Biomedical subjects

Kazukiyo Kobayashi

Publications and source records attributed to Kazukiyo Kobayashi.

At least 19 recordsLinked to original sources

PubMed2006

A globotriaosylceramide (Gb3Cer) mimic peptide isolated from phage display library expressed strong neutralization to Shiga toxins.

A Gb3-trisaccharide mimic peptide was selected with biopanning from a phage display library against anti-Gb3 antibody to neutralize Shiga toxins (Stxs). Biopanning was carried out on a microplate immobilized with a Fab fragment of anti-Gb3 antibody and a subtraction procedure screening was applied to enhance specificity. The selected phage clones showed strong affinity to anti-Gb3 antibody and to Stxs. Among these clones, a 9-mer sequence WHWTWLSEY was determined as the strongest Gb3 mimic peptide and chemically synthesized. The peptide bound strongly to Stx-1 and Stx-2, though the binding was inhibited with Gb3Cer. Surface plasmon resonance (SPR) and fluorescent spectroscopy determined that the affinity of the peptide to both Stxs was strong. Neutralization activity was confirmed by in vitro assay with HeLa cells. The Gb3 mimic peptide potentially has great promise for use against Stxs.

Amino Acid Sequence↗

PubMed2006

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. catarrhalis to human pharyngeal epithelial cells. Furthermore, six different synthetic sulfatides were found to inhibit the attachment of M. catarrhalis significantly at an optimum concentration of 10 microg/ml. Synthetic sulfatides may have the potential to be used as a therapy to prevent M. catarrhalis infections.

Adhesins, Bacterial↗

PubMed2005

One-pot alpha-glycosylation pathway via the generation in situ of alpha-glycopyranosyl imidates in N,N-dimethylformamide.

Divergent pathways are disclosed in the activation of 2-O-benzyl-1-hydroxy sugars by a reagent combination of CBr4 and Ph3P, all of which afford one-pot alpha-glycosylation methods. When this reagent is used in CH2Cl2, the 1-hydroxy sugar is converted to the alpha-glycosyl bromide in a conventional way and leads to the one-pot alpha-glycosylation method based on a halide ion-catalytic mechanism. In either DMF or a mixture of DMF and CHCl3, however, alternative alpha-glycosyl species are generated. From the 1H and 13C NMR study of the products, as well as the reactions using Vilsmeier reagents [(CH3)2N+=CHX]X- (X=Br and Cl), these were identified as cationic alpha-glycopyranosyl imidates having either Br- or Cl- counter ion. The cationic alpha-glycosyl imidate (Br-), derived specifically in the presence of DMF, is more reactive than the alpha-glycosyl bromide and thus is responsible for the accelerated one-pot alpha-glycosylation. The one-pot alpha-glycosylation methodology performed in DMF was assessed also with different types of acceptor substrates including tertiary alcohols and an anomeric mixture of 1-OH sugars.

Carbohydrates↗

PubMed2005

Design of multifunctional peptides expressing both antimicrobial activity and shiga toxin neutralization activity.

We have designed novel short peptides expressing both antimicrobial and Shiga-toxin (Stx) neutralization activities by combining nuclear localization signal (NLS) peptides (RIRKKLR, PKKKRKV, and PRRRK) tandemly with globotriaoside (Gb3) mimic peptide (WHWTWL). These fusion peptides exhibited excellent antimicrobial activity against both gram-positive and gram-negative bacteria. A peptide WHWTWLRIRKKLR (Trp-His-Trp-Thr-Trp-Leu-Arg-Ile-Arg-Lys-Lys-Leu-Arg), especially, exhibited about 100 times higher activity than the original NLS peptide. SPR analysis demonstrated that the binding of this peptide to both Stxs was strong: K(d) = 6.6 x 10(-6) to Stx-1 and 6.8 x 10(-6) to Stx-2. The in vitro assay against Stx-1 using HeLa cells showed that this peptide increased the survival rate of HeLa cells against the infection of Stx-1. The peptide has been found to maintain high antimicrobial activity, Stx neutralization activity, and no cytotoxicity at its concentration of 7.8-31.3 microg/mL (4.2-16.7 microM). The present peptide design has a prospect of developing potent multifunctional drugs to destroy proteinaceous toxin-producing bacteria and to simultaneously neutralize the toxins released by bacteriolysis.

Amino Acid Sequence↗

PubMed2005

Effect of the carbohydrate side-chain on the conformation of a glycoconjugate polystyrene in aqueous solution.

An oligomaltose-carrying polystyrene "glycoconjugate polystyrene" was synthesized by the homopolymerization of 4-vinylbenzylamine oligomaltonic amides, derived from maltose, maltotriose, maltopentaose, and maltoheptaose. The resultant amphiphilic glycoconjugate polystyrenes were dissolved in 0.1 M aqueous urea, and their structures characterized by small-angle X-ray scattering and molecular modeling. "Glycoconjugate polystyrene" was found to behave as a "molecular bottle brush", composed of a large pseudo-helical polystyrene backbone and carbohydrate brushes. A large pseudo-helical polystyrene backbone is formed by a random sequence of TT, TG, and/or TTGG. The results indicate that the cross-section of a backbone chain with smaller oligosaccharide side-chains is obliged to expand more than that with longer side-chains. Even with rigid hydrophilic pendant oligosaccharide chains, the larger pseudo-helix of the main chain could orient the side-chains so as to envelop the hydrophobic backbone in aqueous solution. Thus the conformation of the main chain is determined not only by the chemical nature of an oligosaccharide chain but also by its length.

Computer Simulation↗

PubMed2005

Cooperative lectin recognition of periodical glycoclusters along DNA duplexes: alternate hybridization and full hybridization.

We describe herein the construction of periodically, spatially controlled glycoclusters along DNA duplexes and their cooperative lectin recognition. Site-specifically alpha-mannosylated oligodeoxynucleotide 20-mer (Man-ODN20) was synthesized via the phosphoramidite solid-phase synthesis. Alternate hybridization of the Man-ODN20 with the half-sliding complementary ODN 20-mer (hscODN20) gave an alternately prolonged Man-cluster Man-ODN20/hscODN20. The binding of the Man-cluster to FITC-labeled ConA lectin showed sigmoidal fluorescence dependency on the concentration of Man-ODN, indicating that some mannose residues along the repeating DNA duplex were cooperatively bound to ConA (apparent affinity constant: K(af)=2.4 x 10(4)M(-1) and Hill coefficient: n=3.5). The duplex of Man-ODN20 with full complementary ODN 20-mer (fcODN20) was little bound to ConA. The binding behavior of Man-ODN20/hscODN20 is compared with that of the alternately prolonged Gal-cluster Gal-ODN20/hscODN20 previously reported. Duplexes 20-mer, 40-mer, and 60-mer presenting one, two, and three periodic galactoses were also prepared by full hybridization of 20-mer beta-galactosylated oligodeoxynucleotide (Gal-ODN20) with the periodically repeating full complementary 20-mer, 40-mer, and 60-mer ODNs. RCA(120) lectin was found to little bind the 20-mer and 40-mer duplexes and to bind weakly and non-cooperatively the 60-mer duplex (K(af)=1.1 x 10(4)M(-1)). The cooperative lectin recognition of these glycoclusters in relation with the degree of association (DA) of ODN and the numbers of glycosides along the DNA duplex is discussed.

Chromatography, Gel↗

PubMed2005

Preparation and supramolecular properties of unadulterated glycosyl liposomes from a bis(alpha-D-mannopyranosyl)-[60]fullerene conjugate.

The bis(alpha-D-mannopyranosyl)-[60]fullerene conjugate 3 was prepared by thermal coupling of C60 and either 2-azidoethyl 2,3,4,6-tetra-O-acetyl- or 2,3;4,6-di-O-isopropylidene-alpha-D-mannopyranoside (Scheme). Compound 3 was found to readily self-assemble. Dynamic-light-scattering (DLS) and atomic-force microscopy (AFM) experiments supported that the amphiphilic compound gives rise to nano-sized supramolecular structures during sugar deprotection (Ac-group removal) performed in MeOH/CH2Cl2 solution. Encapsulation studies with an aqueous suspension of 3 showed that the self-assembling structure envelopes Ba2+ and the fluorescent dye Acridine Red during its formation, which indicates that it resembles a bilayer vesicle or an unadulterated liposome with an inner hollow space. In addition to this notable property, the unique molecular geometry of the spatially arranged mannosyl surface residues of 3 gives rise to strong binding of the carbohydrate-recognizing lectin Con A. Hence, the polar amphiphilic end of 3 mimics the structure of 3,6-branched tri-alpha-D-mannoside (6; Fig. 3), a natural ligand of the Con A protein.

Barium↗

PubMed2004

Peptides binding to a Gb3 mimic selected from a phage library.

Peptides binding to a Gb3 mimic were selected from 12-mer peptide library. The self-assembled monolayer (SAM) of a Gb3 mimic was formed on the gold surface, and biopanning was carried out with the phage display peptide library. After three rounds of biopanning, four individual sequences were obtained from 10 phage clones, and the selected peptides having the specific 7-mer sequence (FHENWPS) showed affinities to the Gb3 mimic as strong as to RCA120. Molecular dynamics calculations suggested that the peptides bound to the Gb3 mimic by hydrophobic interaction and hydrogen bonding formation, and the cooperative interactions played an important role in the recognition. The Stx-1 binding was inhibited by the peptides.

Amino Acid Sequence↗

PubMed2004

Sulfatase-catalyzed assembly of regioselectively O-sulfonated p-nitrophenyl alpha-D-gluco- and alpha-D-mannopyranosides.

A chemoenzymic methodology is extended to the library synthesis of regioselectively O-sulfonated pNP D-gluco and D-mannopyranosides. The method involves the sequential reactions of chemical O-sulfonation and sulfatase-catalyzed O-desulfonation. pNP 2,6-di-O-sulfo-alpha-D-glucopyranoside and pNP 3,6-di-O-sulfo-alpha-D-mannopyranoside were obtained as sodium salts using chemical methods by way of dibutylstannylene acetals or tributylstannyl ethers. They were then applied to enzyme reactions using three molluscan enzymes (snail, limpet, and abalone). The sulfatase reactions cleaved a sulfate group at the secondary O-2 or O-3 position to yield the corresponding pNP 6-O-sulfo sugars. Neither pNP 6-O-sulfo-alpha-D-glucopyranoside nor 6-O-sulfo-alpha-D-mannopyranoside became the enzyme substrate. Evidently, the molluscan sulfatases have a tendency to cleave the secondary O-sulfo group with assistance from the 6-O-sulfo group.

Animals↗

PubMed2004

Design of N-acetyl-6-sulfo-beta-d-glucosaminide-based inhibitors of influenza virus sialidase.

Biological activity of N-acetyl-6-sulfo-beta-d-glucosaminides (6-sulfo-GlcNAc 1) having a structural homology to N-acetylneuraminic acid (Neu5Ac 2) and 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (Neu5Ac2en 3) was examined in terms of inhibitory activity against influenza virus sialidase (influenza, A/Memphis/1/71 H3N2). pNP 6-Sulfo-GlcNAc 1a was proved to show substantial activity to inhibit the virus sialidase (IC(50)=2.8 mM), though p-nitrophenyl (pNP) GlcNAc without 6-sulfo group and pNP 6-sulfo-GlcNH(3)(+) 1b without 2-NHAc showed little activity (IC(50) >50 mM). The activity was enhanced nearly 100-fold when the pNP group of 1a was converted to p-acetamidophenyl one 5 (IC(50)=30 microM) or replaced with 1-naphthyl 6 (IC(50)=10 microM) or n-propyl one 8 (IC(50)=11 microM).

Antiviral Agents↗

PubMed2004

Self-organized glycoclusters along DNA: effect of the spatial arrangement of galactoside residues on cooperative lectin recognition.

We describe herein the relationship between the spatial arrangement of self-organized galactose clusters and lectin recognition. beta-Galactose-modified deoxyuridine phosphoramidite was synthesized and applied to solid-phase synthesis to provide 18-, 20-, and 22-mers of site-specifically galactosylated oligodeoxynucleotides (Gal-ODNs). These Gal-ODNs were self-organized through hybridization with the corresponding 18-, 20-, and 22-mers of half-sliding complementary ODNs (hsc-ODNs) to give periodic galactoside clusters. The self-organization of ODNs was confirmed by size exclusion chromatography and gel electrophoresis. The binding of the Gal-clusters to the FITC-labeled RCA(120) lectin was analyzed by monitoring the change in fluorescence intensity. The assembly of 20-mer Gal-ODN with the 20-mer hsc-ODN was strongly and cooperatively recognized by the lectin. The 18-mer assembly was bound more weakly and less cooperatively, and the 22-mer assembly was minimally bound to the lectin. RCA(120) lectin recognized not only the density of galactoside residues, but also the spatial arrangement. The size of the Gal cluster was estimated from the association constant of Gal-ODN with hsc-ODN. The relationship between lectin-recognition and Gal-cluster size is also discussed.

DNA↗

PubMed2004

Stereo- and biochemical profiles of the 5-6- and 6-6-junction isomers of alpha-D-mannopyranosyl [60]fullerenes.

The 5-6- and 6-6-junction isomers of alpha-D-mannopyranosyl [60]fullerene were studied by means of circular dichroism (CD), deuterium labeling, 1H-NMR, molecular-dynamics (MD) calculations, and a lectin-binding assay. The CD spectra of the O-acetylated derivatives allowed clear discrimination of the isomers, while the 1H-NMR spectra, with assistance from deuterium labeling and MD calculations, served to disclose the unique conformation and molecular geometry of each acetylated isomer in chloroform solution. The deprotected 5-6- and 6-6-isomers, which gave colloidal suspensions in aqueous mixtures, displayed marked activity in blocking lectin-induced hemagglutination by concanavalin A.

Carbohydrate Conformation↗

PubMed2004

Analysis of GM3-Gg3 interaction using clustered gycoconjugate models constructed from glycolipid monolayers and artificial glycoconjugate polymers.

Carbohydrate-carbohydrate interactions between clustered GM3 on the Langmuir monolayer and clustered Gg3 trisaccharide along a polystyrene chain were investigated using surface pressure-area (pi-A) isotherms and surface plasmon resonance (SPR). The pi-A isotherm of the GM3 monolayer was expanded substantially and specifically by the Gg3-trisaccharide-bearing glycoconjugate polystyrene [PN(Gg3)] even at 10(-12) M. The PN(Gg3)-induced expansion of the GM3 monolayer required no calcium ion, and the expansion was strongly inhibited in the presence of urea and acetamido sugars. SPR studies of the GM3-Gg3 interaction were carried out to estimate the affinity constant and specificity of the interaction quantitatively. PN(Gg3) was adsorbed onto the GM3 monolayer strongly and specifically with an apparent affinity constant of Ka = 2.5 x 10(6) M(-1). The mechanism of the GM3-Gg3 interaction was discussed on the basis of the relationship between affinity and structure. We found that the NHAc groups of N -acetylneuraminic acid in GM3 and of GalNAc in Gg3 play an important role in the GM3-Gg3 interaction and that PN(Gg3) recognizes not only some specified portions of GM3 but also the trisaccharide as a whole. Published in 2003.

G(M3) Ganglioside↗

PubMed2003

Design and synthesis of C3-symmetric Lewis(x) antigen.

[structure: see text] C(3)-Symmetric glycoconjugates carrying three equivalent Lewis(X) antigens or beta-lactosides were synthesized from p-nitrophenyl glycosides and trimesic acid via regio- and stereocontrolled glycosylation reactions. An (1)H NMR study has shown that the C(3)-symmetric glycoconjugates soluble in water provide useful probes to investigate the Ca(2+)-dependent Lewis(X)-Lewis(X) association.

Calcium↗

PubMed2003

N-Acetyl-6-sulfo-D-glucosamine as a promising mimic of N-Acetyl neuraminic acid.

6-Sulfo-D-GlcNAc with a molecular geometry close to that of N-acetylneuraminic acid (Neu5Ac) was hypothesized to serve as a simple Neu5Ac mimic possessing high potential in biochemical and medicinal applications. The hypothesis was evidenced with a neuraminidase inhibition assay using p-nitrophenyl (pNP) 3-, 4-, and 6-sulfo-beta-D-GlcNAc (4, 5 and 2a) and 6-sulfo-beta-D-Glc 6, in which only pNP 6-sulfo-beta-D-GlcNAc 2a was found to show substantial activity.

Acetylglucosamine↗

PubMed2003

An easy access to halide ion-catalytic alpha-glycosylation using carbon tetrabromide and triphenylphosphine as multifunctional reagents.

The reaction of a 2-O-benzyl-1-hydroxy sugar with CBr4 and Ph3P generates a glycosyl bromide in situ, which is coupled with an acceptor alcohol in the presence of N,N-tetramethylurea to afford an alpha-glycosyl product virtually quantitatively. In a proposed pathway, the reagent combination plays multiple roles such as the generation of a glycosyl donor, the activation of glycosylation, and the dehydration of the reaction system. These roles allow a simple alpha-glycosylation to be performed without special attention to dehydration. Various alpha-glycosyl (D-gluco-, D-galacto- and L-fuco-) products including glycosyl glycerols and cholesterols have been prepared with this method.

Cholesterol↗

PubMed2003

One-pot alpha-glycosylation method using Appel agents in N,N-dimethylformamide.

[reaction: see text] A concept for the development of practical glycosylation is presented and demonstrated by one-pot alpha-glycosylation applying Appel agents for 2-O-benzyl-1-OH hexoses in DMF. The reaction, in situ giving the equilibrium of glycosyl bromides and more reactive O-glycoside intermediates, accomplishes a near-quantitative alpha-glycosylation removing the water molecules.

Dimethylformamide↗

PubMed2003

Synthetic potential of molluscan sulfatases for the library synthesis of regioselectively O-sulfonated D-galacto-sugars.

The substrate specificities of three molluscan sulfatases (E.C. 3.1.6.1; snail, abalone, and limpet origins) were investigated with assorted p-nitrophenyl (pNP) di-O-sulfonated beta-D-galactopyranosides and beta-lactosides [3,6-SO(3) Gal (1), 3',6'-SO(3) Lac (2), 4, 6SO(3) Gal (3), 2,6-SO(3) Gal (4), 3,4-SO(3) Gal (5), and 3,6-SO(3) GalNAc (6); Ac, acetyl; Gal, galactose; Lac, lactose] together with mono-O-sulfonated beta-D-galactopyranoside [pNP 3SO(3)-Gal (7)] and tri-O-sulfonated alpha-D-galactopyranoside [2,3,6-SO(3)-alpha-Gal (11)]. Some notable differences between the substrate specificity of the three sulfatases were disclosed; snail sulfatase hydrolyzed the 3O- and 2O-sulfo groups of 1 and 4, respectively, to afford 6SO(3) Gal (9) in high yields, while the abalone enzyme did not act on 4. Only the limpet enzyme could cleave the 3O-sulfo groups of 7 to give pNP beta-galactoside. In contrast, every enzyme could utilize 11 as a good substrate to afford a mixture of 6SO(3)-alpha-Gal (13) and 2,6-SO(3) alpha-Gal (12). None of the enzymes could cleave the O-sulfo groups of 5 and 6, which indicates that a primary 6O-sulfo group tends to promote the enzymatic hydrolysis of O-sulfo groups at the secondary positions.

Animals↗