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Kiyoshi Suzuki

Publications and source records attributed to Kiyoshi Suzuki.

At least 19 recordsLinked to original sources
PubMed2006

Multistage mass spectrometric sequencing of keratan sulfate-related oligosaccharides.

To establish a universal protocol for sequencing keratan sulfate (KS) using mass spectrometry (MS), systematic electrospray ionization-MSn fragmentation experiments were carried out for 10 KS-related oligosaccharides of defined structure. Under the experimental conditions employed, fully charged molecular-related ions were observed as dominant peaks in all MS(1) spectra, which clearly reflected the number of sulfates and sialic acids in the oligosaccharide structures. In the subsequent MS2, almost all of the oligosaccharides gave fragment ions corresponding to their dehydrated molecular-related ions as well as (0,2)A(r) scission ions (according to the nomenclature developed by Domon and Costello, where "r" represents the reducing end in this study). Further fragmentation of the (0,2)A(r) ions in MS3 predominantly yielded the corresponding (2,4)A(r) ions. Finally, in MS(4), these (2,4)A(r) ions were subjected to extensive glycosidic cleavage. Hence, the MS4 data of KS oligosaccharides provided sufficient information for their sequence determination. In addition, some important features of MSn fragmentation became evident. These findings should lead to the establishment of consensus rules applied for KS oligosaccharides, including those previously unidentified, and also accelerate functional studies on KS, i.e., KS-related glycosaminoglycomics.

Carbohydrate Conformation↗
PubMed2006

Characterization of a GDP-D-mannose 3'',5''-epimerase from rice.

The enzymatic characterization of GDP-d-mannose 3'',5''-epimerase (GME), a key enzyme in the biosynthesis of vitamin C in plants is described. The GME gene (Genbank Accession No. AB193582) in rice was cloned, and expressed as a fusion protein in Escherichia coli. Reaction products from GDP-d-mannose, as produced by GME catalysis, were separated by recycling HPLC on an ODS column, and were determined to be GDP-l-galactose and GDP-l-gulose, based on their NMR spectra and sugar analysis. The reaction catalyzed by GME was inhibited by GDP, and was strongly accelerated by NAD(+) in contrast to the case of GME from Arabidopsis thaliana. This difference in the effect of NAD(+) on GME activity can be attributed to the NAD binding domain which is conserved in the rice gene, but not in the Arabidopsis thaliana gene. The apparent K(m) and k(cat) were determined to be 1.20x10(-5)M and 0.127s(-1), respectively, in the presence of 20microM NAD(+). The fractions of GDP-d-mannose, GDP-l-galactose and GDP-l-gulose, at equilibrium, were approximately 0.75, 0.20 and 0.05, respectively.

Arabidopsis↗
PubMed2006

Systematic identification of N-acetylheparosan oligosaccharides by tandem mass spectrometric fragmentation.

Recently, a useful procedure for the preparation of both even- and odd-numbered series of N-acetylheparosan (NAH) oligosaccharides was established. The present report describes findings when these NAH oligosaccharides were subjected to comparative mass spectrometry (MS)/MS fragmentation analysis by matrix-assisted laser desorption/ionization (MALDI)-LIFT-time-of-flight (TOF)/TOF-MS/MS, and electrospray ionization (ESI) collision-induced dissociation (CID) MS/MS. The resultant fragment ions were systematically assigned to elucidate fragmentation characteristics. In the MALDI-LIFT-MS/MS experiments, all the NAH oligosaccharides underwent unique glycosidic cleavages that included B-Y ion cleavages (nomenclature system of Domon and Costello, Glycoconjugate J. 1988; 5: 397) at the C-1 side, and C-Z ion cleavages at the C-4 side, with respect to glucuronic acid (GlcA). In addition, (0,2)A and/or (0,2)X cross-ring cleavages were observed for relatively small oligosaccharides. The former observation clearly reflects the occurrence of a GlcA-N-acetylglucosamine (GlcNAc) alternating structure of NAH, while the latter feature implies the occurrence of the -beta-1-4-glucuronide linkage. Extensive glycosidic cleavages were also observed in the ESI-CID-MS/MS fragmentation, though cleavage specificity was less evident than in the case of MALDI-LIFT-TOF/TOF-MS/MS. The information obtained in this study should be valuable for understanding both biosynthetic and degradation processes of NAH and its derivatives including heparin and heparan sulfate, as well as artificially modified NAH oligosaccharides.

Gas Chromatography-Mass Spectrometry↗
PubMed2006

Keratan sulfate and chondroitin/dermatan sulfate in maximally recovered hypocellular stromal interface scars of postmortem human LASIK corneas.

PURPOSE: To analyze the amounts and distributions of nonsulfated and sulfated keratan sulfate (KS) and chondroitin/dermatan sulfate (CS/DS) disaccharides in the interface wound of human postmortem LASIK corneas in comparison with normal control corneas. METHODS: Corneal stromal tissue samples from central and paracentral hypocellular primitive stromal interface scars of human LASIK corneas and from similar regions of normal control corneas were collected by laser capture microdissection (LCM) and subsequently were digested with specific glycosidase enzymes. Digests were directly analyzed by electrospray ionization tandem mass spectrometry (ESI-MS/MS). RESULTS: Concentrations of both monosulfated GlcNAc(6S)-beta-1,3-Gal (MSD2) and disulfated Gal (6S)-beta-1,4-GlcNAc(6S) (DSD) KS disaccharides from the LASIK interface scars were significantly lower than in normal control corneal stromas. No significant difference was found for the concentration of nonsulfated (NSD) KS disaccharides in LASIK interface scars compared with normal controls. The concentration of DeltaUA-beta-1,3-GalNAc(6S) (Deltadi-6S) CS/DS disaccharides from the LASIK interface scar was significantly higher than normal corneal stroma, whereas concentrations of DeltaUA-beta-1,3-GalNAc(4S) (Deltadi-4S) and nonsulfated Deltadi-0S CS/DS disaccharides demonstrated no significant differences from normal corneas. CONCLUSIONS: The profiles of KS and CS/DS disaccharides in LASIK interface scars are significantly different from those in normal cornea stromal tissue, as revealed by LCM and ESI-MS/MS.

Chondroitin Sulfates↗
PubMed2005

Microscale preparation of even- and odd-numbered N-acetylheparosan oligosaccharides.

In order to prepare a series of N-acetylheparosan (NAH)-related oligosaccharides, bacterial NAH produced in Escherichia coli strain K5 was partially depolymerized with heparitinase I into a mixture of even-numbered NAH oligosaccharides, having an unsaturated uronic acid (DeltaUA) at the non-reducing end. A mixture of odd-numbered oligosaccharides was derived by removing this DeltaUA in the aforementioned mixture by a 'trimming' reaction using mercury(II) acetate. Each oligosaccharide mixture was subjected to gel-filtration chromatography to generate a series of size-uniform NAH oligosaccharides of satisfactory purity (assessed by analytical anion-exchange HPLC), and their structures were identified by MALDITOF-MS, ESIMS, and 1H NMR analysis. As a result, a microscale preparation of a series of both even- and odd-numbered NAH oligosaccharides was achieved for the first time. The developed procedure is simple and systematic, and thus, should be valuable for providing not only research tools for heparin/heparan sulfate-specific enzymes and their binding proteins, but also precursor substrates with medical applications.

Acetylglucosamine↗
PubMed2005

Osteoclastic function is accelerated in male patients with type 2 diabetes mellitus: the preventive role of osteoclastogenesis inhibitory factor/osteoprotegerin (OCIF/OPG) on the decrease of bone mineral density.

To clarify the pathogenesis of altered bone metabolism in diabetic state and its underlying mechanisms, the bone mineral content and fasting levels of serum intact parathyroid hormone (i-PTH), intact osteocalcin (i-OC), tartrate-resistant acid phosphatase (TRAP) and osteoclastgenesis inhibitory factor/osteoprotegerin (OCIF/OPG) were measured in male type 2 diabetic patients and their age-matched controls. In addition, urine levels of osteoclastic markers, C-telopeptide of type I collagen (CTx), deoxypyridinoline (DPD), and N-telopeptide of type I collagen (NTx) were simultaneously determined. Serum levels of i-PTH and i-OC in diabetic patients were significantly lower than those in the controls. Conversely, serum concentrations of TRAP were significantly elevated in diabetic patients. However, no clear correlation was observed between serum i-OC and TRAP. It was also observed that urinary excretion of CTx, DPD, and NTx was significantly increased in the diabetics as compared with the controls. Unexpectedly, serum levels of OCIF/OPG tended to be higher in the diabetic group, and these values exhibited a significantly positive correlation with those of serum TRAP. There was found a significantly negative correlation between serum TRAP and bone mineral density (BMD) and also between serum OCIF/OPG and bone mineral density. It seems probable that OCIF/OPG has a suppressive role on the increased bone resorption to prevent further loss of the skeletal bone mass in type 2 diabetic patients.

Acid Phosphatase↗
PubMed2005

[A case of nonclostridial gas gangrene of the leg complicated by severe pneumonia].

A 73-year-old man admitted for febrile left leg pain with dyspnea, who had poorly controlled diabetes was found on admission to have severe hypoxia and chest X-ray showed infiltrates in the middle to lower left lung. X-rays of the left leg showed gas around the knee joint. These findings suggested severe pneumonia with gas gangrene, necessitating immediate debridement of the gas gangrene lesion and hyperbaric oxygenation. Antibiotics were also administered intravenously (panipenem/betamipron 0.5 g x 3/day, clindamycin 600 mg x 2/day, and erythromycin 500 mg x 3/day). We conducted fiberoptic bronchoscope daily because consolidation of the whole left lung developed with purulent sputum expectoration. Both pneumonia and gas gangrene gradually ameliorated avoiding amputation of theleg. Gas gangrene was cured without leaving sequelae such as motor dysfunction. Staphylococcus aureus was detected in both pus from the leg and sputum collected by bronchoscopy. Microorganisms showed the same pattern of sensitivity to antibiotics, suggesting a causal relationship between pneumonia and gas gangrene through the blood stream. Gas gangrene was considered the primary infection followed by pneumonia, since pain and swelling of the left leg preceded the airway symptoms. The present case illustrates in compromised hosts including diabetics, gas gangrene may develop taking an opportunity of airway infection, and that in some cases, early debridement of the lesion and optimal use of antibiotics may help cure this disease without aggressive surgery. Hyperbaric oxygenation may also be useful, although its validity must be investigated further.

Aged↗
PubMed2005

Detection and quantification of sulfated disaccharides from keratan sulfate and chondroitin/dermatan sulfate during chick corneal development by ESI-MS/MS.

PURPOSE: To identify and quantify changes in keratan sulfate (KS) and chondroitin/dermatan sulfate (CS/DS) sulfated disaccharides in the developing chick cornea using electrospray ionization tandem mass spectrometry (ESI-MS/MS). METHODS: Cryostat sections of fresh nonfixed corneas were obtained from White Leghorn embryonic day (E)8 to E20 chicks, and from 4- and 70-week-old chickens. Tissue sections on glass slides were incubated with selected glycosidase enzymes. Digest solutions were analyzed directly by ESI-MS/MS. RESULTS: The concentration of KS monosulfated disaccharide (MSD) Gal-beta-1,4-GlcNAc(6S) in E8 cornea equaled that at E20, declined to its lowest level by E10, increased to a second peak by E14, decreased to a second low by E18, peaked again by E20, and remained high in adult corneas. A similar concentration profile was observed for KS disulfated disaccharide (DSD) Gal(6S)-beta-1,4-GlcNAc(6S), and thus also for total sulfated KS disaccharides. The molar percent of DSD was higher than that of MSD from E8 to E18, equivalent at E20, and less than that of MSD in adult corneas. In contrast, total concentration of CS/DS Deltadi-4S plus Deltadi-6S decreases as development progresses and is lowest in adult corneas. Concentration and molar percent of Deltadi-6S is highest at E8, then decreases through development as the concentration and molar percent of Deltadi-4S increases from E8 and exceeds that of Deltadi-6S after E14. CONCLUSIONS: New, rapid, direct chemical analysis of extracellular matrix components obtained from sections from embryonic and adult chick corneas reveals heretofore undetected changes in sulfation characteristics of KS and CS/DS disaccharides during corneal development.

Animals↗
PubMed2004

Characterization of soluble and putative membrane-bound UDP-glucuronic acid decarboxylase (OsUXS) isoforms in rice.

Arabinoxylans in crop plants are the major sugar components of the cell walls, and UDP-xylose is a key substrate in the biosynthesis of xylans. In this study, the six putative UDP-D-glucuronic acid decarboxylase genes from rice (Oryza sativa UDP-xylose synthase; OsUXS) were cloned. Except for the soluble form of OsUXS3 (GenBank Accession No. \AB079064), the remaining five OsUXS enzymes contain a putative membrane-bound region. The six OsUXS genes were classified into three types by phylogenetic analysis and were expressed during the development of rice seeds. The HPLC retention times of the enzyme products and NMR data, indicate that the recombinant OsUXS2 enzyme catalyzes the conversion of UDP-D-glucuronic acid to UDP-D-xylose. Interestingly, the reactions catalyzed by the recombinant OsUXS2 and OsUXS3 enzymes were inhibited by NADP+, and accelerated by NADPH. The catalytic activities of the recombinant OsUXS2 and OsUXS3 enzymes were strongly inhibited by UDP, UTP, TDP, and TTP. The expression levels of OsUXS genes changed in different manners during the development of rice seeds, suggesting that each corresponding OsUXS enzyme plays a significant role in rice seed development at a certain stage. In the present study, we report that the UXS2-type enzyme of rice is not only characterized for the first time but also show significant findings involved in the gene expression of OsUXSs.

Amino Acid Motifs↗
PubMed2004

High-coverage profiling analysis of genes expressed during rice seed development, using an improved amplified fragment length polymorphism technique.

A novel method, based on quantitative PCR and amplified fragment length polymorphism was applied to the analysis of high-coverage gene expression profiles during the development of rice seeds. This represents the first report of the application of this method to plants, which permitted the detection and analysis of approximately 70% of all the genes that are expressed in rice. The method was used to compare gene expression at different developmental stages, subspecies or cultivars, and phyletic lines to identify genes of interest through differences in their level of expression. Using this approach, even novel anonymous genes could be detected. Examples of these include the soluble starch synthase (SS) II-I and the rice branching enzyme 4 (rbe4) genes in the starch synthesis pathway. A profiling database was compiled and the results compared with public data on full-length cDNA sequences of rice. The method enables candidate novel genes to be immediately identified among the large numbers of genes that are expressed during the development of rice seeds. Our results will contribute to a better understanding of comparative transcriptomics in all plant species.

Gene Expression Profiling↗
PubMed2004

Structural characterization and biological activities of an exopolysaccharide kefiran produced by Lactobacillus kefiranofaciens WT-2B(T).

Lactobacillus kefiranofaciens, isolated from kefir grains, produces an extracellular polysaccharide when cultured, not only in PYG10 medium but also in a liquid medium containing a rice hydrolysate that had been previously degraded by treatment with a glucoamylase. The maximum yield of the polysaccharide, using the rice hydrolysate as the medium, was 2.5 g/L after a 7-day culture period at pH 5.0 and 33 degrees C. Compositional analysis, methylation analysis, specific rotation, and (1)H and (13)C NMR spectroscopy revealed that the structures of polysaccharides obtained from these two different culture media are essentially identical. The polysaccharide is composed of a hexasaccharide repeating unit and, thus, is known as kefiran. The weight-average molecular weight and the z-average radius of gyration of a sample, purified from the rice hydrolysate medium, were determined to be 7.6 x 10(5) g/mol and 39.9 nm, respectively, by gel permeation chromatography equipped with a multiangle laser-light-scattering photometer. Changes in blood pressure and serum components were examined in SHRSP/Hos rats, using doses of 100 and 300 mg of kefiran/kg of rat. A suppression in the increase in blood pressure was observed in these rats after 30 days. This activity is discussed in terms of the concentration of serum components of the rat, with emphasis on lipid components such as cholesterols, triglycerides, and free fatty acids.

Animals↗
PubMed2004

Isolation and partial characterization of fucan sulfates from the body wall of sea cucumber Stichopus japonicus and their ability to inhibit osteoclastogenesis.

Two types of fucan sulfate were isolated from chloroform/methanol extract of the body wall of the sea cucumber Stichopus japonicus. One type (type A) contained 3.41 mmol fucose/g and 2.35 mmol sulfate/g, and the molecular mass was determined to be 9 kDa by gel permeation chromatography (GPC). Structural analysis suggested that type A consists of a backbone of (1-->3)-linked fucosyl residues that are substituted at C-4 with fucosyl residues, and that fucosyl residues are sulfated at C-2 and/or C-4. Another type (type B) contained 3.90 mmol fucose/g and 3.07 mmol sulfate/g, and the molecular mass was determined to be 32kDa by GPC. Structural analysis showed that type B is largely composed of unbranched (1-->3)-linked fucosyl residues, and that sulfate substitution(s) occur at C-2 and/or C-4. The potential of both types to inhibit osteoclastogenesis was examined by an in vitro assay system, showing that both types of fucan sulfate inhibit osteoclastogenesis more than 95% at 50 microg/mL concentration. These results suggest that types A and B fucan sulfate from sea cucumber are potent inhibitors of osteoclastogenesis.

Animals↗
PubMed2003

Cloning and expression of a UDP-glucuronic acid decarboxylase gene in rice.

A cDNA fragment was cloned from rice immature seeds by the RT-PCR method. The deduced amino acid sequence of the cDNA showed a high degree of identity with UDP-d-glucuronic acid decarboxylase (UXS) from other plants and was most similar to the soluble UXS from Arabidopsis. The recombinant protein, expressed in an Escherichia coli system, catalysed the conversion of UDP-d-glucuronic acid to UDP-d-xylose, confirming that the gene encoded UXS. The uxs gene was expressed in mature, harvested rice seeds as well as in immature seeds 14 d post-anthesis, suggesting that the uxs gene is necessary at the beginning of the germination period. This is the first report of the cloning of the uxs gene from monocots.

Amino Acid Sequence↗
PubMed2003

Purification, characterization, and molecular cloning of a novel keratan sulfate hydrolase, endo-beta-N-acetylglucosaminidase, from Bacillus circulans.

Keratan sulfate (KS) is degraded by various enzymes including endo-beta-galactosidase, keratanase, and keratanase II, which are used for the structural analysis of KS. We purified a novel KS hydrolase, endo-beta-N-acetylglucosaminidase, from the cell pellet and conditioned medium of Bacillus circulans, by sequential chromatography using DE52 and phenyl-Sepharose columns with approximately 63- and 180-fold purity and 58 and 12.5% recovery, respectively. Like keratanase II of Bacillus sp. Ks36, the enzyme, designated Bc keratanase II, hydrolyzed KS between the 4GlcNAcbeta1-3Gal1 structure (endo-beta-N-acetylglucosaminidase), but not hyaluronan, heparan sulfate, heparin, and chondroitin sulfate C, demonstrating a strict specificity to KS. The enzyme digested shark cartilage KS to disaccharides and tetrasaccharides and bovine cornea KS to hexasaccharide, indicating that it prefers highly sulfated KS. Distinct from keratanase II of strain Ks36, the enzyme digested shark cartilage KS at an optimal temperature of 55 degrees C. Based on partial peptide sequencing of the enzyme, we molecularly cloned the gene, which encodes a protein with a predicted molecular mass of approximately 200 kDa. From the deduced protein sequence, Bc keratanase II contained a domain at the C terminus, homologous to the S-layer-like domain of pullulanase from Thermoanaerobacterium thermosulfurigenes and endoxylanase from Thermoanaerobacterium saccharolyticum, and a carbohydrate-binding domain, which may serve to specifically recognize KS chains. A full-length recombinant enzyme showed keratanase II activity. These results may prove useful for the structural analysis of KS toward achieving an understanding of its function.

Amino Acids↗
PubMed2003

Modification of di- and tetrasaccharides from shark cartilage keratan sulphate by refined anhydromethanolic hydrochloric acid-treatments and evaluation of their specific desulphation.

Highly sulphated keratan di- and tetrasaccharides were prepared from keratan sulphate (KS) of shark cartilage by enzymatic digestion with keratanase II and subsequent chromatography. The tetrasaccharide fraction carrying four sulphate groups was completely desulphated by 100 mM anhydromethanolic hydrochloric acid (MeOH-HCl) treatment at room temperature for 16 h. The conditions for the desulphation reaction by MeOH-HCl treatment were examined using sulphated keratan di- and tetrasaccharides as substrates by means of reversed phase high performance liquid chromatography (HPLC) and/or capillary electrophoresis, followed by the preparation of partially desulphated keratan oligosaccharides. Sulphate substitution patterns of monosulphated keratan disaccharide and trisulphated keratan tetrasaccharide were evaluated by methylation analysis. The results suggested that 6-O-sulphate groups of Gal moieties are cleaved faster than those of GlcNAc moieties under the present conditions adopted for the MeOH-HCl treatment of KS-derived oligosaccharides.

Acetylglucosaminidase↗
PubMed2002

Immunohistochemical localization of hemicelluloses and pectins varies during tissue development in the bamboo culm.

The distribution of hemicelluloses and pectins in bamboo internodes was studied immunocytochemistrically at various stages of development. The ultra-structures of bamboo cell walls have been reported previously at various stages. The internodes were identically classified into three developmental phases: primary wall stage (phase I), unlignified secondary wall stage (phase II) and lignified wall stage (phase III), using the same bamboo culm. (1-->3, 1-->4)-Beta-glucans were distributed in nearly all tissues in an actively elongating stage. Limited amounts of beta-glucans were deposited in primary walls and the middle lamellae, but were limited to the phloem in secondary walls. This suggests that the function of beta-glucans might be different in phloem vis-à-vis other tissues. Highly-substituted xylans were located in nearly all tissues of early phase I, but had disappeared in all tissues immediately prior to lignification. In contrast, low-branched xylan epitopes were present only in the protoxylem in phase I, but were present in all tissues immediately prior to lignification in phase II. In phase III, the epitopes were densely localized in lignified walls, suggesting that the substitution of xylans is closely related to maturation. Methyl-esterified (but not unesterified) pectins were present in all tissues of early phase I. Just before and after lignification, both types of pectins were concentrated in the phloem and protoxylem. Xyloglucans were largely distributed in the phloem and in lignified tissues, suggesting that they might be closely correlated with maturation. This represents the first account of the distribution of hemicelluloses and pectins at the tissue and ultrastructural level in bamboo internodes at various stages of development.

Bambusa↗
PubMed2002

Enhancement of t-PA-mediated plasminogen activation by partially defucosylated glycosaminoglycans from the sea cucumber Stichopus japonicus.

Sea cucumber glycosaminoglycan (SC-GAG) was isolated from the body wall of the sea cucumber Stichopus japonicus. The SC-GAG consists of a chondroitin sulfate E-type core polymer with sulfated fucose branches attaching glycosidically to almost every disaccharide unit of the core polymer at the C-3 position of the GlcA or at C-4 and/or C-6 position(s) of GalNAc. SC-GAG was subjected to mild acid-hydrolysis, which cleaved selectively the glycosidic linkages between the core polymer and the fucose branches, resulting in two types of partially defucosylated SC-GAG derivatives. One type (type A), obtained by 3 h-hydrolysis, contained 33% of the fucose branches and the other type (type B), obtained by 6-h hydrolysis, contained 10% of the fucose branches. The molecular masses of types A and B were determined to be 8 and 4 kDa, respectively, by gel permeation HPLC. A chondroitinase ABC (Chase ABC)-digestion demonstrated that types A and B contained 46 and 66% of digestable disaccharide units, respectively, and both types contained 29% of E-type unsaturated disaccharide units bearing no fucose branches. Intact SC-GAG and types A and B were compared for t-PA-mediated plasminogen activation by an in vitro assay system. Although intact SC-GAG and type B exhibited rather weak activity at 6.25 microg/ml, type A exhibited 5 to 10-fold higher activity than intact SC-GAG and type B at the same concentration. The activity of type A was almost one-third that of purified chondroitin sulfate E (127 kDa containing 64.5% E-type disaccharide units) from squid cartilage at 6.25 microg/ml concentration. These results suggest that t-PA-mediated plasminogen activation requires the presence of E-type disaccharide units bearing no fucose branches and a molecular mass larger than 7.5 kDa in terms of the chondroitin sulfate E structure with or without fucose branching.

Animals↗