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Hybrid dynamic coating using n-dodecyl beta-d-maltoside (DDM) and methyl cellulose (MC) has been developed for suppression of analyte adsorption and electroosmotic flow (EOF) in a poly(methyl methacrylate) (PMMA) channel. The adsorption of APTS-labeled sugars in a PMMA channel was obviously suppressed with DDM dynamic coating; however, EOF was reduced only by a factor of approximately 25%, resulting in irreproducible separations. In contrast, both analyte adsorption and EOF in a PMMA channel were efficiently minimized with MC coating; however, concentrated MC above 0.3% was required to achieve high-performance separations, which greatly increased viscosity of the solution and caused difficulties during buffer loading and rinsing. In addition, n-dodecyltrimethylammonium chloride did not show observable effects on reducing analyte adsorption, although it has the same hydrophobic alkyl chain as DDM. These results strongly indicated that the polysaccharide moiety of surface modifiers has a specific affinity to surface charges and is crucial to achieving efficient and stable dynamic coating on the PMMA surface. Hybrid dynamic coating with 0.25% DDM and 0.03% MC was found to minimize both analyte adsorption and EOF in a PMMA channel to a negligible level, while still keeping a low viscosity of the solution. High-speed and high-throughput profiling of the N-linked glycans derived from alpha1-acid glycoprotein, fetuin, and ribonuclease B was demonstrated in both single-channel and 10-channel PMMA chips using DDM-MC hybrid coating. We propose that DDM-MC hybrid coating might be a general method for suppressing analyte adsorption and EOF in polymer MCE devices. The current MCE-based method might be a promising alternative for high-throughput screening of carbohydrate alterations in glycoproteins.

Most proteins show characteristic biological roles after modification with carbohydrates, and profiling of total carbohydrates (glycans) has been a big target for a full understanding of protein functions. Owing to extreme heterogeneity of the glycans, high sensitivity and high resolving power are required for their analysis. Combination of techniques using high-performance liquid chromatography, capillary electrophoresis and mass spectrometry allows comprehensive analysis of glycans derived from biological sources.:, (� 2006 Elsevier Ltd . All rights reserved.)

Capillary electrophoresis (CE) is an effective tool to analyze carbohydrate mixture derived from glycoproteins with high resolution. However, CE has a disadvantage that a few nanoliters of a sample solution are injected to a narrow capillary. Therefore, we have to prepare a sample solution of high concentration for CE analysis. In the present study, we applied head column field-amplified sample stacking method to the analysis of N-linked oligosaccharides derived from glycoprotein separated by two-dimensional gel electrophoresis. Model studies demonstrated that we achieved 60-360 times concentration effect on the analysis of carbohydrate chains labeled with 3-aminobenzoic acid (3-AA). The method was applied to the analysis of N-linked oligosaccharides from glycoproteins separated and detected on PAGE gel. Heterogeneity of alpha1-acid glycoprotein (AGP), i.e. glycoforms, was examined by 2D-PAGE and N-linked oligosaccharides were released by in-gel digestion with PNGase F. The released oligosaccharides were derivatized with 3-AA and analyzed by CE. The results showed that glycoforms having lower pI values contained a larger amount of tetra- and tri-antennary oligosaccharides. In contrast, glycoforms having higher pI values contained bi-antennary oligosaccharides abundantly. The result clearly indicated that the spot of a glycoprotein glycoform detected by Coomassie brilliant blue staining on 2D-PAGE gel is sufficient for quantitative profiling of oligosaccharides.

Animal colostrum and milk contain complex mixtures of oligosaccharides, which have species-specific profiles. Milk oligosaccharides have various types of structure related to the core structures of glycolipids and N- and O-glycans of glycoproteins and provide a good library to examine the binding of oligosaccharides to various lectins. Recently, we reported a capillary affinity electrophoresis (CAE) method for analyzing the interactions between lectins and complex mixtures of N-linked oligosaccharides prepared from serum glycoproteins. The present paper reports the interactions between 24 milk oligosaccharides and six lectins (PA-I, RCA(120), SBA, WGA, UEA-I, and AAL) analyzed using CAE. Based on the resulting data, we constructed a library that enables us to determine nonreducing terminal monosaccharides, such as Gal, GalNAc, GlcNAc, and Fuc, and to differentiate Gal- or Fuc-linked isomers, such as lacto-N-tetraose, lacto-N-neotetraose, and lacto-N-fucopentaose II and III. In addition, using the library, we show that a combination of the lectins can characterize the neutral oligosaccharides derived from bovine colostrum.

Cell surface glycans and recognition molecules of these glycans play important roles in cellular recognition and trafficking, such as in the inflammation response by sialyl LewisX oligosaccharides. Malignant cells also utilize a similar mechanism during colonization and establishment of tumor tissues in the host. These considerations prompt us to develop a screening method for comprehensive analysis of N-glycans derived from membrane fractions of cancer cells. The method involves two step separations. Initially, N-glycans released from cell membrane fractions with N-glycoamidase F were labeled with 2-aminobenzoic acid and separated based on the number of sialic acid residues attached to the oligosaccharides using affinity chromatography on a serotonin-immobilized stationary phase. Each of the nonretarded fractions containing asialo- and high-mannose type oligosaccharides and mono-, di-, tri-, and tetra-sialooligosaccharide fractions which were desialylated with neuraminidase was analyzed by a combination of HPLC using an Amide-80 column as the stationary phase and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). We analyzed total N-glycan pools of membrane fractions obtained from some cancer cells, and found that U937 cells (Histocytic lymphoma cells) expressed a large amount of oligosaccharides having polylactosamine residues and MKN45 cells (Gastric adenocarcinoma cells) contained hyper-fucosylated oligosaccharides which contained multiple fucose residues. The method described here will be a powerful technique for glycomics studies in cell surface glycoproteins, and will enable one to search marker oligosaccharides characteristically observed in various diseases such as cancer, inflammation, and congenital disorder.

Spontaneous ectopic uterine ovarian tissue was found in 9 of 118 cynomolgus monkeys (Macaca fascicularis), an incidence of 7.6%. These ectopic tissues were minute and were incidentally observed in the parametrium by microscopic examination. They were composed of primordial or primary follicles and ovarian stroma. Ectopic ovarian tissue may result from disturbed migration of the ovarian primordium during embryogenesis.

Water soluble and lectin-recognizable carbohydrate-centered glycoclusters were prepared efficiently by the Huisgen 1,3-cycloaddition reaction of methyl-2,3,4,6-tetra-O-propargyl beta-D-galactopyranoside with 2-azidoethyl glycosides of lactose and N-acetyllactosamine. Their binding by a plant lectin RCA120 was examined by capillary affinity electrophoresis using fluorescence-labeled asialoglycans from human alpha1-acid glycoprotein. The glycoclusters showed 400-fold stronger inhibitory effect than free lactose, manifesting strong multivalency effect.

Characteristics of tear-film may be influenced by contact lens wear, because contact lenses present the habitual, direct rubbing action of the lids upon the covered ocular surface and may cause changes of tear-film. In the present paper, influence of contact lens on proteins in tear samples was studied using carbohydrates attached to the protein as a marker. We found that N-acetylneuraminic acid (Neu5Ac) was significantly decreased in tear samples of volunteers wearing contact lens (wearing, 86.1 +/- 57.7 nmol/ml; normal, 190.2 +/- 121.9 nmol/ml). Analysis by polyacrylamide gel electrophoresis demonstrated that the amounts of major proteins in tear fluids, such as lactoferrin and secretory immunoglobulin A were not changed upon wearing contact lenses. In contrast, cellulose acetate membrane electrophoresis revealed that mucin band in tear samples from contact lens wearers showed significant decrease as examined by lectin staining.

We analyzed some glycosaminoglycans and natural and artificial acidic polysaccharides using microchip electrophoresis in the buffer containing ethidium bromide, and found that they were successfully separated and detected within 150 s with comparable sensitivity with that of conventional electrophoresis using cellulose acetate membrane. We applied the technique to the analysis of glycosaminoglycans in pharmaceutical preparations and also in cultured cancer cells. Rapidness and easy operation of the proposed technique are quite useful for routine analysis of glycosaminoglycans.

We developed a method for the analysis of the interaction between hyaluronan (HA) oligosaccharides and hyaluronan-binding proteins (HABPs) using capillary affinity electrophoresis (CAE). The method is based on high-resolution separation of fluorescent-labeled HA molecules in the presence of hyaluronan-binding proteins at different concentrations by capillary electrophoresis (CE) with laser-induced fluorescent detection. Hyaluronan-binding protein from bovine nasal cartilage interacts strongly with HA decasaccharide or larger oligosaccharides. Effect of the molecular size of HA oligomers clearly showed that longer carbohydrate chains than decasaccharide were required for recognition by HA binding protein. Interestingly, the interaction did not cause retardation of HA oligomers as observed in many binding reactions such as the interaction between pharmaceuticals and serum albumin, but showed disappearance of the oligomer peak. Although we cannot explain the accurate mechanism on the interaction, disappearance is probably due to low equilibrium rate between free and conjugate states. The present technique will be useful to compare the relative binding affinity, and to understand the mechanism on the interaction between hyaluronan and hyaluronan-binding proteins.

Microchip electrophoresis is a powerful tool for fast analysis of nucleic acids and has expanded its applicability to the analysis of various biological materials including proteins and carbohydrates. Glycosaminoglycans have intrinsic negative charges, and are good targets for electrophoretic analysis. In the present paper, we developed a method to analyze oligosaccharides and unsaturated disaccharides derived from some glycosaminoglycans after digestion with specific enzymes followed by derivatization with 2-aminoacrydone (AMAC) by reductive amination. The method described here allowed rapid analysis of oligosaccharides derived from glycosaminoglycans within 150 s with high sensitivity. We show an application of the present technique to the glycosaminoglycan analysis in cultured HeLa cells.

We developed capillary affinity electrophoresis (CAE) to analyze the molecular interaction between carbohydrate chains and proteins in solution state. A mixture of oligosaccharides derived from a glycoprotein was labeled with 8-aminopyrene-1,3,6-trisulfonate (APTS), and used as glycan library without isolation. Interaction of a carbohydrate-binding protein with each oligosaccharide in the mixture could be simultaneously observed, and relative affinities of oligosaccharides toward the protein were accurately determined. In this study, we applied CAE to detect the presence of lectins in some plants (Japanese elderberry bark and tulip bulb). In the crude extract of the elderberry bark, binding activity toward sialo-carbohydrate chains could be easily detected. We also examined the presence of lectins in the crude extract of tulip bulbs and determined the detailed carbohydrate-binding specificity of Tulipa gesneriana agglutinin (TGA), one of the lectins from tulip bulbs. Kinetic studies demonstrated that TGA showed novel carbohydrate-binding specificity and preferentially recognized triantennary oligosaccharides with Gal residues at nonreducing termini and a Fuc residue linked through alpha(1-6) linkage at chitobiose portion of the reducing termini but not tetraantennary carbohydrates. The results described here indicate that CAE will be a valuable method for both screening of lectins in natural sources and determination of their detailed carbohydrate-binding specificities.

We analyzed carbohydrate chains of human, bovine, sheep, and rat alpha1-acid glycoprotein (AGP) and found that carbohydrate chains of AGP of different animals showed quite distinct variations. Human AGP is a highly negatively charged acidic glycoprotein (pKa = 2.6; isoelectic point = 2.7) with a molecular weight of approximately 37,000 when examined by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and contains di-, tri-, and tetraantennary carbohydrate chains. Some of the tri- and tetraantennary carbohydrate chains are substituted with a fucose residue (sialyl Lewis x type structure). In sheep AGP, mono- and disialo-diantennary carbohydrate chains were abundant. Tri- and tetrasialo-triantennary carbohydrate chains were also present as minor oligosaccharides, and some of the sialic acid residues were substituted with N-glycolylneuraminic acid. In rat AGP, very complex mixtures of disialo-carbohydrate chains were observed. Complexity of the disialo-oligosaccharides was due to the presence of N, O-acetylneuraminic acids. Triantennary carbohydrate chains carrying N,O-acetylneuraminic acid were also observed as minor component oligosaccharides. We found some novel carbohydrate chains containing both N-acetylneuraminic acid and N-glycolylneuraminic acid in bovine AGP. Interestingly, triantennary carbohydrate chains were hardly detected in bovine AGP, but diantennary carbohydrate chains with tri- or tetrasialyl residues were abundant. Furthermore the major sialic acid in these carbohydrate chains was N-glycolylneuraminic acid. It should be noted that these sialic acids are attached to multiple sites of the core oligosaccharide and are not present as disialyl groups.

We synthesized a temperature-responsive polymer, N-(isopropylacrylamide)-methacrylic acid copolymer, to which poly-l-lysine was introduced. The synthesized polymer as well as the parent polymer showed reversible soluble-insoluble changes in response to temperature changes across the lower critical solution temperature at 32 degree C in an aqueous solution. We found that the polymer efficiently captured acidic bio-macromolecules such as RNA, glycosaminoglycans and mucin-type glycoproteins in biological samples, and the captured molecules were recovered using aqueous NaCl solutions at high concentration. The target acidic molecules thus obtained will be employed for further studies such as structural analysis after brief desalting procedure. The proposed method does not require any chromatographic separations, but only needs a small volume of an aqueous salt solution for releasing captured molecules. Overall procedures are quite easy and simple, and are completed at least within 1 h. We show a few examples for capturing RNA and glycosaminoglycans from cultured cells using the polymer.

Background and Aims: On activation, protease-activated receptor (PAR)-2 modulates multiple gastric functions and exerts mucosal protection via activation of sensory neurons. The role of PAR-1, a thrombin receptor, in the stomach remains unknown. We thus examined if the PAR-1 agonist could protect against gastric mucosal injury in rats., Methods: Gastric mucosal injury was created by oral administration of ethanol/HCl or absolute ethanol in conscious rats. Gastric mucosal blood flow and acid secretion were determined in anesthetized rats. Immunohistochemical analyses of PAR-1 and cyclooxygenase (COX)-1 were also performed in rat and human stomach., Results: The PAR-1 agonist TFLLR-NH(2), administered intravenously in combination with amastatin, protected against the gastric mucosal injury induced by ethanol/HCl or absolute ethanol. The protective effect of TFLLR-NH(2) was abolished by indomethacin or a COX-1 inhibitor but not by ablation of sensory neurons with capsaicin. TFLLR-NH(2) produced an NO-independent increase in gastric mucosal blood flow that was partially inhibited by blockade of the endothelium-derived hyperpolarizing factor pathway. This inhibitory effect was promoted by indomethacin. TFLLR-NH(2) suppressed carbachol-evoked acid secretion in an indomethacin-reversible manner. Immunoreactive PAR-1 and COX-1 were expressed abundantly in rat gastric muscularis mucosae and smooth muscle, and the former protein was also detectable in blood vessels. Similar staining was observed in human gastric muscularis mucosae., Conclusions: The PAR-1 agonist, given systemically, protects against gastric mucosal injury via COX-1-dependent formation of prostanoids, modulating multiple gastric functions. Our data identify a novel protective role for PAR-1 in gastric mucosa, and the underlying mechanism is entirely different from that for PAR-2.

Hyaluronic acid (hyaluronan) is a ubiquitous extracellular matrix component, and present at high concentrations in skin, joints and cornea. In the skin, it is synthesized primarily by dermal fibroblasts and by epidermal keratinocytes. Hyaluronic acid usually exists as a high molecular mass (600,000-1,000,000) and non-sulfated glycosaminoglycan composed of a disaccharide unit of [bond]3GlcNAc beta 1[bond]4GlcA beta 1[bond]. Hyaluronic acid has been widely used not only for osteoarthritis and ophthalmology but also for cosmetics for skin care. To examine the biological activities of hyaluronic acid, we have to accurately determine the quantity and molecular masses in biological samples. We review recent development in the analysis of hyaluronic acid having various molecular sizes using electrophoretic and chromatographic techniques. Recently, interactions between hyaluronic acid oligomers and hyaluronic acid-binding proteins have attracted the interest for understanding the biological functions. We show some interesting reports on biological interactions of hyaluronic acid and its oligomers with some proteins.

This study examines the neural substrates involved in the recognition of ambiguous facial expressions using functional magnetic resonance imaging. Subjects performed two tasks, one in which they judged facial expressions and another in which they identified gender. Subtraction between ambiguous expression and clear expression conditions revealed the activation of anterior cingulate (ACC), medial frontal (MeFG) and bilateral inferior frontal gyrus (IFG). Structural equation modeling showed that the functional connectivity between these areas was greater with the ambiguous expressions than with the clear ones. The activation of the ACC, MeFG, and right IFG was greater with ambiguous expressions than with ambiguous gender. These results suggest that the neural network involving these frontal regions plays a crucial role in the processing of the ambiguously expressed facial emotions.

In this report, we describe a new lectin from the fungus Rhizopus stolonifer that agglutinates rabbit red blood cells. Agglutinating activity was detected in the extract of mycelium-forming spores cultured on agar plates but not in the mycelium-forming no spores from liquid medium. This lectin, which we designated R. stolonifer lectin (RSL), was isolated by affinity chromatography with porcine stomach mucin-Sepharose. SDS-polyacrylamide gel electrophoresis and mass spectral analysis showed that RSL is approximately 4.5 kDa, whereas gel filtration indicated a mass of 28 kDa. This indicates that the lectin is a hexamer of noncovalently associated RSL monomers. RSL activity was very stable, since it was insensitive to heat treatment at 70 degrees C for 10 min. Analysis of RSL binding specificity by both microtiter plate and precipitation assays showed that N-glycans with l-fucose linked to the reducing terminal GlcNAc residues are the most potent inhibitors of RSL binding, whereas N-glycans without alpha(1-6)-linked fucose residues are approximately 100-fold weaker inhibitors of binding. Oligosaccharides with alpha(1-2, -3, and -4) linkages showed no inhibition of binding in these assays. In a mirror resonance biosensor assay, high affinity binding was observed between RSL and the glycopeptide of bovine gamma-globulin, which has N-glycans with alpha(1-6)-linked fucose residues. However, RSL showed only a weak interaction with the glycopeptide of quail ovomucoid, which lacks fucose residues. Finally, capillary affinity electrophoresis studies indicated that RSL binds strongly to N-glycans with alpha(1-6)-linked fucose residues. Together, these results show that RSL recognizes the core structure of N-glycans with alpha(1-6)-linked l-fucose residues. This specificity could make RSL a valuable tool for glycobiological studies.

N-Glycans in glycoprotein can be liberated either from glycoproteins or from their glycopeptides with glycoamidases. The latter approach is preferable, because it requires a smaller amount of the enzyme, and yields N-glycans in excellent yields. Moreover it alleviates the necessity of removing from the reaction mixture the detergents needed to denature the glycoproteins. On the other hand, this approach necessitates removal of interfering peptidic materials, because some of the peptide peaks often overlap with the peaks of carbohydrate chains in high-performance anion-exchange chromatography (HPAEC). These peptidic materials also hinder labeling of N-glycans by reductive amination. We have tried to remove the interfering peptidic materials by several different methods--octadecyl (C18) silica cartridge, cation-exchange resin column, and graphitized carbon cartridge. Unfortunately, none of these could completely remove the interfering peptidic materials. Therefore, we resorted to modify the amino groups of the peptidic materials with sodium 2,4,6-trinitro-benzene-1-sulfonate (TNBS) to render them more hydrophobic, so that they can be retained more strongly on the C18 or graphitized carbon cartridges. In the model study presented here, we were able to obtain N-glycans for HPAEC analyses without any interfering materials by a combination of TNBS reaction and graphitized carbon treatment.

O-Glycans (mucin type oligosaccharides) are ubiquitously found in various glycoproteins such as mucin-type glycoproteins on the surface of various digestive organs. In the present paper, we propose a method for detecting O-glycanase which catalyzes the hydrolysis of the O-glycan linkage between oligosaccharides and serine/threonine residues of mucin-type glycoprotein. As the substrate for O-glycanases, we chose glycopeptides containing O-glycans derived from bovine fetuin. The present method is divided into two parts. At the initial stage, the presence of O-glycanase was confirmed by observing characteristic ions due to O-glycans and peptides released from the glycopeptide by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. In the second step, europium labeled O-glycosylated peptide permits more detailed analysis such as enzyme kinetics. We demonstrated the usefulness of the present method using O-glycanase (Streptococcus pneumoniae) as model enzyme. The present approach can easily confirm the presence of O-glycanase by detecting both deglycosylated peptides and O-glycans, even if contaminating peptides or glycosidases are present in crude biological samples.

A specific and sensitive method for the determination for difluprednate (DFBA) and its metabolite (deacetylated DFBA, DFB) in aqueous humor was developed. DFBA and DFB were initially absorbed on a Pinkerton-type column, then analyzed by high-performance liquid chromatography using a semi-micro column after column switching. Under the optimized conditions, calibration curves for DFBA and DFB showed good linearity over the range of 1.0-50 and 0.5-50 ng/ml, respectively. We applied the method to the analysis of DFBA and DFB in rabbit aqueous humor, and found that DFBA in rabbit aqueous humor 1 h after instillation of 0.002% DFBA ophthalmic emulsion was not detected, but DFB was present at the concentration of 4.3+/-3.1 ng/ml.

The therapeutic efficacy of KP-103, a novel topical triazole, in a guinea pig tinea unguium model was investigated. Experimental tinea unguium and tinea pedis were produced by inoculation of Trichophyton mentagrophytes SM-110 between the toes of the hind paw of guinea pigs. One percent solution (0.1 ml) of KP-103, amorolfine, or terbinafine was topically applied to the nails and whole sole of an infected foot once daily for 30 consecutive days, and terbinafine was also orally administered at a daily dose of 40 mg/kg of body weight for 30 consecutive days, starting on day 60 postinfection. The fungal burdens of nails and plantar skin were assessed using a new method, which makes it possible to recover infecting fungi by removing a carryover of the drug remaining in the treated tissues into the culture medium. Topically applied KP-103 inhibited the development of nail collapse, significantly reduced the fungal burden of the nails, and sterilized the infected plantar skin. On the other hand, topical amorolfine and topical or oral terbinafine were ineffective for tinea unguium, although these drugs eradicated or reduced the fungal burden of plantar skin. The in vitro activities of amorolfine and terbinafine against T. mentagrophytes SM-110 were 8- and 32-fold, respectively, decreased by the addition of 5% keratin to Sabouraud dextrose broth medium. In contrast, the activity of KP-103 was not affected by keratin because its keratin affinity is lower than those of the reference drugs, suggesting that KP-103 largely exists in the nails as an active form that was not bound to keratin and diffuses in the nail without being trapped by keratin. The effectiveness of KP-103 against tinea unguium is probably due to its favorable pharmacokinetic properties in the nails together with its potent antifungal activity.

We examined the formation of complexes between terbium ion (Tb3+) and carbohydrates labeled with aminobenzene compounds. Of the examined compounds, carbohydrates labeled with 4-aminosalicylic acid showed intense fluorescence with Tb3+ in the presence of cetylpyridinium chloride at pH 6.0. Calibration curves for maltose derivative showed good linearity between 5 pmol and at least 600 pmol, with good reproducibility. We applied the proposed technique to binding studies between manno-oligosaccharides and Concanavalin A.

A highly heterogeneous glycoprotein, alpha1-acid glycoprotein, was resolved into their glycoforms by capillary electrophoresis using a surface-modified capillary in 20 mM acetate buffer (pH 4.2) containing 0.5% (w/v) hydroxypropylmethylcellulose. We collected the fractions containing each glycoform as nearly pure state by capillary electrophoresis, and examined the molecular masses of these glycoforms by matrix assisted laser desorption time-of-flight mass spectrometry. We also analyzed carbohydrate chains after releasing them with N-glycosidase F followed by fluorescent labeling with 8-aminopyrene-1,3,6-trisulfonate. We found that the separation of glycoforms was mostly due to the presence of multiantennary carbohydrate chains. We propose that the present technique is useful for the analysis of post translational modification of proteins with carbohydrate chains.

This review describes the recent development in the analysis of glycoproteins using capillary electrophoresis with various separation techniques, and focuses especially on the analysis of recombinant glycoprotein pharmaceuticals. We include the analysis of glycoprotein multiforms (ie glycoform) as well as glycan analysis. The relationship between glycoprotein multiforms and oligosaccharide distributions in a glycoprotein sample is also discussed. Further, recent development in capillary electrophoresis-mass spectrometry is described., (Copyright 2002 John Wiley & Sons, Ltd.)

1. Protease-activated receptor 2 (PAR2), present in capsaicin-sensitive sensory neurons, induces gastric mucus secretion and mucosal cytoprotection. 2. We studied the possible cross-talk between PAR2 and vanilloid receptor 1 (VR1). The VR1 antagonist capsazepine partially inhibited the PAR2-mediated increase in gastric mucus secretion. 3. Thus, activation of VR1 is responsible, at least in part, for the neurally mediated mucosal cytoprotection following activation of PAR2.

We developed a method for the determination of molecular mass of acidic polysaccharides based on their high-resolution separation by capillary electrophoresis. Polymers of N-acetylneuraminic acid (NeuAc) and polysulfated hyaluronic acid were separated into their molecular species up to 100 mono- and 20 disaccharide units, respectively. The relationship between the molecular mass of NeuAc-polymer and their electrophoretic mobilities showed good linearity, and was applied to the determination of molecular masses of larger NeuAc species unresolved by capillary electrophoresis under the same conditions. In the first step, the standard curve for the determination of molecular mass was constructed from the relationship between electrophoretic mobility and molecular mass. Subsequently, the mobility was extrapolated to the standard curve, and the molecular mass was calculated. Five different preparations of NeuAc polymers having different molecular masses showed smaller values than those determined by conventional chromatographic techniques. Further, molecular mass determined by the present method correlated with number-average molecular mass. The methodology presented here was applied to the determination of molecular mass of polysulfated hyaluronic acid. The data indicated that native hyaluronic acid was extensively degraded during sulfonation reaction., (Copyright 2002 John Wiley & Sons, Ltd.)

Simultaneous analysis of sialic acids has been a challenging target, because sialic acids having N- or O-acetyl, glycolyl, and sulfonic acid ester groups are labile during their release from carbohydrate chains and analytical procedures. In the present paper, we propose a method using high-performance liquid chromatography coupled with eletrospray ionization mass spectrometry (HPLC-ESI-MS). The method was evaluated by applying to the analysis of sialic acids in various tissues, especially digestive organs in mice and rats. The method was based on the in situ precolumn derivatization of sialic acids after releasing them by hydrolysis. The sialic acids were derivatized with 1,2-diamino-4,5-methylenedioxybenzene to form highly fluorescent quinoxaline derivatives. By using two different hydrolysis conditions (i.e., with 2 M acetic acid and with 0.1 M hydrochloric acid), both total sialic acids and sialic acid distributions were easily determined. We found that sialic acids showed characteristic distributions in the tissues of mice and rats. Further, HPLC-ESI-MS revealed that all the tissues examined in mice and rats commonly contained highly acetylated sialic acids and 8-O-sulfated N-acetylneuraminic acid.

Fluorescence polarization has been widely used for the studies on the molecular motion in solution and has been applied to immunoassays for proteins, therapeutic drug monitoring in clinical pharmacy, and assays for environmentally toxic compounds. Because fluorescence polarization is most readily applicable to the kinetic analysis of the binding reaction between a substance having small molecular mass and a receptor molecule, this method is easily applied to the analysis of carbohydrate-lectin binding. In this tutorial Thematic Review, we briefly introduce the principles of fluorescence polarization and some applications of fluorescence polarization technique to glycobiology., (Copyright 2001 Academic Press.)

We examined lactone-formation reaction of oligomers and polymers of N-acetylneuraminic acid in diluted hydrochloric acid solution and found that the time course of the lactonization reaction was easily traced by capillary electrophoresis. The reaction proceeded more rapidly with increasing the molecular mass of oligomers because the conformation of inner units became rigid and more favorable for the formation of lactone linkage. Present results obtained using capillary electrophoresis will be useful in understanding of physical and chemical properties of oligo/polysialic acids.

During high-resolution capillary electrophoresis analysis in an electrolyte solution containing a neutral polymer, small oligomers of regularly arranged acidic polysaccharides such as hyaluronic acid and N-acetylneuraminic acid polymers showed reversal of the migration order. This anomalous migration was well correlated with their reported biological activity. In the present study, we analyzed hyaluronidase action on the purified hyaluronic acid oligomers using capillary electrophoresis and found that hydrolytic and transglycosylation actions by hyaluronidase were dependent on the molecular sizes of hyaluronic acid oligomers, and well correlated to their migration profiles. Furthermore, fluorescent polarization technique was employed for understanding the relationship between molecular size of hyaluronic acid oligomers and their electromigrations.

Protease-activated receptor-2 (PAR-2) acts as a modulator of multiple physiological/pathophysiological functions including salivary exocrine secretion. Given the supersensitivity of endothelial PAR-2 under endotoxaemia, we investigated if endotoxin/lipopolysaccharide (LPS) could alter the sensitivity of PAR-2 in the salivary glands. The in vivo salivation in response to i.v. administration of the PAR-2-activating peptide SLIGRL-NH2, but not of carbachol, gradually decreased 6-20 h after LPS administration in the mice. The LPS-induced hyporeactivity to the PAR-2 agonist was partially reversed by repeated administration of aprotinin, a non-specific protease inhibitor. PAR-2 mRNA levels in the salivary glands, as assessed by the semi-quantitative RT-PCR analysis, remained unchanged following LPS challenge. Our findings indicate that in contrast to the supersensitivity of endothelial PAR-2 as described previously, subsensitivity of PAR-2 in the salivary glands develops during the LPS-induced systemic inflammation, which might involve desensitisation of PAR-2 by endogenous proteases.

alpha1-Acid glycoprotein (AGP) showed multiple peaks on separation using capillary electrophoresis in a chemically modified capillary with dimethylpolysiloxane at slightly acidic conditions. We analyzed glycoforms of AGP species after separation by ion-exchange chromatography, Con A affinity chromatography, and Cu(II)-chelating affinity chromatography. The AGP species thus obtained were digested with N-glycosidase F, and the released carbohydrate chains were analyzed by high-performance liquid chromatography after labeling with 3-aminobenzoic acid. The results afforded basic information on the contribution of carbohydrate chains to the separation mechanism of glycoforms of AGP by capillary electrophoresis. In addition, we describe an easy method for AGP analysis in serum samples using the electrokinetic injection.

Protease-activated receptor-2 (PAR-2), a receptor activated by trypsin/tryptase, modulates smooth muscle tone and exocrine secretion in the salivary glands and pancreas. Given that PAR-2 is expressed throughout the gastrointestinal tract, we investigated effects of PAR-2 agonists on mucus secretion and gastric mucosal injury in the rat. PAR-2-activating peptides triggered secretion of mucus in the stomach, but not in the duodenum. This mucus secretion was abolished by pretreatment with capsaicin, which stimulates and ablates specific sensory neurons, but it was resistant to cyclo-oxygenase inhibition. In contrast, capsaicin treatment failed to block PAR-2-mediated secretion from the salivary glands. Intravenous calcitonin gene-related peptide (CGRP) and neurokinin A markedly elicited gastric mucus secretion, as did substance P to a lesser extent. Specific antagonists of the CGRP1 and NK2, but not the NK1, receptors inhibited PAR-2-mediated mucus secretion. Pretreatment with the PAR-2 agonist strongly prevented gastric injury caused by HCl-ethanol or indomethacin. Thus, PAR-2 activation triggers the cytoprotective secretion of gastric mucus by stimulating the release of CGRP and tachykinins from sensory neurons. In contrast, the PAR-2-mediated salivary exocrine secretion appears to be independent of capsaicin-sensitive sensory neurons.

A virus infection was studied using half of the normal oral dose of amantadine hydrochloride-100 mg/d instead of 200 mg/d. The patients in this study, who visited the clinics during January and February 1999, were confirmed within 48 hours to have influenza A virus infections by the Directigen FluA test. Using a quasi-randomized controlled trial, 26 patients were treated with amantadine hydrochloride in addition to the usual medication, while 23 were treated with only the ordinary medication. There were no significant differences in the mean age, 35.6 years old, or in clinical features between the two groups. The period of fever over 38 degrees C in the amantadine treated group was 1 day while that in the control group was 1.7 days, which shows a significant difference (p = 0.049). There was no significant difference in the duration of aching, such as arthralgia, or of general fatigue. There was no significant difference in the appearance of subsequent new symptoms after the onset of influenza A virus infection. In conclusion, it is expected that oral amantadine hydrochloride, 100 mg/d, together with the ordinary medication, will reduce the duration of the period of fever over 38 degrees C.

We developed a convenient and specific method for the determination of mucin-type glycoproteins using galactose oxidase and horseradish peroxidase on the basis of the contents of galactosyl and N-acetylgalactosaminyl residues in glycoproteins. Galactose and galactosamine residues released from glycoproteins after hydrolysis were oxidized with galactose oxidase and subsequently the resultant hydrogen peroxide was determined by a combination of horseradish peroxidase and 3-(p-hydroxyphenyl) propionic acid as a fluorogenic substrate. The contents of galactose/galactosamine residues in N- and O-glycans, as determined by the galactose oxidase-peroxidase method, were in good agreement with those described in the previous reports. We applied the present method to determine mucin-type glycoproteins secreted from rat gastric mucosa by stimulation with misoprostol, a prostaglandin E(1) analogue in vivo. Thus, the galactose oxidase-peroxidase method is useful for the determination of mucin-type glycoproteins in biological materials., (Copyright 2000 Academic Press.)

The influence of sugar moieties of ovoglycoprotein from chicken egg white (OGCHI) on chiral discrimination of various solutes has been investigated. Partially deglycosylated OGCHI (pd-OGCHI) and completely deglycosylated OGCHI (cd-OGCHI) were obtained by treatments of OGCHI with N-glycosidase, and a mixture of endoglycosidase and N-glycosidase, respectively. The average molecular masses of OGCHI, pd-OGCHI and cd-OGCHI were estimated to be about 30 000, 28 400 and 21 400, respectively, by matrix-assisted laser desorption time-of-flight mass spectrometry. The isoelectric points of OGCHI, pd-OGCHI and cd-OGCHI were in the ranges 4.37-4.51, 4.34-4.44 and 4.17-4.43, respectively, by isoelectric focusing. The OGCHI, pd-OGCHI and cd-OGCHI were bound to aminopropyl-silica gels activated with N,N'-disuccinimidylcarbonate to compare retentive and enantioselective properties of the three columns. It was found that pd-OGCHI showed excellent chiral recognition abilities comparable to OGCHI, and that the retentivity and enantioselectivity of basic solutes tested on the pd-OGCHI column were higher than those on the OGCHI column, while those of acidic solutes tested on the pd-OGCHI column were lower. Further, cd-OGCHI still showed chiral recognition abilities for various solutes tested. These results reveal that the chiral recognition site(s) for OGCHI exists on the protein domain of OGCHI.

The effect of sialic acid (N-acetyl neuraminic acid), sialic acid dimer, sialic acid polymers (colominic acid) and sulfated colominic acid on the activity of hyaluronidase, on the dispersion of cumulus cells by mouse sperm and on in vitro mouse fertilization (sperm penetration of zona pellucida) were evaluated. Bovine testicular hyaluronidase activity was significantly inhibited by colominic acid and sulfated colominic acid, but not by sialic acid and its dimer. The dispersion of cumulus cells from eggs by mouse sperm was also inhibited by colominic acid and sulfated colominic acid. In vitro fertilization of mouse gametes was inhibited by sulfated colominic acid. The IC50 value of sulfated colominic acid-induced inhibition of fertilization was 0.3 mg/ml (ca. 0.9 mM). The value changed from 0.9 mM for cumulus-surrounded egg to 1.5 mM for cumulus free-egg. On the other hand, colominic acid showed little or no inhibitory effect on mouse in vitro fertilization at 0.5 mg/ml (ca. 1.6 mM). This antifertility activity by sulfated colominic acid did not appear to be due to an effect on sperm motility or on the oocytes. These results suggest that (1) the cumulus cells surrounding the eggs were dispersed by sperm hyaluronidase, (2) hyaluronidase was inhibited by colominic acid and by sulfated colominic acid, (3) sulfated colominic acid inhibits sperm penetration of zona pellucida by the inhibition of hyaluronidase and/or some enzymes required for mouse gametes fertilization.

Protease-activated receptor-2 (PAR-2) is distributed throughout the gastrointestinal systems. The present study investigated the role for PAR-2 in the rat salivary glands. PAR-2 mRNA was detected in the sublingual, submaxillary, and parotid glands by a reverse-transcriptase polymerase chain reaction. In the isolated sublingual gland that exhibited the strongest signal for PAR-2, Ser-Leu-Ile-Gly-Arg-Leu-NH(2), a PAR-2-activating peptide, and trypsin, a PAR-2-activating enzyme, but not thrombin that can activate PARs 1, 3, and 4, triggered secretion of N-acetylneuraminic acid, an indicator of mucin, that was a unique major sialic acid detectable after hydrolysis of the sublingual mucin with 0.1 N HCl. The PAR-2-mediated secretion of mucin was attenuated by genistein, a tyrosine kinase inhibitor, but not by inhibitors of protein kinase C and phosphatidyl inositol 3'-kinase. Thus, PAR-2 is expressed by the three distinct salivary glands in the rat, and sublingual PAR-2 appears to play a role in triggering mucin secretion, at least in part, via activation of tyrosine kinase., (Copyright 2000 Academic Press.)

The aim of the present research is to characterize the leakage of intestinal constituents induced by beta-cyclodextrin (beta-CyD) derivatives using an in situ perfusion and an in vitro everted sac. The efficacy of 6-O-alpha-D-glucosyl (G1)- and 6-O-alpha-D-maltosyl (G2)-beta-CyDs as oral carriers was also compared with that of 2-hydroxypropyl-(HP1; average molar degree of substitution, 0.9) and 2,6-di-O-methyl (DM)-beta-CyDs. In the in situ studies, phenol red (PR) penetration and the release profiles of intestinal constituents for G2-beta-CyD were fairly close to those for HP1-beta-CyD. However, the ability of G2-beta-CyD to include cholesterol was greater than that of HP1-beta-CyD. To characterize the release of intestinal constituents induced by modified beta-CyDs, the capability of including cholesterol was held constant between DM- and branched beta-CyDs. The everted sac study showed that the amount of DM-beta-CyD transferred to the serosal side was not significantly different from the branched beta-CyDs. On the serosal side, the amount of cholesterols released was approximately 3 times higher for DM-beta-CyD than for the branched beta-CyDs at 60 min. The cumulative amounts of cholesterols for DM-beta-CyD increased approximately 6 times at 60 min compared with at 30 min, predominating over the leakage (average 2.6-fold) on the mucosal side. In contrast, the exposure of the branched beta-CyDs resulted in an insignificant increase over the period of this experiment. The present study suggests that permeable beta-CyD derivatives play an important role in the leakage of intestinal components. G2-beta-CyD is preferably recommended as a drug solubilizer in oral formulations as well as HP1-beta-CyD, based on the lower release of intestinal constituents.

Colominic acid is an alpha2,8-linked sialic acid polymer produced by Escherichia coli. We found that synthetic sulfated-colominic acids (SC) remarkably inhibited the cytotoxicity of bee and snake venom toward mouse fibroblast cells, but colominic acids showed no inhibition themselves, indicating the important role of sulfate groups in the inhibitory activity of SC. Other sulfated carbohydrates such as chondroitin sulfates, heparin and heparan sulfate showed no inhibition. SC also exhibited potent inhibition of melittin, a highly basic peptide, which is a major cytotoxic component of bee venom. SC did not inhibit phospholipase A2 activity in bee venom. This suggests that the inhibition of bee and snake venom by SC is due to inhibition of melittin and cardiotoxin, which is a cytolytic peptide in snake venom, respectively. SC with a higher sulfur content and a larger molecular mass showed more potent activity. The interaction between SC and melittin basically seems an ionic one, however, the conformation of SC is also likely important. For the binding of SC to melittin leading loss of its cytotoxic activity, the sulfate groups of SC must be properly arranged to interact with lysine and arginine residues of melittin molecules, which play an important role in the cytolytic activity. A higher molecular mass of SC substituted with more sulfate groups is required for more obvious inhibition of the cytotoxic activity.

A fluorometric binding assay for lectin and yeast cells using the avidin-biotin system was previously reported (Y. Oda, M. Kinoshita, and K. Kakehi, Anal. Biochem. 254, 41-48, 1997). However, the true amount of bound lectin could not be determined by this method due to difficulty in determination of the number of bound biotin molecules. In the present study, we have developed a method for assaying the binding reaction between fluorescent lectin and cells using a flow injection technique, which allows estimation of the amount of lectin bound to cells. An aliquot of the cell suspension was directly analyzed by injection into a flow injection system after the binding between the fluorescently labeled lectin and cells. The labeled lectins showed good linearity, at least over a range of 20-1000 ng as the injected amount. The intrinsic fluorescence of the labeled lectins did not change upon the binding. The binding reaction of the hydroxycoumarin-labeled lectins with yeast cells was rapid and reached an equilibrium state within 10 min. Scatchard analysis showed that Saccharomyces cerevisiae cells contained approximately 1. 3-1.6 x 10(8) binding sites per cell for Concanavalin A, Lycoris radiata agglutinin, and Tulipa gesneriana lectin with affinity constants of 3.2-4.7 x 10(6) M-1. The present method was applied to the study of binding between lectins and bacteria and mouse spleen cells. The assay method described here is highly sensitive and will be an alternative to assays using lectins labeled with radioisotopes. The procedure is quite simple and can be completed within 1 h., (Copyright 1999 Academic Press.)

Stabilities of chloral hydrate in an aqueous solution and its medicated syrup were examined by high performance capillary electrophoresis. Analysis of the concentration of chloral hydrate indicated that there was no obvious change in the concentration of chloral hydrate both in the aqueous solution and in the syrup preparation after keeping them for 3 months at room temperature or at 60 degrees C. The lowering of pH was more obvious in the syrup solution than in the aqueous solution, and this tendency was estimated to be due to the formation of hydrochloric acid. We propose that the stabilities of the preparation of chloral hydrate should be monitored by observing pH changes.

High-resolution analysis of polymers of N-acetylneuraminic acid and hyaluronic acid was performed by capillary electrophoresis in a buffer containing a neutral polymer. Both polysaccharides having more than 100 monosaccharide residues were well separated into their molecular species by capillary electrophoresis using a combination of a chemically modified capillary and a buffer containing poly(ethylene glycol) as an additive. During optimization of the separation conditions, small oligomers of both polysaccharides were observed to migrate in the reverse order of their molecular masses on the electropherograms. However, oligomers larger than pentamer and decamer for N-acetylneuraminic acid polymers and hyaluronic acid, respectively, migrated in the order of their molecular masses. We propose that these unusual migration patterns are closely related to the stereochemical structures and the oligomer migrating the fastest is the minimum unit that forms the regular three-dimensional structure required for the biological function.

beta-1-O- (NAG) and 2-O-glucuronides (2-isomer) of (S)-naproxen (NA) were prepared to determine which positional isomer(s) of the acyl glucuronide of NA is responsible for forming covalent adducts with human serum albumin (HSA). Their comparative stability and covalent binding adduct formation with HSA were investigated at pH 7.4 and at 37 degreesC. NA and its acyl glucuronides were simultaneously determined by HPLC. Three positional isomers were formed successively after incubation of NAG in the buffer only. However, when NAG was incubated with HSA (30 mg/mL), isomers other than the 2-isomer were formed in little or negligible quantities. In HSA solution, NAG (kd = 2.08 +/- 0.08 h-1) was four times less stable than 2-isomer (kd = 0.51 +/- 0.02 h-1). NAG was degraded by hydrolysis (khyd = 1.01 +/- 0.10 h-1) and isomerization (kiso = 1.07 +/- 0.07 h-1) to the same extent; however, hydrolysis was predominant for the 2-isomer (kd = 0.51 +/- 0.02 h-1). The incubation of both NAG and 2-isomer with HSA led to the formation of a covalent adduct; however, the adduct formation from the 2-isomer proceeded more slowly than that from NAG. The present results suggest that the covalent binding of NA to HSA via its acyl glucuronides proceeds through both transacylation (direct nucleophilic displacement) and glycation mechanisms; NAG rapidly forms an adduct that may be unstable, and the protein adduct from the 2-O-acyl glucuronide is as important for the covalent binding as those from the 1-O-acyl glucuronides.

The fluorescence polarization (FP) technique was evaluated to determine molecular interaction between plant lectins and polysaccharides, yeast cells and glycopeptide after labeling the lectins with fluorescein isothiocyanate. Use of Lycoris radiata agglutinin allowed determination of the molecular interactions with large biomolecules containing mannose oligomers and polymers. Another example using a fluorescein-labeled glycopeptide also indicated that use of the FP method would allow easy observation of the molecular interactions on the quantitative base. The present technique is highly sensitive and facile because it does not require any washing procedures before measurement.

A fluorometric assay of lectin binding to yeast cells is reported. The relative amount of biotinylated lectins bound to the yeast cells was estimated by enzyme activity using 4-methylumbelliferyl-beta-D-galactoside as a substrate for the lectin-bound beta-galactosidase through biotin-avidin interaction. Binding properties of 4 mannose-specific and 3 glucose/mannose-specific lectins to 22 different species of yeast cells were studied. The binding reaction of biotinylated lectins to the yeast cells was rapid and became constant within 10 min. Each lectin showed its characteristic binding specificity to each yeast species. The relative fluorescent intensities observed for 4-methylumbelliferone released by the action of bound beta-galactosidase were good indicators for the classification of yeast cells in quantitative base. We found that the yeast cells of the Saccharomyces genus can be classified into three groups, and those of Pichia were grouped into two groups. The present method can examine many samples simultaneously and be completed within 3 h., (Copyright 1997 Academic Press.)