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Brown algae contain a polysaccharide-rich cell wall, mainly composed of alginate and fucoidan which have been extensively studied for their individual structure and bioactivities. Particularly, the cell wall of Cladosiphon okamuranus is rich in fucoidan rather than alginate. However, little is known about its arrangement or interlinking with other polysaccharides such as cellulose in the cell wall. To determine its structure in detail, the cell wall was sequentially fractionated into five fractions: hot water (HW), ammonium oxalate, hemicellulose-I (HC-I), HC-II, and cellulose. Almost 80% of the total cell wall recovered from alcohol insoluble residue in C. okamuranus consisted of HW and HC-I, which mainly contained fucoidan composed of fucose, glucuronic acid, and sulfate in molar ratios of 1.0:0.3:0.9 and 1.0:0.2:0.3, respectively. Methylation analysis revealed that fucoidan in HW and HC-I structurally differed in terms of content of sulfate, and sugar residue which was 1,4-linked xylose and 1,4-linked fucose. Small angle X-ray scattering measurements also showed distinct conformational differences between HW and HC-I. These structural heterogeneities of fucoidan may be related to their localization, and fucoidan in HC-I may be involved in reinforcing cell wall structure by cross-linking to cellulose.

Root-knot nematodes (RKNs) are plant parasites and major agricultural pests. RKNs are thought to locate hosts through chemotaxis by sensing host-secreted chemoattractants; however, the structures and properties of these attractants are not well understood. Here, we describe a previously unknown RKN attractant from flaxseed mucilage that enhances infection of Arabidopsis and tomato, which resembles the pectic polysaccharide rhamnogalacturonan-I (RG-I). Fucose and galactose sidechains of the purified attractant were found to be required for attractant activity. Furthermore, the disaccharide α-l-galactosyl-1,3-l-rhamnose, which forms the linkage between the RG-I backbone and galactose sidechains of the purified attractant, was sufficient to attract RKN. These results show that the α-l-galactosyl-1,3-l-rhamnose linkage in the purified attractant from flaxseed mucilage is essential for RKN attraction. The present work also suggests that nematodes can detect environmental chemicals with high specificity, such as the presence of chiral centers and hydroxyl groups.

The brown alga, Cladosiphon okamuranus (Okinawa mozuku), is economically one of the most important edible seaweeds, and is cultivated for market primarily in Okinawa, Japan. C. okamuranus constitutes a significant source of fucoidan, which has various physiological and biological activities. To facilitate studies of seaweed biology, we decoded the draft genome of C. okamuranus S-strain. The genome size of C. okamuranus was estimated as ∼140 Mbp, smaller than genomes of two other brown algae, Ectocarpus siliculosus and Saccharina japonica. Sequencing with ∼100× coverage yielded an assembly of 541 scaffolds with N50 = 416 kbp. Together with transcriptomic data, we estimated that the C. okamuranus genome contains 13,640 protein-coding genes, approximately 94% of which have been confirmed with corresponding mRNAs. Comparisons with the E. siliculosus genome identified a set of C. okamuranus genes that encode enzymes involved in biosynthetic pathways for sulfated fucans and alginate biosynthesis. In addition, we identified C. okamuranus genes for enzymes involved in phlorotannin biosynthesis. The present decoding of the Cladosiphon okamuranus genome provides a platform for future studies of mozuku biology.

Plant cell walls are composed of polysaccharides such as cellulose, hemicelluloses, and pectins, whose location and function differ depending on plant type. Arabinose is a constituent of many different cell wall components, including pectic rhamnogalacturonan I (RG-I) and II (RG-II), glucuronoarabinoxylans (GAX), and arabinoxyloglucan (AXG). Arabinose is found predominantly in the furanose rather than in the thermodynamically more stable pyranose form. The UDP-arabinopyranose mutases (UAMs) have been demonstrated to convert UDP-arabinopyranose (UDP-Arap) to UDP-arabinofuranose (UDP-Araf) in rice (Oryza sativa L.). The UAMs have been implicated in polysaccharide biosynthesis and developmental processes. Arabinose residues could be a component of many polysaccharides, including branched (1→5)-α-arabinans, arabinogalactans in pectic polysaccharides, and arabinoxyloglucans, which are abundant in the cell walls of solanaceous plants. Therefore, to elucidate the role of UAMs and arabinan side chains, we analyzed the UAM RNA interference transformants in tobacco (Nicotiana tabacum L.). The tobacco UAM gene family consists of four members. We generated RNAi transformants (NtUAM-KD) to down-regulate all four of the UAM members. The NtUAM-KD showed abnormal leaf development in the form of a callus-like structure and many holes in the leaf epidermis. A clear reduction in the pectic arabinan content was observed in the tissue of the NtUAM-KD leaf. The arabinose/xylose ratio in the xyloglucan-rich cell wall fraction was drastically reduced in NtUAM-KD. These results suggest that UAMs are required for Ara side chain biosynthesis in both RG-I and AXG in Solanaceae plants, and that arabinan-mediated cell wall networks might be important for normal leaf expansion.

A novel α-glucan substituted rare 6-deoxy-D-altropyranose was isolated from edible fruiting bodies of a mushroom (Lactarius lividatus) grown in Okinawa, Japan. The polysaccharide consists of D-glucose, D-galactose and 6-deoxy-D-altrose in a molar ratio of 3.0:1.0:1.0. The specific rotation [α](589) was estimated as +64.3° (0.2% in water) at 25 °C. Based on results of IR, NMR ((1)H, (13)C, 2D-COSY, 2D-HMQC, 2D-ROESY and 2D-HMBC), and methylation analyses, the structure of the polysaccharide was determined as [formula, see text] This work is the first demonstration of rare 6-deoxy-D-altropyranose moiety on polysaccharides.

β-Glucosidases designated MoCel3A and MoCel3B were successfully overexpressed in Magnaporthe oryzae. MoCel3A and MoCel3B showed optimal activity at 50 °C and pH 5.0-5.5. MoCel3A exhibited higher activity on higher degree of polymerization (DP) oligosaccharides and on β-1,3-linked oligosaccharides than on β-1,4-linked oligosaccharides. Furthermore, MoCel3A could liberate glucose from polysaccharides such as laminarin, 1,3-1,4-β-glucan, phosphoric acid-swollen cellulose, and pustulan, of which laminarin was the most suitable substrate. Conversely, MoCel3B preferentially hydrolyzed lower DP oligosaccharides such as cellobiose, cellotriose, and laminaribiose. Furthermore, the synergistic effects of combining enzymes including MoCel3A and MoCel3B were investigated. Depolymerization of 1,3-1,4-β-glucan by M. oryzae cellobiohydrolase (MoCel6A) enhanced the production of glucose by the actions of MoCel3A and MoCel3B. In these reactions, MoCel3A hydrolyzed higher DP oligosaccharides, resulting in the release of glucose and cellobiose, and MoCel3B preferentially hydrolyzed lower DP oligosaccharides including cellobiose. On the other hand, MoCel3A alone produced glucose from laminarin at levels equivalent to 80% of maximal hydrolysis obtained by the combined action of MoCel3A, MoCel3B, and endo-1,3-β-glucanase. Therefore, MoCel3A and MoCel3B activities yield glucose from not only cellulosic materials but also hemicellulosic polysaccharides.

沖縄県北中城村で養殖されているヒトエグサ（Mono-stroma nitidum）より多糖を抽出し，化学および構造特性を明らかにした．精製多糖の全糖量，ウロン酸含有量，灰分，水分および硫酸含量はそれぞれ67.2％，11.8％，27.3％，3.5％および24.7％であった．加水分解物の液体クロマトグラフィーの結果より，L－ラムノースを主構成糖とする多糖であることが確認された．化学分析の結果よりラムナン硫酸の構成糖と硫酸基の比はL－Rha : D－GlcA : D－Xyl : D－Glc : D－Gal : SO3＝7.0 : 1.0 : 0.5 : 0.1 : 0.5 : 5.0と算出された．また，メチル化分析により2, 3，4－トリ－O－メチル－D－グルクロン酸， 3, 4－ジ－O－メチル－L－ラムノース， 2, 4－ジ－O－L－ラムノース， 2－モノ－L－ラムノースおよび4－モノ－O－メチル－L－ラムノースを同定した． 1Hおよび13C－NMRスペクトルよりL－ラムノースとD－グルクロン酸の存在が確認され，養殖ヒトエグサより分離した多糖はラムナン硫酸であると同定した．メチル化分析および1H－NMR解析により，養殖ヒトエグサより分離したラムナン硫酸の化学構造は，α－1, 3結合のL－ラムノースを主鎖とし， 側鎖にα－1, 2結合のL－ラムノース（2モル）およびD－グルクロン酸（1モル）が置換することが考えられた． また， 硫酸基はL－ラムノース残基のC4（主鎖）とC2（側鎖）に置換することが明らかになった．ラムナン硫酸の化学構造を推定した．

Three GH-6 family cellobiohydrolases are expected in the genome of Magnaporthe grisea based on the complete genome sequence. Here, we demonstrate the properties, kinetics, and substrate specificities of a Magnaporthe oryzae GH-6 family cellobiohydrolase (MoCel6A). In addition, the effect of cellobiose on MoCel6A activity was also investigated. MoCel6A contiguously fused to a histidine tag was overexpressed in M. oryzae and purified by affinity chromatography. MoCel6A showed higher hydrolytic activities on phosphoric acid-swollen cellulose (PSC), β-glucan, and cellooligosaccharide derivatives than on cellulose, of which the best substrates were cellooligosaccharides. A tandemly aligned cellulose binding domain (CBD) at the N terminus caused increased activity on cellulose and PSC, whereas deletion of the CBD (catalytic domain only) showed decreased activity on cellulose. MoCel6A hydrolysis of cellooligosaccharides and sulforhodamine-conjugated cellooligosaccharides was not inhibited by exogenously adding cellobiose up to 438 mM, which, rather, enhanced activity, whereas a GH-7 family cellobiohydrolase from M. oryzae (MoCel7A) was severely inhibited by more than 29 mM cellobiose. Furthermore, we assessed the effects of cellobiose on hydrolytic activities using MoCel6A and Trichoderma reesei cellobiohydrolase (TrCel6A), which were prepared in Aspergillus oryzae . MoCel6A showed increased hydrolysis of cellopentaose used as a substrate in the presence of 292 mM cellobiose at pH 4.5 and pH 6.0, and enhanced activity disappeared at pH 9.0. In contrast, TrCel6A exhibited slightly increased hydrolysis at pH 4.5, and hydrolysis was severely inhibited at pH 9.0. These results suggest that enhancement or inhibition of hydrolytic activities by cellobiose is dependent on the reaction mixture pH.

Background : A brown seaweed, Saccharina longissima ( Laminaria angustata var. longissima ) has been consumed in Okinawa for 300 years. It is native to the southernmost part of Japan, in Okinawa, but grows on the Pacific coast (Kushiro and Nemuro) in Hokkaido in the northernmost part of Japan. Previously, we reported the structural characteristics from S. longissima . The fucoidan consisted of L-fucose, D-galactose, D-glucose, D-xylose, D-glucuronic acid, and sulfuric acid. This fucoidan activated the RAW 264.7 murine macrophage cell line. The aim of this study was to investigate the effects of a fucoidan isolated from Saccharina longissima on cell anti-proliferation and apoptosis in the U937 human leukemia monocyte lymphoma cell line. Methods : U937 cells were incubated with four fucoidan concentrations. The degree of apoptosis was determined using the APOPercentage APOPtosis assay, caspaer-3 activity assay and Western blotting analysis assay. Results : The treatment of cell with a fucoidan highly substituted with sulfate, in addition to L-fucose residues, inhibited cell growth. Apoptosis increased in U937 cells in a dose-dependent manner following the addition of fucoidan. However, the fucoidan did not induce apoptosis in the presence of caspase inhibitor (z-VAD-FMK). Conclusions : The fucoidan from Saccharina longissima induced apoptosis in U937 cells. Accordingly, the kelp containing this fucoidan may contribute to the observed longevity of the Okinawan population. Keywords: fucoidan, Saccharina longissimi, brown seaweed, traditional food, human leukemia cells, apoptosis

Plants use UDP-arabinofuranose (UDP-Araf) to donate Araf residues in the biosynthesis of Araf-containing complex carbohydrates. UDP-Araf itself is formed from UDP-arabinopyranose (UDP-Arap) by UDP-arabinopyranose mutase (UAM). However, the mechanism by which this enzyme catalyzes the interconversion of UDP-Arap and UDP-Araf has not been determined. To gain insight into this reaction, functionally recombinant rUAMs were reacted with UDP-Glc or UDP-Araf. The glycosylated recombinant UAMs were fragmented with trypsin, and the glycopeptides formed were then identified and sequenced by LC-MS/MS. The results of these experiments, together with site-directed mutagenesis studies, suggest that in functional UAMs an arginyl residue is reversibly glycosylated with a single glycosyl residue, and that this residue is required for mutase activity. We also provide evidence that a DXD motif is required for catalytic activity.

The non-Newtonian behavior and storage modulus of an alginate isolated from Nemacystus decipiens which had been harvested in Uruma City (Okinawa, Japan) in aqueous solutions were investigated. Gelation occurred at a concentration of 0.5% of the alginate on addition of a mixed solution of NaCl (0.1%), KCl (0.1%) and CaCl2 (0.1%) at room temperature (25°C). The flow curves, at 35°C, of the alginate showed plastic behavior at various concentrations. The storage modulus of the alginate increased with an increase in concentration and decreased gradually with an increase in temperature up to 50°C, which was estimated to be a transition temperature; then it, on the contrary, increased gradually with a further increase in the temperature and showed the maximum value at a temperature of 60, 70 and 65°C at 0.3, 0.5 and 0.7%, respectively. The storage modulus of the alginate showed a large value even in the presence of urea (4.0 M). The storage modulus of the alginate showed a very large value at the pH range of 6.0-7.2, but very small at pH 4.0. However, a comparable large storage modulus was observed in the alkaline range (pH 9.0-11.0). Intra- and intermolecular Ca2+-bridges may play a dominant role in the gel formation of alginate molecules in the presence of Ca2+ ions with ionic bonding and electrostatic forces of attraction. The hydrophobic interaction may take place on the L-guluronic acid-rich homopolymeric blocks of the alginate molecules in the high temperature range.

The 1H-nuclear magnetic resonance spectrum showed that the l-rhamnosyl residues of native gellan gum were coinvolved in both a small number of 4C1-pyranose conformations and a large number of 1C4-pyranose conformations, whereas for deacylated polymer, almost of the residues were involved in 4C1-pyranose conformation. The flow curves of native gellan gum showed plastic behavior above 0.2%. The elastic modulus stayed at a constant value with increase in temperature up to 40 °C, then decreased rapidly. The elastic modulus increased with addition of CaCl2 (6.8 mM) and stayed constant value with increase in temperature up to 65 °C, then decreased rapidly. The stronger elastic modulus was observed in deacylated gellan gum with addition of CaCl2. The elastic modulus of native gellan gum showed larger value than that in aqueous solution in the presence of urea (4.0 M). Intra- and intermolecular associations of native gellan gum molecules in the presence of Ca+2 were proposed.

The non-Newtonian behavior and dynamic viscoelasticity of Halberd wheat starch (23.0% amylose content) pastes were determined with a rheogoniometer. The flow curves, at 25°C, of Halberd starch pastes showed plastic behavior at concentrations of more than 4.0%. The shear viscosity of the starch pastes decreased gradually with an increase in temperature at concentrations less than 4.0%, but remained constant at a concentration of 5.0%. The storage modulus of the starch pastes increased with increasing concentration and remained high during increase in temperature up to 80°C. Almost the same storage modulus was observed upon addition of urea (4.0 M) to the paste at low temperature (0°C), and the modulus stayed at a large value with increase in temperature up to 60°C, which was estimated to be a transition temperature; then it decreased rapidly with further temperature increase. A transition temperature of 60°C was also observed in the dynamic viscosity. A very large storage modulus and dynamic viscosity were observed in alkaline solution (0.05 M NaOH) at low temperature (0°C), both quantities decreased slightly with increase in temperature up to 25°C, which was estimated to be a transition temperature, then decreased rapidly with further increase in temperature. A possible intermolecular hydrogen bonding between amylose and amylopectin molecules is proposed.

Plant cell walls undergo dynamic changes during plant growth and development. Although the cell wall remodeling is an essential feature of plant growth and development, the biosynthesis mechanisms are poorly understood. Arabinan is a pectic polysaccharide which is linked by arabinofuranosyl (Araƒ) residues with α-(1,5) linkage. Arabinofuranosyl residues are a quantifiably important constituent of plant primary and secondary cell walls. Plants use UDP-arabinofuranose (UDP-Araƒ) to synthesize Araƒ regions of the polysaccharides containing Araƒ residues including proteoglycans and glycoproteins. However, it is unknown how UDP-Araƒ is synthesized in plant cells. We succeeded to, for the first time, determine UDP-arabinopyranose mutase (UAM) activity and clone the gene of the enzyme from rice seedlings. UAM catalyzed the interconversion of UDP-arabinopyranose (UDP-Arap) to UDP-Araƒ. Microbial UDP-galactose mutases require reduced FAD for activity, however, the plant UAM do not require the cofactor. Thus, the plant mutase must have different catalytic mechanism. Three proteins were identified from partial amino acid sequence of UAM, which are encoded by Os03g40270, Os04g56520 and Os07g41360. These proteins have more than 80% sequence identity with reversibly glycosylated polypeptide. UAM genes are present in Chlamydomonas, Physcomitrella and pine, suggesting that UAM is widespread in green plants.

A pectin was extracted from the peel of Citrus tankan with a yield of 2.75%. The uronic acid content was 80.0%, and the degree of methoxylation was 63.2%. The pectin was composed of D-GalA, D-Gal, L-Ara and L-Rha in the molar ratio of 100:11.3:3.6:2.6. The molecular weight was estimated to be approximately 9.2 x 10(4). The pectin formed a gel by conventional procedures.

A polysaccharide was extracted with hot HCl from the endocarp of Citrus depressa grown in Okinawa, Japan. The yield was 4.1% on a fresh weight basis. Composition of the polysaccharide was 89.3% total carbohydrate, 79.2% uronic acid, 4.1% ash and 8.8% moisture. The degree of methoxylation was estimated to be 66.2%. The polysaccharide was composed of d -GalA, d -Gal, l -Ara and l -Rha in the ratio of 100:10.3:1.53:0.94, respectively. The molecular mass was estimated to be approximately 4.1 × 10 4 . NMR spectra indicated that the polysaccharide was mainly composed of (1→4)-linked α- d -GalA and (1→4)-linked β- d -Gal. Methylation analysis results identified 1→2 and 1→2,4 linked Rha, 1→4, 1→3, 1→6 and 1→3,6 linked Gal. The results indicated that the polysaccharide was a pectin, which was relatively simpler than other pectins. The polysaccharide was classified as high-methoxyl pectin. The pectin turned into a gel by the generalised method.

We investigated the structure of fucoidan from Hizikia fusiformis. Using DEAE-Sepharose column chromatography, fucoidan was fractionated into four groups. F4, main fraction of fucoidan, contained 38 % (w/w) of ester sulfate and had the simplest sugar component (L-Fuc: D-Gal=7:3). The structure of F4 was discussed using de-sulfation, 2D-NMR and methylation analysis and proposed as [→3)-α-L-Fucp-(1→3)-α-L-Fucp-(1→3)-α-L-Fucp-(1→3)-α-L-Fucp-(1→]nR=α-L-Fucp-(1→4)-α-L-Fucp-(1→ (50%a) or α-D-Galp-(1→4)-α-D-Galp-(1→ (50 %a).58 % in molar ratio of the hydroxyl group is sulfated.

Common reed (Phragmites australis) is a well known salt-tolerant plant and it is suggested that reeds recover Na(+) in the xylem sap of the shoot base (basal part of the shoot), store it temporarily in the shoot base, release it into the phloem sap, and then retranslocate it to the roots. To investigate whether Na(+) is retained in the shoot base of reeds, confocal laser scanning microscope (CLSM) observations were conducted using an intracellular Na(+)-specific fluorescent probe. The CLSM observations revealed that reeds produced a large number of the starch granules at the shoot base when salt-stressed, and that the fluorescence indicating the location of intracellular free Na(+) was observed in the same position as the starch granules. The Na content of starch granules was considerably greater than that of the shoot base, whereas the potassium (K) contents of the granules was only slightly greater than that of the shoot base. Reeds produced Na(+)-binding starch granules in the parenchyma cells of the shoot base when salt-stressed; these starch granules may decrease intracellular free Na(+). It is proposed that the site-specific production of Na(+)-binding starch granules constitutes a novel salt tolerance mechanism.

Fucoidan from Cladosiphon okamuranus and its sulfate derivatives were prepared. Sulfate contents of native and oversulfated fucoidan were estimated to be 13.5% and 32.8%, respectively. The results of 1 H NMR suggest that 2,4-di- O -sulfo-, 2-mono- O -sulfo- and 4-mono- O -sulfo- l -fucopyranose were involved in oversulfated fucoidan and 4-mono- O -sulfo- l -fucopyranose was involved in native fucoidan. The oversulfated fucoidan reduced the proliferation of U937 cells in a dose-dependent manner, but the activity of native fucoidan was weak. The sulfate content and substituting position of sulfate group might be important factors of anti-proliferative activity in U937 cells. To examine whether the anti-proliferative activity of oversulfated fucoidan was caused by induction of apoptosis, apoptosis assay, caspase-3 activity assay and Western blotting analysis were performed. These results indicated that the oversulfated fucoidan induced apoptosis via caspase-3 and -7 activation-dependent pathway.

Plant cell walls constitute the bulk of the earth renewable source of energy and are a component in the diet of humans and herbivores. l-Arabinofuranosyl (Araf) residues are a quantifiably important constituent of these walls. Plants use uridine diphosphate (UDP)-l-arabinofuranose (UDP-Araf) to donate Araf residues in the biosynthesis of Araf-containing polysaccharides, proteoglycans, and glycoproteins. However, little is known about the formation of UDP-Araf. We now describe the purification and partial characterization of a rice UDP-arabinopyranose mutase (UAM) that catalyzes the formation of UDP-Araf from UDP-arabinopyranose (UDP-Arap). The reaction is reversible and at thermodynamic equilibrium the pyranose form is favored over the furanose form (90 : 10). Three related proteins that are encoded by rice gene loci Os03g40270, Os04g56520, and Os07g41360 were identified from partial amino acid sequences of UAM. These proteins have >80% sequence identity with polypeptides that are reversibly glycosylated in the presence of UDP-sugars. The rice mutase and two functionally active recombinant mutases were shown to be reversibly glycosylated in the presence of UDP-Glc. The cofactor, flavin-adenine-dinucleotide (FAD), is required for the catalytic activity of UDP-galactose mutases of prokaryotes, fungi, and protozoa. The plant mutases, which do not require a cofactor, must therefore have a different catalytic mechanism. Putative UAM-encoding genes are present in the green algae Chlamydomonas reinhardtii, the moss Physcomitrella patens, the gymnosperm Pinus taeda (loblolly pine), and in numerous dicots and monocots, indicating that UAMs are widespread in green plants.

Arabinofuranosyltransferase activity was identified in Golgi membranes obtained from mung bean (Vigna radiata) hypocotyls. The enzyme transfers the arabinofuranosyl (Araf) residue from UDP-β-l-arabinofuranose to exogenous (1, 5)-linked α-l-arabino-oligosaccharides labeled at their reducing ends with 2-aminobenzamide. The transferred residue was shown, using 1H-nuclear magnetic resonance spectroscopy and α-l-arabinofuranosidase treatment, to be α-l-Araf and to be linked to O-5 of the nonreducing terminal Araf residue of the acceptor oligosaccharide. The enzyme was nonprocessive because only a single Araf residue was added to the acceptor molecule. Arabino-oligosaccharides with a degree of polymerization between 3 and 8 were acceptor substrates. The 2-aminobenzamide-labeled arabino-tetra- and pentasaccharides were the most effective acceptor substrates analyzed. The enzyme has a pH optimum between 6.5 and 7.0 and its activity is stimulated by Mn2+ and Co2+ ions. The apparent Km and Vmax values of the arabinofuranosyltransferase for UDP-arabinofuranose are 243 μm and 243 pmol min−1 mg protein−1, respectively. The highest enzyme activity was detected in the elongating regions of mung bean hypocotyls. The data show that UDP-arabinofuranose is the donor molecule for the generation of arabino-oligosaccharides composed of Araf residues.

A xyloglucan was isolated from the cell-walls of eggplant. This xyloglucan was hydrolyzed with a xyloglucan specific endo-1,4-β-D-glucanase (xyloglucanase) from Penicillium sp. M451. The resulting hydrolyzate was fractionated by Bio-Gel P-2 chromatography. Hexa-, hepta- and octa-saccharide fractions were obtained in a ratio of 35:43:22 as major oligosaccharide fractions. The xyloglucan oligosaccharide fractions were separately subjected to sugar composition and methylation analyses, high-performance anion exchange chromatography with pulsed amperometric detection, and matrix-assisted laser-desorption ionization time of the flight mass spectrometry before and after treatment with an isoprimeverose-producing oligoxyloglucan hydrolase purified from Eupenicillium sp. M9 or with β-D-galactosidase from Bacillus circulans. Hexa-, hepta- and octa-saccharide fractions might consist mainly of XXGG, of XLGG, LXGG and XSGG, and of LSGG and LLGG, respectively. In addition, the oligosaccharide units of xyloglucans from some solanaceous vegetables were comparatively analyzed. Xyloglucans from the cell-walls of sweet pepper, red pepper and tomato belonging to solanaceous vegetables were shown to be composed of hexa-, hepta- and octa-saccharide fractions in a ratio of 40:37:23, 41:34:25, and 45:37:18, respectively.

Arabidopsis genome projects have led to the discovery of 46 cellulose-synthase-like genes. We believe that some of these genes encode other glucosyltransferases, such as xyloglucan 4-β-glucosyltransferase. In order to define and to identify the gene products, we developed a heterogeneous expression system in Pichia pastoris and COS cells. This system is useful for directly determining the activity of xyloglucan and cellulose synthases from the recombinant proteins. The expression vectors contained a complementary T base for TA cloning at the cloning site and signals for secretion with antibody recognition attached to the structural gene inserted. The gene products of sucrose synthase (sucrose UDP-glucose glucosyltransferase) cDNA was detected by Western blot analysis and by measuring sucrose synthase activity.

Callose is formed from exogenous sucrose by cotton fiber microsomal membranes that contain both sucrose synthase and callose synthase activity. Although the coupled reaction between sucrose and callose synthases occurs predominantly to channel glucose from sucrosederived uridine diphosphate (UDP)-glucose into callose in the membranes, there is no difference in the UDP-glucose-forming/sucrose-forming activity ratios between the soluble and membrane-bound forms of sucrose synthase. The consumption of UDP-glucose from sucrose into callose probably leads to UDP-glucose formation rather than sucrose formation despite the lower affinity of sucrose synthase for sucrose than for UDP-glucose. Callose formation is markedly stimulated by the addition of either recombinant Glu11 (S11E) or in vitro phosphorylated Ser11 mung bean sucrose synthase but not by the wild-type nonphosphorylated Ser11 enzyme. We propose that a negative charge (by phosphorylation or mutagenesis) at Ser11 in sucrose synthase causes the enzyme to promote a coupled callose-forming reaction.

Chlamydomonas reinhardtii cells are surrounded by a mixture of hydroxyprolin-rich glycoproteins consisting of L-arabinose, D-galactose, D-glucose, and D-mannose residues. The L-arabinose residue is thought to be attached by a transfer of UDP-L-arabinofuranose (UDP-Araf), which is produced from UDP-L-arabinopyranose (UDP-Arap) by UDP-arabinopyranose mutase (UAM). UAM was purified from the cytosol to determine the involvement of C. reinhardtii UAM (CrUAM) in glycoprotein synthesis. CrUAM was purified 94-fold to electrophoretic homogeneity by hydrophobic and size-exclusion chromatography. CrUAM catalyzed the reversible conversion between UDP-Arap and UDP-Araf and exhibited autoglycosylation activity when UDP-D-[(14)C]glucose was added as substrate. Compared to the properties of native and recombinant CrUAM overexpressed in Escherichia coli, native CrUAM showed a higher affinity for UDP-Arap than recombinant CrUAM did. This increased affinity for UDP-Arap might have been caused by post-translational modifications that occur in eukaryotes but not in prokaryotes.

The green seaweed, Monostroma nitidum, is widespread in Japan. In Okinawa Prefecture, the production of seaweed is performed using culture-nets that are seeded artificially. Algae contain a soluble polysaccharide, rhamnan sulfate. To estimate its applicability, the anticoagulant activity of rhamnan sulfate was investigated. Rhamanan sulfate was fractionated by ion-exchange chromatography on a DEAE-sepharose column, and two fractions (A and B) were obtained. Partially hydrolyzed rhamnan sulfates with different molecular mass (C1, C2 and C3) were also prepared. The activated partial thromboplastin time (APTT) test, prothrombin time (PT) and thrombin time (TT) were applied using human plasma and compared with standard heparin (174 units/mg). The native rhamnan sulfate (molecular mass, 630 kDa; sulfuric acid content, 22.7%), fraction A (12.4%) and fraction B (27.8%) showed approximately 73% APTT activity in comparison with that of standard heparin, but fraction C2 (molecular mass, 450 kDa) had a higher activity than that of the standard (107%). On the other hand, in the PT assay, all fractions except fraction C2 and C3 (370 kDa) showed higher activity approximately 120-155% greater than that of standard heparin. The TT activity of rhamnan sulfate depended on the sulfate content, and that of fraction B, which has high sulfuric acid content (27.8%), was 135-173% greater than that of heparin. The sulfate groups of L-rhamnosyl residues and carboxyl group of D-glucuronosyl residue on the trisaccharide side chains of the rhamnan sulfate might interact strongly with the active site of thrombin molecules. The results and discussion suggested that rhamnan sulfate from commercially cultured Monostroma nitidum could be a potential anticoagulant polysaccharide.

6-Deoxy- l -altrose is well known as a constituent sugar moiety of lipopolysaccharides in Gram-negative bacteria. However, its isomer, 6-deoxy- d -altrose, is little known. Identification of 6-deoxy- d -altrose isolated from a polysaccharide extracted from an edible mushroom ( Lactarius lividatus ), its comparison with chemically synthesized 6-deoxy- d -altrose using 1 H and 13 C NMR including COSY, HMQC spectroscopy, and investigation of its specific optical rotation were all conducted in this study. The 6-deoxy-hexose isolated from acid hydrolysate of the polysaccharide extracted from L. lividatus was involved in four anomeric isomers (α-pyranose and β-pyranose, and α-furanose and β-furanose), as was chemically synthesized 6-deoxy- d -altrose in an aqueous solution because of mutarotation. Almost all signals of 1D ( 1 H NMR and 13 C NMR) and 2D (COSY and HMQC)-NMR spectra agreed with those of the authentic 6-deoxy- d -altrose. The specific optical rotation [ α ] 589 of 6-deoxy-sugar showed a value of +18.2°, which was in agreement with that of authentic 6-deoxy- d -altrose. Consequently, 6-deoxy-hexose was identified as the 6-deoxy- d -altrose. This work is the first complete identification of 6-deoxy- d -altrose in a natural environment.

The flow behavior, dynamic viscoelasticity, and optical rotation of aqueous solutions of wheat amylose were measured using a rheogoniometer and a polarimeter. The amylose solutions, at 25 °C, showed shear-thinning behavior at a concentration of 1.2%, but plastic behavior at 1.4 and 1.6%, the yield values of which were estimated to be 0.6 and 1.0 Pa, respectively. The viscosity of the wheat amylose increased a little with increase in temperature up to 10 or 20 °C at 1.2% or 1.4 and 1.6%, which was estimated to be a transition temperature. The elastic modulus increased with increase in concentration, and increased with increasing temperature up to 20, 25 and 30 °C, which was estimated to be a transition temperature, respectively, then decreased gradually but stayed at a large value even at high temperature (80 °C). A very low elastic modulus of the wheat amylose was observed upon addition of urea (4.0 M) and in alkaline solution (0.05 M NaOH) even at low temperature. The optical rotation of wheat amylose solution increased a little with decreasing temperature down to 25 °C, then increased rapidly with further decrease in the temperature. The mode of gelation mechanism of amylose molecules, which was previously proposed, was confirmed and a retrogradation mechanism of wheat amylose was proposed.

We investigated a structural characteristics of acetyl fucoidan (CAF) isolated from commercially cultured Cladosiphon okamuranus. The CAF-induced macrophage activation and its signaling pathways in murine macrophage cell line, RAW 264.7 were also investigated. From the results of methylation analysis, CAF consisted of alpha-1--3 linked L: -fucosyl residues and substituted sulfate and acetyl groups at C-4 on the main chain. CAF induced production of nitric oxide (NO), tumor necrosis factor-alpha and interleukin-6 in RAW 264.7 cells. Sulfate and acetyl groups of CAF involved in CAF-induced NO production. Neutralizing anti-Toll-like receptor 4 (TLR4), anti-CD14 and anti-scavenger receptor class A (SRA) but not anti-complement receptor type 3 monoclonal antibodies decreased CAF-induced NO production. The results of immunoblot analysis indicated that CAF activated mitogen-activated protein kinases (MAPKs) such as p38 MAPK, extracellular signal-regulated kinase (ERK)1/2 and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK). SB203580 (p38 MAPK inhibitor) and SP600125 (SAPK/JNK inhibitor), but not U0126 (MAPK/ERK kinase 1/2 inhibitor) decreased CAF-induced NO production. The results suggested that CAF induced macrophage activation through membrane receptors TLR4, CD14 and SRA, and MAPK signaling pathways.

Many bacterial genomes contain a cellulose synthase operon together with a cellulase gene, indicating that cellulase is required for cellulose biosynthesis. In higher plants, there is evidence that cell growth is enhanced by the overexpression of cellulase and prevented by its suppression. Cellulase overexpression could modify cell walls not only by trimming off the paracrystalline sites of cellulose microfibrils, but also by releasing xyloglucan tethers between the microfibrils. Mutants for membrane-anchored cellulase (Korrigan) also show a typical phenotype of prevention of cellulose biosynthesis in tissues. All plant cellulases belong to family 9, which endohydrolyzes cellulose, but are not strong enough to cause the bulk degradation of cellulose microfibrils in a plant body. It is hypothesized that cellulase participates primarily in repairing or arranging cellulose microfibrils during cellulose biosynthesis in plants. A scheme for the roles of plant cellulose and cellulases is proposed.

Arabinopyranosyltransferase (Ara p T) activity that results in the transfer of a single arabinopyranose (Ara p ) residue from UDP-β- l -arabinopyranose (UDP-Ara p ) to exogenous (1 → 5)-linked α- l -arabino-oligosaccharides labeled with 2-aminobenzamide (2-AB) at their reducing ends was identified in a particulate preparation obtained from 3-day-old mung bean ( Vigna radiate L. Wilezek) hypocotyls. The transferred Ara residue was shown to be β-(1 → 3)-linked to O -3 of the non-reducing terminal Ara f residues of the oligosaccharide using nuclear magnetic resonance spectroscopy together with glycosyl composition and glycosyl linkage composition analyses. The 2AB-labeled arabino-octasaccharide was the most effective acceptor substrate analyzed, although arabino-oligosaccharides with a degree of polymerization between 4 and 7 were also acceptor substrates. Maximum Ara p T activity was obtained at pH 6.5–7.0, and 20 °C in the presence of 25 mM Mn 2+ and 0.5% Triton X-100.

Sucrose (Suc) synthase (SuSy) is believed to function in channeling UDP-Glc from Suc to various β-glucan synthases. We produced transgenic poplars (Populus alba) overexpressing a mutant form (S11E) of mung bean (Vigna radiata) SuSy, which appeared in part in the microsomal membranes of the stems. Expression of SuSy in these membranes enhanced the incorporation of radioactive Suc into cellulose, together with the metabolic recycling of fructose (Fru), when dual-labeled Suc was fed directly into the phloem of the leaf. This overexpression also enhanced the direct incorporation of the glucosyl moiety of Suc into the glucan backbone of xyloglucan and increased recycling of Fru, although the Fru recycling system for cellulose synthesis at the plasma membrane might differ from that for xyloglucan synthesis in the Golgi network. These findings suggest that some of the Suc loaded into the phloem of a poplar leaf is used directly by SuSys associated with xyloglucan and cellulose synthases in the stem. This may be a key function of SuSy because the high-energy bond between the Glc and Fru moieties of Suc is conserved and used for polysaccharide syntheses in this sink tissue.

A method for rapidly identifying six of the most commonly found xyloglucan oligosaccharide units, XXXG, XLXG, XXLG, XLLG, XXFG, and XLFG was developed by high-performance anion exchange chromatography (HPAEC) with pulsed amperometric detection (PAD) before and after digestion with purified isoprimeverose-producing oligoxyloglucan hydrolase (IPase). Using this method, the compositions of oligosaccharide units of soybean and mung bean xyloglucans were re-examined. Significant amounts of oligosaccharides that have not previously been reported to be oligosaccharide units of soybean and mung bean xyloglucans were found.

Higher plants efficiently conserve energy ATP in cellulose biosynthesis by expression of sucrose synthase, in which the high free energy between glucose and fructose in sucrose can be conserved and used for the synthesis of UDP-glucose. A mixture of sucrose synthase and bacterial cellulose synthase proceeded to form UDP-glucose from sucrose plus UDP and to synthesize 1,4-β-glucan from the sugar nucleotide. The mutant sucrose synthase, which mimics phosphorylated sucrose synthase, enhanced the reaction efficiency ( V max / K m ) on 1,4-β-glucan synthesis, in which the incorporation of glucose from sucrose was increased at low concentrations of UDP. Because UDP formed after glucosyl transfer can be directly recycled with sucrose synthase, UDP-glucose formed appears to show high turnover with cellulose synthase in the coupled reaction. The expression of sucrose synthase in Acetobacter xylinum not only changed sucrose metabolism but also enhanced cellulose production, in which UDP-glucose was efficiently formed from sucrose. Although the level of UDP-glucose in the transformant with mutant sucrose synthase cDNA was only 1.6-fold higher than that in plasmid-free cells, the level of UDP was markedly decreased in the transformant. The results show that sucrose synthase serves to channel carbon directly from sucrose to cellulose and recycles UDP, which prevents UDP build-up in cellulose biosynthesis.

Bud dormancy is an adaptive strategy that perennials use to survive unfavorable conditions. Gentians (Gentiana), popular alpine flowers and ornamentals, produce overwintering buds (OWBs) that can persist through the winter, but the mechanisms regulating dormancy are currently unclear. In this study, we conducted targeted metabolome analysis to obtain clues about the metabolic mechanisms involved in regulating OWB dormancy. Multivariate analysis of metabolite profiles revealed metabolite patterns characteristic of dormant states. The concentrations of gentiobiose [β-d-Glcp-(1→6)-d-Glc] and gentianose [β-d-Glcp-(1→6)-d-Glc-(1→2)-d-Fru] significantly varied depending on the stage of OWB dormancy, and the gentiobiose concentration increased prior to budbreak. Both activation of invertase and inactivation of β-glucosidase resulted in gentiobiose accumulation in ecodormant OWBs, suggesting that gentiobiose is seldom used as an energy source but is involved in signaling pathways. Furthermore, treatment with exogenous gentiobiose induced budbreak in OWBs cultured in vitro, with increased concentrations of sulfur-containing amino acids, GSH, and ascorbate (AsA), as well as increased expression levels of the corresponding genes. Inhibition of GSH synthesis suppressed gentiobiose-induced budbreak accompanied by decreases in GSH and AsA concentrations and redox status. These results indicate that gentiobiose, a rare disaccharide, acts as a signal for dormancy release of gentian OWBs through the AsA-GSH cycle.