Syuichi Yoshihara

A method for the identification of sulfated glycopeptide by two-dimensional electrophoresis on cellulose acetate membrane

Two-dimensional electrophoresis on cellulose acetate membrane permits the clean separation of sulfated glycopeptide in a mixture of acidic glycans (glycosaminoglycans and acidic glycopeptides). Two systems were used. In system 1, 0.1 M pyridine-0.47 M formic acid buffer (pH 3.0) was used in the first and 0.1 M barium acetate (pH 8.0) in the second dimension. In system 2, 0.1 M pyridine-0.47 M formic acid buffer (pH 3.0) was used in the first and 0.1 M HCl in the second dimension. All of the acidic glycans on electrophoretogram were stained with alcian blue in 70% ethanol. On the other hand, sulfated glycans alone were made visible with alcian blue in 0.1 M HCl. Alcian blue in 70% ethanol or 0.1 M HCl, when combined with periodic acid-Schiffs reagent identified sulfated glycopeptides on cellulose acetate membrane.

Effect of proteoglycan on experimental colitis

Abstract The effect of proteoglycan (PG) on colitis was examined in animal experiments using mice. The PG used was extracted from nasal cartilage of salmon head with 4% acetic acid and prepared by precipitation with ethanol followed by dialysis. The PG contained about 7% protein, and had a molecular mass of 344 kDa on SDS/PAGE. The glycosaminoglycan (GAG) sugar chains of the PG were composed of hexosamine, uronic acid and sulfate at a molar ratio of 1.0:1.0:0.7. The mice were divided into a control group and an administration group. The control group was given free access to drinking water containing dextran sulfate sodium salt (DSS) to induce colitis. On the other hand, the administration group was given free access to drinking water containing DSS and PG. Then, the time course of survival rates in both groups were measured. In the administration group, the survival rate increased significantly in comparison with that of the control group. The difference in the survival rates indicated that the onset of mouse colitis induced by DSS was inhibited by administration of the PG.

Laparoscopic treatment for biliary ascariasis.

Biliary ascariasis is one of the most common types of ascaris infections. The current treatments are helminthic drug therapy, endoscopic extraction, and surgical extraction. A case of biliary ascariasis and cholecystocholedocholithiasis was successfully treated by laparoscopic extraction of the living worm and biliary stones. This procedure was found to be very effective for biliary ascariasis with biliary stones, and it holds promise for similar cases in the future.

Plasma Exchange-based Plasma Recycling Dialysis System as an Artificial Liver Support

Abstract:  We developed a plasma recycling dialysis (PRD) system based on plasma exchange (PE). In this system, rapid reduction of toxic substances and restitution of deficient essential substances are performed by PE and subsequent blood purification is performed by dialysis between separated plasma recycled over a purification device and the patients blood across the membrane of a plasma separator. To demonstrate the safety and efficacy of this system, we used a pig model of fulminant hepatic failure (FHF) and anion exchange resin, activated charcoal and hemodialysis for the purification device. FHF was induced by intraportal administration of α‐amanitine (0.1 mg/kg) and lipopolysaccharides (1 µg/kg) in pigs. Three groups of animals were studied: group 1, diseased controls (N = 4); group 2, PE group (N = 4), 16 h after drug infusion the pigs underwent PE of approximately 1.2 L for 2 h; and group 3, PE + PRD group (N = 4), the pigs underwent PE followed by PRD for 6 h. The hemodynamic status of all animals was stable during the procedure. In group 3, the values of ammonia, total bile acid and total bilirubin continuously decreased and were significantly lower than those of the animals in group 2 24 h after the induction of FHF. The Fischer ratio was significantly higher than in group 2 after 24 h. Group 3 pigs maintained a higher level of consciousness and survived longer than group 2 pigs. Safety of this PE‐based PRD system was demonstrated and the removal of toxic substances was significant. This study confirmed the clinical utility of this system as an artificial liver support.

Enzymatic Attachment of Glycosaminoglycan Chain to Peptide Using the Sugar Chain Transfer Reaction with Endo-β-xylosidase

Endo-β-xylosidase from the mid-gut gland of the molluscus Patinopecten is an endo-type glycosidase that hydrolyzes the xylosyl serine linkage between a core protein and a glycosaminoglycan (GAG) chain, releasing the intact GAG chain from proteoglycan. In this study, we investigated GAG chain transfer activity of this enzyme, in order to develop a method for attaching GAG chains to peptide. Peptidochondroitin sulfate (molecular mass of sugar chain, 30 kDa) from bovine tracheal cartilage as a donor and butyloxycarbonyl-leucyl-seryl-threonyl-arginine-(4-methylcoumaryl-7-amide) as an acceptor were incubated with endo-β-xylosidase. As a result, a reaction product with the same fluorescence as the acceptor peptide was observed. High pressure liquid chromatography analysis, cellulose acetate membrane electrophoresis, and enzymatic digestion showed that this reaction product had the chondroitin sulfate (ChS) from the donor. Furthermore, the acceptor peptide was released from this reaction product after hydrolysis by endo-β-xylosidase. Therefore, it was confirmed that the ChS chain released from the donor was transferred to the acceptor peptide by the GAG chain transfer reaction of endo-β-xylosidase. The optimal pH for hydrolysis by this enzyme was found to be about 4.0, whereas that for this reaction was about 3.0. Not only the ChS but also the dermatan sulfate and the heparan sulfate were transferred to the acceptor peptide by this reaction. By using this reaction, the GAG chain could be attached to the peptide in one step. The GAG chain transfer reaction of endo-β-xylosidase should be a significant glycotechnological tool for the artificial synthesis of proteoglycan.

Characterization of glycosaminoglycans in regenerating canine liver

BACKGROUND/AIMS Liver regeneration after partial hepatectomy is accompanied by hepatocyte proliferation and alteration of the extracellular matrix. Glycosaminoglycans, which are components of the extracellular matrix, interact with other matrix components, and are related to hepatocyte growth. The aim of this study was to investigate the relationship between hepatocyte proliferation and changes in glycosaminoglycan. METHODS Hepatocyte proliferation and changes in glycosaminoglycan were investigated in dogs after 55% partial hepatectomy. Hepatocyte mitosis was investigated by immunohistochemistry using anti-proliferating cell nuclear antigen antibody. The amount of glycosaminoglycan was determined by the carbazole-sulfuric acid method. We used a new method for analysis of glycosaminoglycan chains, involving endo-beta-xylosidase digestion and fluorescence labelling, to investigate the components of glycosaminoglycan. RESULTS Hepatocyte mitosis was increased after hepatectomy, reaching a peak at postoperative day 7. The total amount of hepatic glycosaminoglycan reached a maximum at 1 to 2 weeks afer hepatectomy, and the ratio of the components showed a concomitant change, the amount of heparan sulfate increasing, and that of chondroitin sulfate/dermatan sulfate decreasing. Increased heparan sulfate has shorter chains at 1 to 2 weeks after hepatectomy. CONCLUSIONS These results suggest that the transient changes in heparan sulfate with a decreased chain length and chondroitin sulfate/dermatan sulfate and observed during liver regeneration are associated with hepatocyte proliferation.

Chemical structure of the carbohydrate moiety of fucose-rich glycopeptides from human pancreatic juice

SummaryHuman pancreatic juice, obtained from nine patients after partial excision of the pancreas for bile duct cancer, was fractionated in order to isolate its glycopeptides. Three glycopeptides were purified employing ion-exchange chromatography and gel filtration. All the glycopeptides were found to be free of sialic acid and galactosamine but to have an unusually high content ofl-fucose. The chemical structures of the three glyco-peptides were determined using 500-MHz [1H]-NMR spectroscopy. One of them, glycopeptide, GP-4, possessed a biantennary structure with threel-fucose residues. The second glycopeptide, GP-3, had a triantennary structure with fourl-fucose residues, and the third one, G-2, had a tetra-antennary structure with fivel-fucose residues. The chemical compositions of these glycopeptides, including the absence of sialic acid and the highl-fucose content, indicate that they represent a new class of glycopeptide present in the normal human pancreas.

Enzymic determination of acidic glycoconjugates in human pancreatic juice

SummaryAcidic glycoconjugates (glycosaminoglycans, sulfated glycopeptide, and sialoglycopeptide) were isolated by precipitation with cetylpyridinium chloride from human pancreatic juice after digestion with pronase. The acidic glycoconjugates were found exclusively in the proteinaceous precipitate that occurred during dialysis against a buffer of low ionic strength. The concentration of the acidic glycoconjugates in normal pancreatic juice was about 2.4 mg/L. The acidic glycoconjugates were characterized by electrophoresis on cellulose acetate membrane and chemical analysis before and after digestion withStreptomyces hyaluronidase, chondroitinase AC, chondroitinase ABC, and heparitinase. It was found that the major acidic glycoconjugates were heparan sulfate (39.3%), sulfated glycopeptide (34.4%), chondroitin sulfate (14.2%), and the minor ones hyaluronic acid (6.4%) and sialoglycopeptide (5.7%).

Quantitative determination of sulfated glycopeptide by two-dimensional electrophoresis on cellulose acetate membrane

Quantitative determination of the sulfated glycoproteins present in tissue and secretion fluid was performed. After digestion of the specimen with pronase in order to convert glycoproteins to glycopeptides, the sulfated glycopeptides were separated from a mixture of acidic glycans (glycosaminoglycans, sialoglycopeptides and sulfated glycopeptides) by two-dimensional electrophoresis on cellulose acetate membrane [(1986) J. Biochem. Biophys. Methods 12, 239-246]. After staining with alcian blue, the spot of sulfated glycopeptide on the cellulose acetate membrane was cut out, and then only the dye bound to the sulfated glycopeptide was extracted with a 5% cetylpyridinium chloride solution at 100 degrees C for 15 min. The extract was then measured by absorbance at 615 nm using an authentic sulfated glycopeptide as a standard. This method facilitated the determination of sulfated glycopeptides, which were separated from other acidic glycans, within the range 0-25 micrograms.

Possible role of hepatic bile mucus glycoprotein in development of intrahepatic gallstones

To study the role played by hepatic bile mucus glycoprotein in the development of hepatolithiasis, mucus glycoprotein, isolated from the bile of patients with intrahepatic gallstones by gel filtration and ultracentrifugation, was examined for precipitability in control hepatic bile obtained postopertively from patients successfully treated for cholecysto-and/or choledocho-lithiasis. When the mucus glycoprotein was incubated at 38°C for 48h in the control hepatic bile in the presence of calcium inons, massive precipitation was produced. The precipitation was inhibited by treating the mucus glycoprotein with acid, alkali, a reducing reagent, or protease, the inhibition being most effective with acid, which splits up carbohydrate chains. This suggests that the precipitability of the mucus glycoprotein resides mainly in its carbohydrate chains. These observations imply that the development of intrahepatic gallstones, calcium bilrubinate stones in particular, could be prevented by degrading mucus glycoprotein in hepatic bile.