revealed the microbial ligands, that is, muramyl pentapeptide–DAP , muramyl pentapeptide–LYS, muramyl tripeptide–DAP, muramyl tripeptide–Lys, muramyl tetrapeptide–DAP, muramyl tetrapeptide–LYS and tracheal cytotoxin interacts with the conserved amino acids of the ligand recognition site comprised of β1, α2, α4, β4 and loops connecting β1 ? α2, α2 ? β2, β3 ? β4 and α4 ? α5. Conserved His31, His32, Ala34, Ile35, Pro36, Lys38, Asp60, Trp61, Trp63, Ala89, His90, Asp106, His143 and Arg144 are predicted to essential for binding and provides stability to these zPGRP–PGN complexes. Our study provides basic molecular information for further research on the immune mechanisms of PGRP’s in Zebrafish. The plasticity of the zPGRP’s binding site revealed by these microbial ligands suggests an intrinsic capacity of the innate immune system to rapidly evolve specificities to meet new microbial challenges in the future.

Communicated by Ramaswamy H. Sarma     

固有无序蛋白与蛋白质相互作用位点残基特征分析

本文对固有无序蛋白(IDPs)与其他蛋白质相互作用位点残基特征进行了研究.首先在数据库中选出满足条件的109条IDPs蛋白质链及与其他配体蛋白形成的299个IDPs-蛋白质复合物,然后提取复合物中作为相互作用位点的IDPs-蛋白质残基.这109条IDPs链中共含有50 031个氨基酸残基,其中处于作用位点的残基有4 822个.通过分析发现,20种氨基酸在形成IDPs-蛋白质相互作用位点残基时具有不同的倾向性,根据形成作用位点残基的倾向性,20种氨基酸可分成三大类:倾向型氨基酸(ILE、LEU、ARG、PHE、TYR、MET、TRP)、中间型氨基酸(GLN、GLU、THR、LYS、VAL、ASP、HIS)、非倾向型氨基酸(PRO、SER、GLY、ALA、ASN、CYS).研究结果还进一步表明,不同氨基酸在有序区域与无序区域形成IDPs-蛋白质作用位点残基的倾向性不同.其中,氨基酸TRP、LEU、ILE、CYS在有序和无序区域形成作用位点残基的差异性尤为明显,而氨基酸GLU、PHE、HIS、ALA则基本没有多大差别.对IDPs-蛋白质相互作用位点残基理化特征进行分析发现:疏水性强、侧链净电荷量较少、极性较小、溶剂可及性表面积较大、侧链体积较大、极化率较大的氨基酸比较倾向于形成作用位点残基.主成分分析结果显示,残基的极化率、侧链体积和溶剂可及表面积对作用位点残基影响最大.     

Glial uptake of neurotransmitter glutamate from the extracellular fluid studied in vivo by microdialysis and (13)C NMR

Glial uptake of neurotransmitter glutamate (GLU) from the extracellular fluid was studied in vivo in rat brain by (13)C NMR and microdialysis combined with gas-chromatography/mass-spectrometry. Brain GLU C5 was (13)C enriched by intravenous [2,5-(13)C]glucose infusion, followed by [(12)C]glucose infusion to chase (13)C from the small glial GLU pool. This leaves [5-(13)C]GLU mainly in the large neuronal metabolic pool and the vesicular neurotransmitter pool. During the chase, the (13)C enrichment of whole-brain GLU C5 was significantly lower than that of extracellular GLU (GLU(ECF)) derived from exocytosis of vesicular GLU. Glial uptake of neurotransmitter [5-(13)C]GLU(ECF) was monitored in vivo through the formation of [5-(13)C,(15)N]GLN during (15)NH(4)Ac infusion. From the rate of [5-(13)C,(15)N]GLN synthesis (1.7 +/- 0.03 micromol/g/h), the mean (13)C enrichment of extracellular GLU (0.304 +/- 0.011) and the (15)N enrichment of precursor NH(3) (0.87 +/- 0.014), the rate of synthesis of GLN (V'(GLN)), derived from neurotransmitter GLU(ECF), was determined to be 6.4 +/- 0.44 micromol/g/h. Comparison with V(GLN) measured previously by an independent method showed that the neurotransmitter provides 80-90% of the substrate GLU pool for GLN synthesis. Hence, under our experimental conditions, the rate of 6.4 +/- 0.44 micromol/g/h also represents a reasonable estimate for the rate of glial uptake of GLU(ECF), a process that is crucial for protecting the brain from GLU excitotoxicity.     

Effects of 4-Aminopyridine on Extracellular Concentrations of Glutamate in Striatum of the Freely Moving Rat

4-aminopyridine (4-AP) is a voltage-sensitive K⁺-channel blocker extensively used in in vitro experiments as a depolarizing agent for the release of glutamate (GLU). This research investigated whether 4-AP could be used in in vivo experiments using microdyalisis. For that, the effects of 4-AP on the extracellular concentrations of glutamate (GLU), glutamine (GLN), taurine (TAU) and citrulline (CIT) in striatum of the freely moving rat were investigated. The effects of 4-AP were compared with those produced by perfusion with a high K⁺ (100 mM) medium. Intrastriatal perfusion with 4-AP (1, 5 and 10 mM) produced no effects on extracellular [GLU], [TAU] and [CIT], but decreased extracellular [GLN]. Perfusion with a high K⁺ (100 mM) medium increased extracellular [GLU] and [TAU], decreased extracellular [GLN], and had no effects on [CIT]. To test whether the lack of effects of 4-AP on extracellular [GLU] was due to GLU uptake mechanisms, 4-AP was perfused after a previous inhibition of GLU uptake with L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC). Under the effects of PDC (1 mM), 4-AP (1 mM) had no effects on extracellular [GLU], [TAU] and [CIT], but decreased extracellular [GLN]. These results show that 4-AP decreased extracellular [GLN] but failed to produce a significant release of GLU in striatum of the freely moving rat. Thus, 4-AP can not be used as a depolarizing agent for stimulating the release of GLU in in vivo studies using microdialysis.     

Simultaneous determination of kinetic parameters for the binding of cholera toxin to immobilized sialic acid and monoclonal antibody using an array biosensor

Interactions between protein toxins and carbohydrate receptors are often semi-selective processes and the kinetic parameters that define the binding of a receptor to different toxins may vary with each interaction. In this study, we have determined the affinity constants for binding of cholera toxin (CT) to immobilized sialic acid and to anti-CT antibody (as a simultaneous reference) by measuring real-time binding processes using an array biosensor. N-Acetylneuraminic acid (Neu5Ac), a member of the sialic acid family, was covalently immobilized onto maleimide-activated planar waveguides via a thiol-terminated linker attached to the anomeric carbon of the sugar. Control antibodies were immobilized using two different approaches: covalent attachment onto maleimide-activated slides via the thiol on cysteine residues and non-covalent attachment using a biotin-NeutrAvidin linkage. Cy5-labeled CT was flowed over the immobilized receptors and the fluorescent intensity of the bound CT-receptor complex was recorded as a function of time. The association constants for CT binding to covalently attached Neu5Ac, to covalently attached anti-CT monoclonal antibody, and to antibody tethered by biotin-NeutrAvidin interactions were determined to be 1.3 x 10(8), 2.1 x 10(8) and 5.7 x 10(8)M(-1), respectively.     

低智儿童与正常儿童中氨基酸的比较研究

我们对 30例低智儿童血清中TAU ,SER ,GLU ,GLY ,ALA ,VAL ,CYS ,MET ,ILE ,LEU ,TYR ,PHE ,TRP ,HIS ,ORN ,LYS ,ARG ,PRO ,18种游离氨基酸进行了测定。研究结果显示 :患儿血清中 11种游离氨基酸降低分别为 :TAU ,SER ,VAL ,MET ,ILE ,LEU ,TRY ,PHE ,ORN ,LYS ,TRP .氨基酸的失衡 ,儿童的蛋白质合成就将会受到严重的影响 ,因此将导致大脑的分化 ,发育受阻 ,引起智力低下。