Teruko Konishi

Structural characteristics and in vitro macrophage activation of acetyl fucoidan from Cladosiphon okamuranus

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 α-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-α 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.

The Gentio-Oligosaccharide Gentiobiose Functions in the Modulation of Bud Dormancy in the Herbaceous Perennial Gentiana

This study examines the mechanisms regulating dormancy in gentians, a group of alpine perennials that produce overwintering buds. Multivariate analysis of metabolite profiles revealed that gentiobiose, a rare disaccharide, acts as a signal for dormancy release of overwintering buds through the ascorbate-glutathione cycle, a pathway that detoxifies reactive oxygen species. 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.

Biochemical characterization of Magnaporthe oryzae β-glucosidases for efficient β-glucan hydrolysis

Abstractβ-Glucosidases designated MoCel3A and MoCel3B were successfully overexpressed in Magnaporthe oryzae. MoCel3A and MoCel3B showed optimal activity at 50xa0°C and pHxa05.0–5.5. 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.

Identification of rare 6-deoxy-D-altrose from an edible mushroom (Lactarius lividatus).

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.

A draft genome of the brown alga, Cladosiphon okamuranus, S-strain: a platform for future studies of 'mozuku' biology.

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.

Isolation and Characterization of Pectin from Peel of Citrus tankan

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 9.2×104. The pectin formed a gel by conventional procedures.

Structure of a novel α-glucan substitute with the rare 6-deoxy-D-altrose from Lactarius lividatus (mushroom).

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.

Purification and Characterization of UDP-Arabinopyranose Mutase from Chlamydomonas reinhardtii

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-[14C]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.

UDP-arabinopyranose mutase gene expressions are required for the biosynthesis of the arabinose side chain of both pectin and arabinoxyloglucan, and normal leaf expansion in Nicotiana tabacum

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.