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K Takagaki

Publications and source records attributed to K Takagaki.

At least 37 records · Page 2Linked to original sources
PubMed1998

Ion-spray mass spectrometry for identification of the nonreducing terminal sugar of glycosaminoglycan.

Various oligosaccharides from hyaluronic acid, which have glucuronic acid or N- acetylglucosamine at the nonreducing terminal, were prepared by digestion with a combination of testicular hyaluronidase and beta-glucuronidase. These oligo saccharides were analyzed by negative-mode ion-spray mass spectrometry (MS) with an atmospheric pressure ion source. Introduction of collisionally activated dissociation tandem mass spectrometry (CAD-MS/MS) produced ions derived from cleavage of the glycosidic bonds, allowing the structure to be analyzed. The CAD-MS/MS spectrum showed an intense and characteristic fragment ion at m/z 193 for oligosaccharides having glucuronic acid at the nonreducing terminal. On the other hand, this ion was not observed in the spectra of oligosaccharides having N- acetylglucosamine at the nonreducing terminal. Therefore, the fragmentation pattern revealed by CAD-MS/MS provides useful information for distinguishing glucuronic acid and N- acetylglucosamine at the nonreducing terminal of oligosaccharides derived from hyaluronic acid and other glycosaminoglycans. This ion-spray CAD-MS/MS technique was also applied successfully to the characterization of glycosaminoglycans reconstructed by glycotechnology.

Animals↗
PubMed1998

A novel 4-methylumbelliferyl-beta-D-xyloside derivative, sulfate-O-3-xylosylbeta1-(4-methylumbelliferone), isolated from culture medium of human skin fibroblasts, and its role in methylumbelliferone-initiated glycosaminoglycan biosynthesis.

Human skin fibroblasts were incubated in the presence of 4-methylumbelliferyl-beta-D-xyloside (Xyl-MU). The culture medium was recovered and Xyl-MU derivatives which were initiated by the Xyl-MU acting as a primer were purified. As a result, a novel Xyl-MU derivative was isolated, in addition to previously reported Xyl-MU derivatives such as glycosaminoglycan-MU, Gal-Gal-Xyl-MU, Gal-Xyl-MU, SA-Gal-Xyl-MU, Xyl-Xyl-MU, GlcA-Xyl-MU, and sulfate-GlcA-Xyl-MU. This Xyl-MU derivative was subjected to carbohydrate composition analysis, enzyme digestion, ion-spray mass spectrometric analysis, and Smith degradation. The results indicated that it was sulfate- O -3-Xyl-MU. When Xyl-MU was incubated with [35S]PAPS using a homogenate prepared from the same cultured skin fibroblasts, [35S]sulfate- O -3-Xyl-MU was produced. Moreover, when Xyl-MU was incubated with UDP-[3H]Gal, [3H]galactose was transferred to Xyl-MU, but when sulfate- O -3-Xyl-MU was incubated with UDP-[3H]Gal, [3H]galactose was not transferred. These results indicate that chain elongation from Xyl-MU is inhibited by sulfation of Xyl-MU, and that Xyl-MU sulfation is involved in the control of Xyl-MU-initiated glycosaminoglycan biosynthesis.

Cells, Cultured↗
PubMed1998

Proteoglycans in the nucleus pulposus of canine intervertebral discs after chondroitinase ABC treatment.

Experimental chemonucleolysis of the canine intervertebral disc with chondroitinase ABC and chymopapain was compared during a 52-week period. Roentgenograms and magnetic resonance imaging were used to examine changes in disc space and water content, respectively. Disc space narrowing and reductions in disc water content after chondroitinase ABC treatment were less than that after chymopapain. High-performance liquid chromatography was performed to measure changes in proteoglycans. Similarly to chymopapain, chondroitinase ABC degrades proteoglycans in the nucleus pulposus and decreases their quantity. However, large differences in the molecular weight and acidity of the resynthesized proteoglycans and in the chain length of the resynthesized glycosaminoglycans were observed between the two enzymes. The difference in disc space narrowing and the changes in disc water content between the two enzymes might result from differences in the characteristics of the resynthesized proteoglycans.

Acids↗
PubMed1997

Regulation of hyaluronate metabolism by progesterone in cultured fibroblasts from the human uterine cervix.

The effects of progesterone, dehydroepiandrosterone sulfate and 17beta-estradiol on the synthesis and degradation of hyaluronate were investigated using human uterine cervix fibroblasts. The cells were incubated with [3H]glucosamine in the presence of the hormones and then [3H]hyaluronate was isolated from the medium. The changes in the radioactivity of [3H]hyaluronate showed that progesterone suppressed hyaluronate synthesis by 22% of the control levels, while dehydroepiandrosterone sulfate and 17beta-estradiol enhanced it by 22% and 12% of the control levels, respectively. Furthermore, progesterone induced degradation of high-molecular-weight [14C]hyaluronate into low-molecular-weight hyaluronate (Mr approximately 40000). These results suggest that in cultured fibroblasts from the human uterine cervix progesterone converts hyaluronate metabolism from the synthesis phase to the degradation phase.

Cells, Cultured↗
PubMed1997

Effect of 4-methylumbelliferone on cell-free synthesis of hyaluronic acid.

Human skin fibroblasts were cultured in the presence of 0.5 mM 4-methylumbelliferone for 12 h, and cell-free synthesis of hyaluronic acid was performed using membrane-rich fraction from the cells. The preincubation of the cells with 4-methylumbelliferone reduced hyaluronic acid synthesis to 15% of that of non-preincubated cells, although its chain length was not changed. On the other hand, without preincubation of the cells with 4-methylumbelliferone, hyaluronic acid synthesis was not changed even when 4-methylumbelliferone was added directly to the reaction mixture. These results suggest that 4-methylumbelliferone represses the expression of hyaluronic acid synthase on the cell surface.

Cell Membrane↗
PubMed1997

Effect of monensin on the synthesis of beta-D-xyloside-initiated glycosaminoglycan and its linkage region oligosaccharides in human skin fibroblasts.

Human skin fibroblasts were cultured with a fluorogenic xyloside, 4-methylumbelliferyl-beta-D-xyloside (Xyl-MU) as an initiator, and the effects of monensin, which destroys the normal structure of the Golgi complex, on the synthesis of Xyl-MU-initiated glycosaminoglycan (GAG-MU) and its linkage region oligosaccharides were investigated. When the cells were incubated with Xyl-MU in the presence of monensin, the synthesis of GAG-MU was inhibited. In addition, the synthesis of Galbeta1-3Galbeta1-4Xylbeta1-MU as an intermediate of GAG-MU was inhibited, whereas the synthesis of Galbeta1-4Xylbeta1-MU, which is formed prior to Galbeta1-3Galbeta1-4Xylbeta1-MU, was not. These results indicate that inhibition of GAG-MU synthesis by monensin occurs at the point where the second galactose is joined to Galbeta1-4Xylbeta1-MU.

Cells, Cultured↗
PubMed1997

Ability of intrauterine bacterial lipopolysaccharide to cause in situ uterine contractions in pregnant rabbits.

BACKGROUND: To investigate the ability of bacterial lipopolysaccharide delivered by the intra-uterine route to cause uterine contractions in rabbits, and to assess the suppressive effect of urinary trypsin inhibitor on them. METHODS: Both pregnant and non-pregnant rabbits were chronically implanted with a force-transducer to make it possible to record isometric uterine contractions under unanesthetized and unrestrained conditions. Lipopolysaccharide (10 micrograms/animal) was administered via a catheter to their uteri; and then, after confirmation of lipopolysaccharide-induced uterine contractions, urinary trypsin inhibitor (3,000 or 10,000 units/animal/time) or saline solution was injected through the catheter, 5 times for pregnant animals or 3 times for non-pregnant animals at 1-hour intervals in both cases. Their uterine contractions were continuously recorded for 3 to 5 hours. Effects of lipopolysaccharide (10 micrograms/ml) and urinary trypsin inhibitor (100 and 1,000 units/ml) on the contraction of isolated uteri from pregnant mice were also measured, as was their production of prostaglandin E2 and prostaglandin F2 alpha by an enzyme immunoassay method. RESULTS: Lipopolysaccharide augmented the in situ uterine contractions in both pregnant and non-pregnant rabbits, as well as the in vitro contractions of isolated uteri from pregnant mice. Lipopolysaccharide also increased the uterine prostaglandin production. Urinary trypsin inhibitor inhibited significantly the lipopolysaccharide-induced uterine contractions and the prostaglandin production. CONCLUSIONS: Lipopolysaccharide enhanced uterine contractions through, at least partly, a direct mechanism via uterine prostaglandin production, which action could explain the onset of preterm delivery due to intrauterine bacterial infection. As urinary trypsin inhibitor suppressed the lipopolysaccharide-induced uterine contractions, this inhibitor may be a hopeful candidate of a drug for prevention of preterm delivery.

Animals↗
PubMed1996

Characterization of mucin in whole-gut lavage fluid obtained from patients with inflammatory bowel disease.

Whole-gut lavage fluid, collected by administering an electrolyte lavage solution orally, was found to be an excellent and easily collectable source of abundant mucin. Furthermore, the biochemical features of the mucin from patients with ulcerative colitis and Crohn's disease were investigated. The mucin was separated into four fractions by Sepharose CL-4B, Sepharose CL-2B, and DEAE Sephacel chromatography. Compared with healthy subjects, the total yields of mucin from ulcerative colitis patients were low due to a deficiency of neutral mucin, whereas those from Crohn's disease patients were high, which was attributable mainly to high-molecular-weight mucin. The fucose and sulfate contents were low in ulcerative colitis, but only the former was low in Crohn's disease. The different biochemical features of the mucin obtained from whole gut lavage fluid appear to reflect mucosal pathological changes associated with inflammatory bowel disease.

Adult↗
PubMed1996

Characterization and biological significance of sialyl alpha 2-3galactosyl beta 1-4xylosyl beta 1-(4-methylumbelliferone) synthesized in cultured human skin fibroblasts.

Human skin fibroblasts were incubated in the presence of a fluorogenic xyloside, 4-methyl-umbelliferyl-beta-D-xyloside (Xyl-MU), then the cultured medium was recovered, concentrated with a lyophilizer, and dialyzed against distilled water. The structures of the Xyl-MU derivatives purified from the dialyzable fraction were investigated. In addition to established glycosaminoglycans-MU (GAGs-MU), Gal-Gal-Xyl-MU, Gal-Xyl-MU, sulphate-GlcA-Xyl-MU, GlcA-Xyl-MU, and Xyl-Xyl-MU, which were induced by Xyl-MU, an oligosaccharide having fluorescence was purified using a combination of gel filtration, ion-exchange chromatography and high-performance liquid chromatography, then subjected to carbohydrate composition analysis, enzyme digestion, Smith degradation, 1H-NMR, and ion-spray mass spectrometric analysis. From the data obtained, the oligosaccharide was considered to have the structure SA alpha 2-3Gal beta 1-4Xyl beta 1-MU. The amount of MU-oligosaccharide in the cell culture increased with time and was dependent on the amount of Xyl-MU added. Its production was also different from that of Gal-Gal-Xyl-MU and Gal-Xyl-MU, which are biosynthetic intermediates of GAG-MU. Addition of CDP, an inhibitor of sialytransferase, to the cell culture medium increased the secretion of GAG-MU. These results suggest that SA-Gal-Xyl-MU production may be related to the regulation of GAG-MU biosynthesis.

Carbohydrate Sequence↗
PubMed1995

Identification of glycosaminoglycans using high-performance liquid chromatography on a hydroxyapatite column.

Glycosaminoglycans (heparin, heparan sulfate, dermatan sulfate, chondroitin sulfate, and hyaluronic acid) were labeled with a fluorescent reagent, 2-aminopyridine. The fluoro-labeled glycosaminoglycans were subjected to high-performance liquid chromatography on a hydroxyapatite column. The binding property of each glycosaminoglycan to hydroxyapatite was different. The structural properties of glycosaminoglycans bound to hydroxyapatite were then investigated using chemical desulfated or enzymic depolymerized glycosaminoglycans. This revealed that the sulfate content and molecular weight of the glycosaminoglycans correlated with their binding properties to hydroxyapatite. Desulfated dermatan sulfate but not desulfated chondroitin 6-sulfate bound to the hydroxyapatite. These data indicate that iduronic acid residues of glycosaminoglycans are important for the binding property. The method described which uses hydroxyapatite columns facilitates rapid separation and microanalysis of the glycosaminoglycans, especially dermatan sulfate and chondroitin sulfate.

Aminopyridines↗
PubMed1995

HNK-1-reactive novel oligosaccharide, sulfate-O-3GlcA beta 1-4Xyl beta 1-(4-methylumbelliferone), synthesized by cultured human skin fibroblasts.

4-Methylumbelliferyl-beta-D-xyloside (Xyl-MU) was added to the medium of cultured human skin fibroblasts. After incubation, the culture medium was pooled, and the Xyl-MU-induced oligosaccharides in the medium were purified by gel filtration chromatography. A novel Xyl-MU derivative was obtained, in addition to the previously reported Xyl-MU derivatives such as Gal-Gal-Xyl-MU, Gal-Xyl-MU, Sia-Gal-Xyl-MU, GlcA-Xyl-MU, and Xyl-Xyl-MU. The novel Xyl-MU derivative was purified using gel-filtration chromatography and high performance liquid chromatography and then subjected to carbohydrate composition analysis, enzymic digestion, Smith degradation, and ion spray mass spectrometric analysis. The results indicated that it was sulfate-O-3GlcA beta 1-4Xyl beta 1-MU. The structure of the nonreducing terminal of this Xyl-MU-induced oligosaccharide was the same as that of the oligosaccharide chain of a human peripheral nerve-derived glycolipid, reactive with the mouse monoclonal antibody HNK-1, and this Xyl-MU-induced oligosaccharide also reacted with HNK-1. These results suggest that the oligosaccharide, which is structurally identical to that of human peripheral nerve-derived glycolipid synthesized by nervous tissue and related to cell adhesion, is synthesized also by mesenchymal cells.

Antigens, CD↗
PubMed1995

Cloning, functional expression and tissue distribution of human alpha 1c-adrenoceptor splice variants.

We report the cloning and characterization of two isoforms of human alpha 1c-adrenoceptor cDNA (alpha 1c-2, alpha 1c-3). These isoforms are generated by alternative splicing and differ from the clone we previously isolated (alpha 1c-1) in their length and sequences of the C-terminal domain. Tissue distribution of mRNAs showed that these variants co-express with alpha 1c-1 in the human heart, liver, cerebellum and cerebrum. Despite the structural differences, functional experiments in transfected CHO cells showed that the three isoforms have similar ligand binding properties, and all couple with phospholipase C/Ca2+ signaling pathway.

Alternative Splicing↗
PubMed1995

Hyaluronic-acid-deficient extracellular matrix induced by addition of 4-methylumbelliferone to the medium of cultured human skin fibroblasts.

The effects of xylosyl-beta-D-(4-methylumbelliferone) and its aglycone, 4-methylumbelliferone, on hyaluronic acid synthesis were investigated in cultured human skin fibroblasts. Xylosyl-beta-D-(4-methylumbelliferone) added to the medium of cultured cell reduced the synthesis of hyaluronic acid. Furthermore, 4-methylumbelliferone reduced the production of hyaluronic acid markedly. In addition, 4-methylumbelliferone had hardly any effect on proteoglycan synthesis, whereas xylosyl-beta-D-(4-methylumbelliferone) produced a large amount of glycosaminoglycan chains. The present results indicate that cells cultured with 4-methylumbelliferone produce a hyaluronic-acid-deficient extracellular matrix, which will be useful for functional studies of hyaluronic acid.

Cells, Cultured↗
PubMed1995

Enzymic reconstruction of glycosaminoglycan oligosaccharide chains using the transglycosylation reaction of bovine testicular hyaluronidase.

The reconstruction of glycosaminoglycan chains using the transglycosylation reaction of testicular hyaluronidase was investigated. First, the optimal conditions for the transglycosylation reaction catalyzed by the enzyme were determined by incubation with the enzyme, using hyaluronic acid (M(r) = 800,000) as a donor and pyridylaminated hyaluronic acid hexasaccharide having glucuronic acid at the nonreducing terminal as an acceptor. The carbohydrate chains as reaction products were determined by high performance liquid chromatography and mass spectrometry. The optimal pH for hydrolysis by the enzyme was found to be about 5.0, whereas that for the transglycosylation reaction was about 7.0. Sodium chloride in the reaction medium inhibited the transglycosylation reaction. Under the optimal conditions, the carbohydrate chains were sequentially transferred along with disaccharide units to the nonreducing terminal of the acceptor and elongated up to docosasaccharide from the acceptor, pyridylaminated hexasaccharide. Using a combination of hyaluronic acid, chondroitin, and chondroitin 4- and 6-sulfate as an acceptor and a donor, it was possible to reconstruct hybrid chains, which were natural or unnatural types of glycosaminoglycan chains. Therefore, it is highly likely that application of the transglycosylation reaction using testicular hyaluronidase would facilitate artificial reconstruction of glycosaminoglycans having some physiological functions.

Animals↗
PubMed1994

A novel oligosaccharide, GlcA beta 1-4Xyl beta 1-(4-methylumbelliferone), synthesized by human cultured skin fibroblasts.

Human skin fibroblasts were cultured in the presence of 4-methylumbelliferyl-beta-D-xyloside (Xyl-MU) using a mass-culture system with a microcarrier. The structures of Xyl-MU-induced sugars purified from the dialysable fraction of the incubation medium were investigated. In addition to glycosaminoglycans the elongation of which initiated by Xyl-MU has already been reported, and oligosaccharides similarly initiated by Xyl-MU, such as Gal-Gal-Xyl-MU, Gal-Xyl-MU and SA-Gal-Xyl-MU, a novel Xyl-MU-induced oligosaccharide was detected. This oligosaccharide was identified as GlcA beta 1-4Xyl beta 1-(4-methylumbelliferone) using sugar composition analysis, enzyme digestion, mass spectrometry and Smith degradation. Using this culture system, the amount of the new oligosaccharide produced increased with the incubation time, even after the production of glycosaminoglycan initiated by Xyl-MU and Gal-Xyl-MU had reached a plateau. These results suggest that this oligosaccharide may be involved in terminating the elongation of glycosaminoglycan chains that is initiated by Xyl-MU.

Benzoates↗
PubMed1994

Hyaluronate depolymerization activity induced by progesterone in cultured fibroblasts derived from human uterine cervix.

High-molecular-weight [14C]hyaluronate was incubated with cultured fibroblasts from human uterine cervix and skin, and then the depolymerization of the hyaluronate was investigated. [14C]Hyaluronate in the medium of skin fibroblasts was depolymerized into a constant molecular weight (M(r) about 40,000), whereas that of cervix fibroblasts was not depolymerized, irrespective of incubation period. However, when progesterone was added to the medium of cervix fibroblasts, hyaluronate was depolymerized to the same extent as that in skin fibroblasts. The reducing terminal sugar of the depolymerized hyaluronate was N-acetylglucosamine. These results suggest that a hyaluronate-depolymerizing enzyme, endo-beta-N-acetylglucosaminidase, was induced by progesterone in cultured fibroblasts derived from human uterine cervix.

Cells, Cultured↗
PubMed1994

Characterization of hydrolysis and transglycosylation by testicular hyaluronidase using ion-spray mass spectrometry.

Various oligosaccharides from hyaluronic acid, which were fluorescence-labeled and blocked by pyridylamination at the reducing terminal, were incubated as substrates or acceptors with bovine testicular hyaluronidase. Fluorescence-labeled reaction products in the reaction mixture were monitored selectively and directly by ion-spray mass spectrometry without chemical derivatization. As a result, several features of the relationship between oligosaccharides, substrates, and testicular hyaluronidase were clarified. When hexasaccharides or larger oligosaccharides having D-glucuronic acid at the nonreducing terminal were used as substrates, they were hydrolyzed sequentially to disaccharides from the nonreducing terminal, and these disaccharides were then transferred to other hexasaccharides. On the other hand, when heptasaccharides or larger oligosaccharides having N-acetyl-D-glucosamine at the nonreducing terminal were used as substrates, trisaccharides were released from the nonreducing terminal, and then also transferred to other hexasaccharides, thus forming nonasaccharides. Thus, the relationship between hydrolysis and transglycosylation reactions with testicular hyaluronidase was characterized using ion-spray mass spectrometry.

Animals↗