Bovine κ-casein inhibits human rotavirus (HRV) infection via direct binding of glycans to HRV.

Human rotavirus (HRV) is a major etiologic agent of severe infantile gastroenteritis. κ-Casein (κ-CN) from both human and bovine mature milk has been reported to have anti-HRV activity; however, the mechanism of this activity is poorly understood. The present study examined the molecular basis for the protective effect of bovine κ-CN derived from late colostrum (6-7 d after parturition) and from mature milk. Among the components of casein, κ-CN is the only glycosylated protein that has been identified. Therefore, we investigated whether the glycan residues in κ-CN were involved in the anti-HRV activity. Desialylated CN obtained by neuraminidase treatment exhibited anti-HRV activity, whereas deglycosylated CN obtained by o-glycosidase treatment lacked antiviral activity, indicating that glycans were responsible for the antiviral activity of CN. Furthermore, an evanescent-field fluorescence-assisted assay showed that HRV particles directly bound to heated casein (at 95°C for 30 min) in a viral titer-dependent manner. Although the heated κ-CN retained inhibitory activity in a neutralization assay, the activity was weaker than that observed before heat treatment. Our findings indicate that the inhibitory mechanism of bovine κ-CN against HRV involves direct binding to viral particles via glycan residues. In addition, heat-labile structures in κ-CN may play an important role in maintenance of κ-CN binding to HRV.

Anomalous migration of hyaluronic acid oligomers in capillary electrophoresis: correlation to susceptibility to hyaluronidase.

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.

Lactone formation of N-acetylneuraminic acid oligomers and polymers as examined by capillary electrophoresis.

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.

Fluorescence polarization: analysis of carbohydrate-protein interaction.

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.

Lipopolysaccharide-induced subsensitivity of protease-activated receptor-2 in the mouse salivary glands in vivo.

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.

Capillary electrophoresis of sialic acid-containing glycoprotein. Effect of the heterogeneity of carbohydrate chains on glycoform separation using an alpha1-acid glycoprotein as a model.

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.

The protease-activated receptor-2 agonist induces gastric mucus secretion and mucosal cytoprotection.

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.

Specific distribution of sialic acids in animal tissues as examined by LC-ESI-MS after derivatization with 1,2-diamino-4,5-methylenedioxybenzene.

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.

Chromatographic and capillary electrophoretic methods for the analysis of nicotinic acid and its metabolites.

Methods for the assay of nicotinic acid (NiAc) and its metabolites in biological fluids using high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) are reviewed. Most of the references cited in this review concern HPLC methods. A few CE methods that have been recently reported are also included. As these compounds are relatively polar and have a wide range of physico-chemical properties, the sample pre-treatment or clean-up process prior to analysis is included. Most HPLC methods using an isocratic elution system allow determination of a single or few metabolites, but gradient HPLC methods enable simultaneous determination of five to eight compounds. Simultaneous determination of NiAc including many metabolites in a single run can be achieved by CE. We also discuss the pharmacokinetics of NiAc and some of its metabolites.

Separation of enantiomers on a chiral stationary phase based on ovoglycoprotein. VIII. Chiral recognition ability of partially and completely deglycosylated ovoglycoprotein.

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.

Fluorometric determination of mucin-type glycoproteins by the galactose oxidase-peroxidase method.

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.

Inhibition of in vitro fertilization of mouse gametes by sulfated sialic acid polymers.

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.

Crocus sativus lectin recognizes Man3GlcNAc in the N-glycan core structure.

Crocus sativus lectin (CSL) is one of the truly mannose-specific plant lectins that has a unique binding specificity that sets it apart from others. We studied sugar-binding specificity of CSL in detail by a solution phase method (fluorescence polarization) and three solid phase methods (flow injection, surface plasmon resonance, and microtiter plate), using a number of different glycopeptides and oligosaccharides. CSL binds the branched mannotriose structure in the N-glycan core. Substitution of the terminal Man in the Manalpha(1-3)Man branch with GlcNAc drastically decreases binding affinity much more than masking of the terminal Man in the Manalpha(1-6)Man branch. Most interestingly, the beta-Man-linked GlcNAc in N-glycan core structure contributes greatly to the binding. The effect of this GlcNAc is so strong that it can substantially offset the negative effect of substitution on the nonreducing terminal Man residues. On the other hand, the GlcNAc that is usually attached to Asn in N-glycans and the l-Fuc linked at the 6-position of the GlcNAc are irrelevant to the binding. A bisecting GlcNAc neither contributes to nor interferes with the binding. This unique binding specificity of CSL offers many possibilities of its use in analytical and preparative applications.

Activation of protease-activated receptor-2 (PAR-2) triggers mucin secretion in the rat sublingual gland.

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.

Comparative studies on the analysis of glycosylation heterogeneity of sialic acid-containing glycoproteins using capillary electrophoresis.

Comparative studies concerning glycoform analysis of sialoglycoproteins by capillary electrophoresis were performed using a few separation modes hitherto reported. Glycoprotein samples examined in the present study were successfully separated to their respective glycoforms using surface-modified capillaries commercially available for capillary gas chromatography in the running buffer near their isoelectric points. The analysis times were less than 50 min and reproducibilities in migration times were excellent (less than 2.0% RSD for both run-to-run and day-to-day analyses). We present a method for the glycoform analysis of alpha1-acid glycoprotein in sera by simple pre-treatment as an application. The present technique will become one of the general methods for the evaluation of glycosylation heterogeneity of commercially available glycoprotein drugs.

[Therapeutic efficacy of amantadine hydrochloride in patients with epidemic influenza A virus infection].

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.

3-Aminobenzamide and 3-aminobenzoic acid, tags for capillary electrophoresis of complex carbohydrates with laser-induced fluorescent detection.

The efficiencies in derivatization of reducing carbohydrates were compared by capillary electrophoresis using maltose as a model with nine monoaminobenzene derivatives by reductive amination in the presence of sodium cyanoborohydride. We found that aminobenzene derivatives substituted at the 3-position showed good reactivity with reducing carbohydrates as expected from the reaction mechanism, although the fluorescence intensities and molar absorptivities of these derivatives were not as high as those of 2- and 4-aminobenzene derivatives. The reagents, 3-aminobenzamide and 3-aminobenzoic acid, which showed the highest reactivity, were applied to the labeling of carbohydrate chains obtained from some sialic acid-containing glycoprotein samples, and also high-mannose and hybrid-type oligosaccharides. Capillary electrophoresis of these labeled carbohydrate chains in an inner surface-modified capillary with (50% phenyl)methylpolysiloxane allowed excellent separation of sialic acid-containing carbohydrate chains derived from fetuin and thyroglobulin as well as high mannose-type and hybrid-type carbohydrates derived from bovine pancreas ribonuclease B, soybean agglutinin and hen ovalbumin. The lower limit of calibration was as low as the 10(-16) mol (injected amount) with helium-cadmium laser induced detection.

Release characteristics of endogenous constituents by exposure of small intestine to modified beta-cyclodextrins.

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.

[Examination of the stability of chloral hydrate and its preparation by capillary electrophoresis].

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.