An Advanced Sensing Approach to Biological Toxins with Localized Surface Plasmon Resonance Spectroscopy Based on Their Unique Protein Quaternary Structures.

Botulinum neurotoxins (BoNTs), ricin, and many other biological toxins are called AB toxins possessing heterogeneous A and B subunits. We propose herein a quick and safe sensing approach to AB toxins based on their unique quaternary structures. The proposed approach utilizes IgG antibodies against their A-subunits in combination with those human cell-membrane glycolipids that act as the natural ligands of B-subunits.

Novel Glycolipid Chips with a Double Layer of Au Nanoparticles for Biological Toxin Detection.

Glycolipid chips having a double layer of Au nanoparticles are proposed for detection of biological toxins. The sugar-modified chips constitute an under and an upper layer of Au nanoparticles of 20-80 nm diameter on glass plates, and Au nanoparticles of each layer are linked with 1,8-octanedithiol by a self-assembled monolayer (SAM) technique. A tris-sialo glycosphingolipid, ganglioside GT1b, having lipoic amide at the sphingosine part was immobilized on the Au outside surface of the upper layer, and botulinum toxin (type A heavy chain) was detected by localized surface plasmon resonance (LSPR).

H-NMR Karplus Analysis of Molecular Conformations of Glycerol under Different Solvent Conditions: A Consistent Rotational Isomerism in the Backbone Governed by Glycerol/Water Interactions.

Glycerol is a symmetrical, small biomolecule with high flexibility in molecular conformations. Using a H-NMR spectroscopic Karplus analysis in our way, we analyzed a rotational isomerism in the glycero backbone which generates three kinds of staggered conformers, namely gt (-), gg (-), and tg (-), at each of -1,2 and -2,3 positions. The Karplus analysis has disclosed that the three rotamers are consistently equilibrated in water keeping the relation of 'gt:gg:tg = 50:30:20 (%)' at a wide range of concentrations (5 mM~540 mM).

Assembly of Glycochips with Mammalian GSLs Mimetics toward the On-site Detection of Biological Toxins.

According to our previously proposed scheme, each of three kinds of glycosphingolipid (GSL) derivatives, that is, lactosyl ceramide [Lac-Cer ()] and gangliosides [GM1-Cer () and GT1b-Cer ()], was installed onto the glass surface modified with Au nanoparticles. In the present study, we tried to apply microwave irradiation to promote their installing reactions. Otherwise, this procedure takes a lot of time as long as a conventional self-assembled monolayer (SAM) technique is applied.

Silicon nitride sugar chips for detection of Ricinus communis proteins and Escherichia coli O157 Shiga toxins.

Lactosides having either an amino-triethylene glycol or an azido-triethylene glycol were designed and synthesized, and the two derivatives were immobilized onto silicon nitride (SiN) surfaces. When a click reaction was applied for the immobilization of the azido-sugar, a Ricinus communis lectin (RCA) was detected with a higher response by reflectometric interference spectroscopy (RIfS). When an N-hydroxysuccinimide (NHS) method was applied for the sugar immobilization, the response was less than that of the click one.

Remarkable functions of -3 hydroxy and phosphocholine groups in 1,2-diacyl--glycerolipids to induce clockwise (+)-helicity around the 1,2-diacyl moiety: Evidence from conformation analysis by H NMR spectroscopy.

Cell-membrane glycerolipids exhibit a common structural backbone of asymmetric 1,2-diacyl--glycerol bearing polar head groups in the -3 position. In this study, the possible effects of -3 head groups on the helical conformational property around the 1,2-diacyl moiety in the solution state were examined. H NMR Karplus relation studies were carried out using a series of 1,2-dipalmitoyl--glycerols bearing different -3 substituents (namely palmitoyl, benzyl, hydrogen, and phosphates).

Thermophilic Flavin Adenine Dinucleotide-Dependent Glucose Dehydrogenase Bioanode for Biosensor and Biofuel Cell Applications.

Flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase (GDH) was identified and cloned from thermophilic filamentous fungi using the homology cloning method. A direct electron transfer bioanode composed of FAD-GDH and a single-walled carbon nanotube was produced. Enzymes from thermophilic microorganisms generally have low activity at ambient temperature; however, the FAD-GDH bioanode exhibits a large anodic current due to the enzymatic reaction (1 mA cm) at ambient temperature.

Bis(β-lactosyl)-[60]fullerene as novel class of glycolipids useful for the detection and the decontamination of biological toxins of the Ricinus communis family.

Glycosyl-[60]fullerenes were first used as decontaminants against ricin, a lactose recognition proteotoxin in the Ricinus communis family. A fullerene glycoconjugate carrying two lactose units was synthesized by a [3 + 2] cycloaddition reaction between C60 and the azide group in 6-azidohexyl β-lactoside per-O-acetate. A colloidal aqueous solution with brown color was prepared from deprotected bis(lactosyl)-C60 and was found stable for more than 6 months keeping its red color.

Interaction of 70-kDa heat shock protein with glycosaminoglycans and acidic glycopolymers.

Interaction of Hsp70 with natural and artificial acidic glycans is demonstrated based on the native PAGE analysis. Hsp70 interacts with acidic glycopolymers that contain clustered sulfated and di-sialylated glycan moieties on a polyacrylamide backbone, but not with neutral or mono-sialylated glycopolymers. Hsp70 also interacts and forms a large complex with heparin, heparan sulfate, and dermatan sulfate that commonly contain 2-O-sulfated iduronic acid residues, but not with other types of glycosaminoglycans (GAGs).

Localized surface plasmon resonance detection of biological toxins using cell surface oligosaccharides on glyco chips.

We have detected biological toxins using localized surface plasmon resonance (LSPR) and synthetic glycosyl ceramides (β-lactoside, globosyl trisaccharide (Gb3), or GM1 pentasaccharide) attached to gold (Au) nanoparticles. The particle diameters ranged from 5-100 nm. The detection sensitivity for three toxins (ricin, Shiga toxin, and cholera toxin) was found to depend not only on the attached glycoside but also on the diameter of the Au nanoparticles.

Glycotechnology for decontamination of biological agents: a model study using ricin and biotin-tagged synthetic glycopolymers.

Two types of biotin-tagged glycopolymers carrying lactose or glucose in clusters along the polyacrylamide backbone were prepared and subjected to decontamination analyses with the plant toxin ricin. A buffer solution containing the toxin was treated with one glycopolymer followed by streptavidin-magnetic particles. Supernatant solutions were analyzed with surface plasmon resonance and capillary electrophoresis, and revealed that the lactose glycopolymer "captured" this toxin more effectively than the glucose polymer.

Molecular design, synthesis and bioactivity of glycosyl hydrazine and hydrazone derivatives: notable effects of the sugar moiety.

Assuming that the water solubility of our previous hydrazone derivatives would improve after modification with sugars while keeping or modulating their notable biological activities, we designed and synthesized some glycosyl hydrazine and hydrazone derivatives. Bioassay results indicated that the antitumor activity of our previously prepared hydrazones reduced or disappeared after modification with sugars. On the contrary, some glycosyl derivatives displayed much better antifungal activity against selected fungi.

Determination of ricin by nano liquid chromatography/mass spectrometry after extraction using lactose-immobilized monolithic silica spin column.

Ricin is a glycosylated proteinous toxin that is registered as toxic substance by Chemical Weapons convention. Current detection methods can result in false negatives and/or positives, and their criteria are not based on the identification of the protein amino acid sequences. In this study, lactose-immobilized monolithic silica extraction followed by tryptic digestion and liquid chromatography/mass spectrometry (LC/MS) was developed as a method for rapid and accurate determination of ricin.

Preparation and evaluation of lactose-modified monoliths for the adsorption and decontamination of plant toxins and lectins.

A series of sugar-modified porous silica monoliths with different sugar ligands (β-lactoside, β-N-acetyllactosaminide, β-d-galactoside, β-d-N-acetylgalactosaminide and β-d-glucoside) and linkers were prepared and evaluated using plant toxins and lectins including ricin and a Ricinus communis agglutinin (RCA(120)). Among these sugar monoliths, a lactose monolith carrying a triethylene glycol spacer adsorbed ricin and RCA(120) with the highest efficiency. The monolith showed no binding with albumin, globulin, and lectins from Jack beans, Osage orange, Amur maackia and wheat germ.

Use of lactose against the deadly biological toxin ricin.

Developing a technology for detecting and decontaminating biological toxins is needed. Ricin from Ricinus communis is a highly poisonous toxin; it was formerly used for an assassination in London and in postal attacks in the United States. Ricin is readily available from castor beans and could be used as a biological agent.

Library assembly of mono-, di- and tri-O-sulfated beta-D-xylopyranosides; effect of O-sulfation on pyranose ring conformation.

With molluscan sulfatase-catalyzed de-O-sulfation reactions, a series of mono-, di- and tri-O-sulfated p-nitrophenyl beta-D-xylopyranosides were assembled and applied to a 1H NMR study to examine the effect of O-sulfate groups on the equilibration between pyranose 4C1 and 1C4 conformations.

Multivalent galacto-trehaloses: design, synthesis, and biological evaluation under the concept of carbohydrate modules.

Galacto-trehalose (GT) is a novel class of 1,1'-linked nonreducing disaccharide having an α-galactoside epitope. In this study, a pair of α,α- and α,β-GT isomers were prepared in one pot with our α-glycosylation method, converted into vinyl monomers and then subjected to radical copolymerization with a second sugar (4-acrylamidophemyl β-Glc or β-GlcNAc) in the presence of acrylamide. The derived glycopolymers were assayed with α-galactoside-specific proteins (BSI-B(4) lectin and Shiga toxin-1) to show the results that both α,α- and α,β-isomers are recognized by these carbohydrate-binding proteins more strongly in forms of the GT polymers.

[Highly sensitive detection technology for biological toxins applying sugar epitopes].

The Shiga toxin is a highly poisonous protein produced by enterohemorrhagic Escherichia coli O157. This bacterial toxin causes the hemolytic uremic syndrome. Another plant toxin from castor beans, ricin, is also highly toxic.

A novel sugar-probe biosensor for the deadly plant proteinous toxin, ricin.

Because of the illegal use of highly toxic ricin from the castor-oil plant, Ricinus communis, in bioterrorism and suspected white powder cases, anti-terrorism measures for the toxin are urgently required. Here we demonstrate a facile and sensitive detection method using synthetic analogues of beta-lactosyl- and beta-d-galactosyl ceramides as the ligands based on the fact that ricin binds cell-surface oligosaccharides. Sugar-probes having lipoic acids as anchor functions were synthesized via either a chemical or chemoenzymatic way and were immobilized on the sensor chips by a self-assembled monolayer technique.

Glycochips from polyanionic glycopolymers as tools for detecting Shiga toxins.

An alternating layer-by-layer adsorption methodology was applied to the assembly of glycochips by using synthetic polyanionic glycopolymers. Three glycochips carrying globobioside (Gb(2)), beta-lactoside (beta-Lac), or alpha-D-mannoside (alpha-Man) residues were prepared, and used for the detection of Shiga toxins, Stx-1 and Stx-2, by using surface plasmon resonance (SPR). Using this method, we could confirm that both Stx-1 and Stx-2 show binding specificity for the Gb(2) glycochip as well as a weak affinity for the beta-Lac glycochip.

Synthesis of p-nitrophenyl sulfated disaccharides with beta-D-(6-sulfo)-GlcNAc units using beta-N-acetylhexosaminidase from Aspergillus oryzae in a transglycosylation reaction.

When alpha-D-GlcNAc-OC(6)H(4)NO(2) -p and beta-D-(6-sulfo)-GlcNAc-OC(6)H(4)NO(2)-p (2) were used as substrates, beta-N-acetylhexosaminidase from Aspergillus oryzae transferred the beta-D-(6-sulfo)-GlcNAc(unit from 2 to alpha-D-GlcNAc-OC(6)H(4)NO(2) -p to afford beta-D-(6-sulfo)-GlcNAc-(1-->4)-alpha-D-GlcNAc-OC(6)H(4)NO(2)-p (3) in a yield of 94% based on the amount of donor, 2, added. beta-D-(6-sulfo)-GlcNAc-(1-->4)-alpha-D-Glc-OC(6)H(4)NO(2)-p (4) was obtained with alpha-D-Glc-OC(6)H(4)NO(2) -p as acceptor in a similar manner. With a reaction mixture of 2 and beta-D-GlcNAc-OC(6)H(4)NO(2)-p (1) in a molar ratio of 6:1, the enzyme mediated the transfer of beta-D-GlcNAc from 1 to 2, affording disaccharide beta-D-GlcNAc-(1-->4)-beta-(6-sulfo)-D-GlcNAc-OC(6)H(4)NO(2)-p (5) in a yield of 13% based on the amount of 1 added.

Effective chemoenzymatic synthesis of p-aminophenyl glycosides of sialyl N-acetyllactosaminide and analysis of their interactions with lectins.

A convenient chemoenzymatic procedure for the synthesis of p-aminophenyl glycosides of sialyl N-acetyllactosaminide has been developed from p-nitrophenyl N-acetyl-beta-D-glucosaminide as starting material through three steps: synthesis of p-nitrophenyl N-acetyllactosaminide with beta-D-galactosidase, chemical reduction of the p-nitrophenyl group, and sialylation with sialyltransferase. The p-aminophenyl glycosides were then successfully biotin-labeled through the coupling with N-(+)-biotinyl-6-aminohexanoic acid to afford biotinylated oligosaccharides with an aminohexanosyl group and phenyl group as the spacers between the biotin and glycan. Furthermore, the biotin-labeled sugars were shown to be useful for immobilization and assay of the carbohydrate-lectin interactions by an optical biosensor based on surface plasmon resonance.

Sulfatide and its synthetic analogues recognition by Moraxella catarrhalis.

Moraxella catarrhalis is one of the major pathogens of respiratory and middle ear infections. Attachment of this bacterium to the surface of human pharyngeal epithelial cells is the first step in the pathogenesis of infections. This study revealed that sulfatide might act as a binding molecule for the attachment of M.

Substrate specificity of N-acetylhexosaminidase from Aspergillus oryzae to artificial glycosyl acceptors having various substituents at the reducing ends.

The substrate specificity of N-acetylhexosaminidase (E.C. 3.

Inhibition of PrPSc formation by synthetic O-sulfated glycopyranosides and their polymers.

Sulfated glycosaminoglycans (GAGs) and sulfated glycans inhibit formation of the abnormal isoform of prion protein (PrPSc) in prion-infected cells and prolong the incubation time of scrapie-infected animals. Sulfation of GAGs is not tightly regulated and possible sites of sulfation are randomly modified, which complicates elucidation of the fundamental structures of GAGs that mediate the inhibition of PrPSc formation. To address the structure-activity relationship of GAGs in the inhibition of PrPSc formation, we screened the ability of various regioselectively O-sulfated glycopyranosides to inhibit PrPSc formation in prion-infected cells.