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Sugar chains are biopolymers that play various important roles for organisms, and their structures are complex and diverse. Enzymes acting on sugar chains have undergone a wide variety of molecular evolutions. Nevertheless, the limited availability of sugar chains often prevents the investigation of related enzymes, even among enzymes that act on sugar chains comprising only glucose, an essential monosaccharide in nature. Glucans such as starch and cellulose represent a large amount of biomass, and enzymes acting on these glucans have been investigated extensively. In contrast, there is much less known about enzymes acting on glucans with β-1,2-glucosidic linkages (β-1,2-glucans), especially those enzymes involved in degrading β-1,2-glucans. Research on such degrading enzymes has only been undertaken for approximately a decade. In this review, β-1,2-glucan-associated enzymes will be introduced, especially β-1,2-glucan-degrading enzymes such as a glycoside phosphorylase acting on β-1,2-glucooligosaccharides and the identification of two novel β-1,2-glucanases and related enzymes, along with the distribution and physiological role of β-1,2-glucans in nature. [ABSTRACT FROM AUTHOR]

Ossification of the posterior longitudinal ligament of the spine (OPLL) is a common intractable disease that causes spinal stenosis and myelopathy. We have previously conducted genome-wide association studies for OPLL and identified 14 significant loci, but their biological implications remain mostly unclear. Here, we examined the 12p11.22 locus and identified a variant in the 5′ UTR of a novel isoform of CCDC91 that was associated with OPLL. Using machine learning prediction models, we determined that higher expression of the novel CCDC91 isoform was associated with the G allele of rs35098487. The risk allele of rs35098487 showed higher affinity in the binding of nuclear proteins and transcription activity. Knockdown and overexpression of the CCDC91 isoform in mesenchymal stem cells and MG-63 cells showed paralleled expression of osteogenic genes, including RUNX2 , the master transcription factor of osteogenic differentiation. The CCDC91 isoform directly interacted with MIR890 , which bound to RUNX2 and decreased RUNX2 expression. Our findings suggest that the CCDC91 isoform acts as a competitive endogenous RNA by sponging MIR890 to increase RUNX2 expression. Nakajima et al. identified a functional variant in the 5′ UTR of a novel non-coding isoform of CCDC91 within the known 12p11.22 genome-wide association study locus for ossification of the posterior longitudinal ligament of the spine. The isoform directly interacts with MIR890 , which binds to RUNX2 and regulates osteogenesis. [ABSTRACT FROM AUTHOR]

A large amount of β-1,2-glucan was produced enzymatically from quite a small amount of sophorose as an acceptor material through three synthesis steps using a sucrose phosphorylase and a 1,2-β-oligoglucan phosphorylase. The first synthesis step was performed in a 200 μL of a reaction solution containing 5 mM sophorose and 1.0 M sucrose. β-1,2-Glucan in a part of the resultant solution was hydrolyzed to β-1,2-glucooligosaccharides by a β-1,2-glucanase. The second synthesis was performed in 25 times the volume for the first synthesis. The hydrolysate solution (1% volume of the reaction solution) was used as an acceptor. After treatment with the β-1,2-glucanase again, the third synthesis was performed 200 times the volume for the second synthesis (1 L). The reaction yield of β-1,2-glucan at each synthesis was 93%, 76% and 91%. Finally, more than 140 g of β-1,2-glucan was synthesized using approximately 20 μg of sophorose as the starting acceptor material. Abbreviations: DPs: degrees of polymerization; SOGP: 1,2-β-oligoglucan phosphorylase; Sopns: β-1,2-glucooligosaccharides with DP of n; Glc1P: α-glucose 1-phosphate; SucP: sucrose phosphorylase from Bifidobacterium longum subsp. longum; SGL: β-1,2-glucanase; CaSGL: Chy400_4174 protein; TLC: thin layer chromatography; GOPOD: glucose oxidase/peroxidase; PGM: phosphoglucomutase; G6PDH: glucose 6-phosphate dehydrogenase β-1,2-Glucan was synthesized by repetition of elongation and degradation of the β-1,2-glucan using only a small amount of sophorose. [ABSTRACT FROM AUTHOR]

Abstract Sophorose (Sop 2) is known as a powerful inducer of cellulases in Trichoderma reesei , and in recent years 1,2-β-D-oligoglucan phosphorylase (SOGP) has been found to use Sop 2 in synthetic reactions. From the structure of the complex of SOGP with Sop 2 , it was predicted that both the 3-hydroxy group at the reducing end glucose moiety of Sop 2 and the 3′-hydroxy group at the non-reducing end glucose moiety of Sop 2 were important for substrate recognition. In this study, three kinds of 3- and/or 3′-deoxy-Sop 2 derivatives were synthesized to evaluate this mechanism. The deoxygenation of the 3-hydroxy group of D-glucopyranose derivative was performed by radical reduction using a toluoyl group as a leaving group. The utilization of a toluoyl group that plays two roles (a leaving group for the deoxygenation and a protecting group for a hydroxy group) resulted in efficient syntheses of the three target compounds. The NMR spectra of the two final compounds (3-deoxy- and 3,3′-dideoxy-Sop 2) suggested that the glucose moiety of the reducing end of Sop 2 can easily take on a furanose structure (five-membered ring structure) by deoxygenation of the 3-hydroxy group of Sop 2. In addition, the ratio of the five- and six-membered ring structures changed depending on the temperature. The SOGPs exhibited remarkably lower specific activity for 3′-deoxy- and 3,3′-dideoxy-Sop 2 , indicating that the 3′-hydroxy group of Sop 2 is important for substrate recognition by SOGPs. Graphical abstract Image 1 Highlights • The 3- and/or 3′-deoxy-sophorose derivatives were synthesized using a toluoyl group. • Some 3-deoxy-sophorose derivatives readily take the furanose structure. • Both 3- and 3′-OH in sophorose are important for 1,2-β-oligoglucan phosphorylase. [ABSTRACT FROM AUTHOR]

A simple HPLC method was developed for the simultaneous analysis of nine preservatives in food including benzoic acid, sorbic acid, dehydroacetic acid, methyl paraben, ethyl paraben, isopropyl paraben, propyl paraben, isobutyl paraben and butyl paraben. Samples were extracted with 60 v/v% methanol containing poly-aluminium chloride (PAC) and sodium hydroxide prior to analysis. PAC, which is normally used as a coagulant, was successfully applied to remove interfering substances from the samples. The method showed good linearity with coefficients of determination higher than 0.999 over the range of 0.2–5 µg ml–1for all target preservatives. LOQs of the method were in the range of 0.002–0.008 g kg–1. Method performance was evaluated in a variety of foods demonstrated to have quantitative recoveries of 81.7–102.5% with satisfactory intra-day precision of < 3.7% and inter-day precision of < 6.5%. The method also demonstrated applicability to real foods containing preservatives. [ABSTRACT FROM AUTHOR]

Ossification of the posterior longitudinal ligament of the spine (OPLL) is a common spinal disorder that results from ectopic ossification of the posterior longitudinal ligament and causes intractable myelopathy and radiculopathy. In a previous genome-wide association study (GWAS), we found six loci associated with OPLL; however, susceptibility genes in these loci have not been identified yet. Here, we examined one of the GWAS loci and identified RSPO2 (encoding R-spondin 2) as a susceptibility gene for OPLL. R-spondin 2 is a secreted agonist of canonical Wnt-β-catenin signaling. RSPO2 was decreased in the early stage of chondrocyte differentiation. R-spondin 2 inhibited expression of genes encoding early chondrocyte differentiation markers by activating Wnt-β-catenin signaling. rs374810, the most significantly associated SNP in the GWAS locus in chromosomal region 8q23.1 was located in the chondrocyte promoter region of RSPO2 . A transcription factor, CCAAT-enhancer-binding protein β (C/EBPβ), specifically bound to the RSPO2 core promoter region containing rs374810 and increased RSPO2 expression. The risk allele of rs374810 affected the binding of the promoter with C/EBPβ and decreased the RSPO2 transcription in vitro and in vivo. Our genetic and functional data indicate that RSPO2 is a susceptibility gene for OPLL. [ABSTRACT FROM AUTHOR]

Despite the presence of β-1,2-glucan in nature, few β-1,2-glucan degrading enzymes have been reported to date. Recently, the Lin1839 protein from Listeria innocua was identified as a 1,2-β-oligoglucan phosphorylase. Since the adjacent lin1840 gene in the gene cluster encodes a putative glycoside hydrolase family 3 β-glucosidase, we hypothesized that Lin1840 is also involved in β-1,2-glucan dissimilation. Here we report the functional and structural analysis of Lin1840. A recombinant Lin1840 protein (Lin1840r) showed the highest hydrolytic activity toward sophorose (Glc-β-1,2-Glc) among β-1,2-glucooligosaccharides, suggesting that Lin1840 is a β-glucosidase involved in sophorose degradation. The enzyme also rapidly hydrolyzed laminaribiose (β-1,3), but not cellobiose (β-1,4) or gentiobiose (β-1,6) among β-linked gluco-disaccharides. We determined the crystal structures of Lin1840r in complexes with sophorose and laminaribiose as productive binding forms. In these structures, Arg572 forms many hydrogen bonds with sophorose and laminaribiose at subsite +1, which seems to be a key factor for substrate selectivity. The opposite side of subsite +1 from Arg572 is connected to a large empty space appearing to be subsite +2 for the binding of sophorotriose (Glc-β-1,2-Glc-β-1,2-Glc) in spite of the higher Km value for sophorotriose than that for sophorose. The conformations of sophorose and laminaribiose are almost the same on the Arg572 side but differ on the subsite +2 side that provides no interaction with a substrate. Therefore, Lin1840r is unable to distinguish between sophorose and laminaribiose as substrates. These results provide the first mechanistic insights into β-1,2-glucooligosaccharide recognition by β-glucosidase. [ABSTRACT FROM AUTHOR]

We have studied the structure and optical properties of CdS:O films deposited on SLG (soda lime glass) substrates by cathode sputtering in the atmosphere of oxygen/argon gases. According to X-ray diffraction (XRD) and confocal Raman scattering data, the films annealed in vacuum at temperatures above 300 °C exhibited crystalline structure of CdS. On the other hand, XRD, confocal Raman scattering, AFM (atomic force microscopy) and TEM (transmission electron microscopy) data indicated amorphous structure nano-crystallization in CdS:O films annealed in vacuum at 400 °C. For the films, the emissions located at 3.35 eV were evident from photoluminescence measurements. The origin of this energy gap is discussed by taking into account redistribution of electronic density of states in the amorphous phase and quantum-size effect. (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [ABSTRACT FROM AUTHOR]

Lin1840 is a putative β-glucosidase that is predicted to be involved in 1,2-β-glucan metabolism since the lin1839 gene encoding a 1,2-β-oligoglucan phosphorylase and the lin1840 gene are located in the same gene cluster. Here, Lin1840 was crystallized. The crystals of Lin1840 diffracted to beyond 1.8 Å resolution. The crystal belonged to space group I121, with unit-cell parameters a = 89.75, b = 95.10, c = 215.00 Å, α = 90.00, β = 96.34, γ = 90.00°. [ABSTRACT FROM AUTHOR]

Ossification of the posterior longitudinal ligament of the spine (OPLL) is a common spinal disorder among the elderly that causes myelopathy and radiculopathy. To identify genetic factors for OPLL, we performed a genome-wide association study (GWAS) in ∼8,000 individuals followed by a replication study using an additional ∼7,000 individuals. We identified six susceptibility loci for OPLL: 20p12.3 (rs2423294: P = 1.10 × 10−13), 8q23.1 (rs374810: P = 1.88 × 10−13), 12p11.22 (rs1979679: P = 4.34 × 10−12), 12p12.2 (rs11045000: P = 2.95 × 10−11), 8q23.3 (rs13279799: P = 1.28 × 10−10) and 6p21.1 (rs927485: P = 9.40 × 10−9). Analyses of gene expression in and around the loci suggested that several genes are involved in OPLL etiology through membranous and/or endochondral ossification processes. Our results bring new insight to the etiology of OPLL. [ABSTRACT FROM AUTHOR]

Currently, research on the construction of vascular-like tubular structures is a hot area of tissue engineering, since it has potential applications in the building of artificial blood vessels. In this paper, we report a fluidic self-assembly method using cell embedded microstructures to construct vascular-like microtubes. A novel 4-layer microfluidic device was fabricated using polydimethylsiloxane (PDMS), which contains fabrication, self-assembly and extraction areas inside one channel. Cell embedded microstructures were directly fabricated using poly(ethylene glycol) diacrylate (PEGDA) in the fabrication area, namely on-chip fabrication. Self-assembly of the fabricated microstructures was performed in the assembly area which has a micro well. Assembled tubular structures (microtubes) were extracted outside the channel into culture dishes using a normally closed (NC) micro valve in the extraction area. The self-assembly mechanism was experimentally demonstrated. The performance of the NC micro valve and embedded cell concentration were both evaluated. Fibroblast (NIH/3T3) embedded vascular-like microtubes were constructed inside this reusable microfluidic device. [ABSTRACT FROM AUTHOR]

In this paper, we report on a microfluidic device with a multi-valve system to conduct several exposure tests on Caenorhabditis elegans (C. elegans) simultaneously. It has pneumatic valves and no-moving-parts (NMP) valves. An NMP valve is incorporated with a chamber and enables the unidirectional movement of C. elegans in the chamber; once worms are loaded into the chamber, they cannot exit, regardless of the flow direction. To demonstrate the ability of the NMP valve to handle worms, we made a microfluidic device with three chambers. Each chamber was used to expose worms to Cd and Cu solutions, and K-medium. A pair of electrodes was installed in the device and the capacitance in-between the electrode was measured. When a C. elegans passed through the electrodes, the capacitance was changed. The capacitance change was proportional to the body volume of the worm, thus the body volume change by the heavy metal exposure was measured in the device. Thirty worms were divided into three groups and exposed to each solution. We confirmed that the different solutions induced differences in the capacitance changes for each group. These results indicate that our device is a viable method for simultaneously analyzing the effect of multiple stimuli on C. elegans. [ABSTRACT FROM AUTHOR]

We characterized recombinant Lin1839 protein (Lin1839r) belonging to glycoside hydrolase family 94 from Listeria innocua. Lin1839r catalyzed the synthesis of a series of 1,2-β-oligoglucans (Sopn: n denotes degree of polymerization) using sophorose (Sop2) as the acceptor and α-d-glucose 1-phosphate (Glc1P) as the donor. Lin1839r recognized glucose as a very weak acceptor substrate to form polymeric 1,2-β-glucan. The degree of polymerization of the 1,2-β-glucan gradually decreased with long-term incubation to generate a series of Sopns. Kinetic analysis of the phosphorolytic reaction towards sophorotriose revealed that Lin1839r followed a sequential Bi Bi mechanism. The kinetic parameters of the phosphorolysis of sophorotetraose and sophoropentaose were similar to those of sophorotriose, although the enzyme did not exhibit significant phosphorolytic activity on Sop2. These results indicate that the Lin1839 protein is a novel inverting phosphorylase that catalyzes reversible phosphorolysis of 1,2-β-glucan with a degree of polymerization of ≥3. We propose 1,2-β-oligoglucan: phosphate α-glucosyltransferase as the systematic name and 1,2-β-oligoglucan phosphorylase as the short name for this Lin1839 protein. [ABSTRACT FROM AUTHOR]

The nematode Caenorhabditis elegans (C. elegans) receives attention as a bioindicator, and the C. elegans condition has been recently analyzed using microfluidic devices equipped with an imaging system. To establish a method without an imaging system, we have proposed a novel microfluidic device with which to analyze the condition of C. elegans from the capacitance change using a pair of micro-electrodes. The device was designed to culture C. elegans, to expose C. elegans to an external stimulus, such as a chemical or toxicant, and to measure the capacitance change which indicates the condition of C. elegans. In this study, to demonstrate the capability of our device in a toxic aqueous environment, the device was applied to examine the effect of cadmium on C. elegans. Thirty L4 larval stage C. elegans were divided into three groups. One group was a control group and the other groups were exposed to cadmium solutions with concentrations of 5% and 10% LC50 for 24 h. The capacitance change and the body volume of C. elegans as a reference were measured four times and we confirmed the correlation between them. It shows that our device can analyze the condition of C. elegans without an imaging system. [ABSTRACT FROM AUTHOR]

β-(1 → 2)-Glucans can be synthesized by 1,2-β-oligoglucan phosphorylase using β-(1 → 2)-glucooligosaccharides as acceptors and α- d -glucose 1-phosphate as a donor. Using phosphorolysis of sucrose as a source of α- d -glucose 1-phosphate, we generated β-(1 → 2)-glucans with degrees of polymerization (DPs) up to approximately 280. Average DPs up to approximately 1000 were obtained using β-(1 → 2)-glucan with average DP of 160 as an acceptor and pure α- d -glucose 1-phosphate as a donor. A colorimetric assay of the β-glucosidase activity against the β-(1 → 2)-glucan products was used to determine their DPs. [Display omitted] • β-(1→2)-Glucan with a degree of polymerization of 280 was synthesized using sucrose. • A degree of polymerization of 1000 was achieved by avoiding use of sucrose as a donor. • Degrees of polymerization were evaluated using a colorimetric β-glucosidase assay. [ABSTRACT FROM AUTHOR]

Proteoglycans (PGs) are a major component of the extracellular matrix in many tissues and function as structural and regulatory molecules. PGs are composed of core proteins and glycosaminoglycan (GAG) side chains. The biosynthesis of GAGs starts with the linker region that consists of four sugar residues and is followed by repeating disaccharide units. By exome sequencing, we found that B3GALT6 encoding an enzyme involved in the biosynthesis of the GAG linker region is responsible for a severe skeletal dysplasia, spondyloepimetaphyseal dysplasia with joint laxity type 1 (SEMD-JL1). B3GALT6 loss-of-function mutations were found in individuals with SEMD-JL1 from seven families. In a subsequent candidate gene study based on the phenotypic similarity, we found that B3GALT6 is also responsible for a connective tissue disease, Ehlers-Danlos syndrome (progeroid form). Recessive loss-of-function mutations in B3GALT6 result in a spectrum of disorders affecting a broad range of skeletal and connective tissues characterized by lax skin, muscle hypotonia, joint dislocation, and spinal deformity. The pleiotropic phenotypes of the disorders indicate that B3GALT6 plays a critical role in a wide range of biological processes in various tissues, including skin, bone, cartilage, tendon, and ligament. [Copyright &y& Elsevier]

Ossification of the posterior longitudinal ligament of the spine (OPLL) is a common musculoskeletal disease among people after middle age. The OPLL presents with serious neurological abnormalities due to compression of the spinal cord and nerve roots. The OPLL is caused by genetic and environment factors; however, its etiology and pathogenesis still remain to be elucidated. To determine the susceptibility loci for OPLL, we performed a genome-wide linkage study using 214 affected sib-pairs of Japanese. In stratification analyses for definite cervical OPLL, we found loci with suggestive linkage on 1p21, 2p22-2p24, 7q22, 16q24 and 20p12. Fine mapping using additional markers detected the highest non-parametric linkage score (3.43, P = 0.00027) at D20S894 on chromosome 20p12 in a subgroup that had no complication of diabetes mellitus. Our result would shed a new light on genetic aspects of OPLL. [ABSTRACT FROM AUTHOR]

A glycoside hydrolase responsible for laminarin degradation was partially purified to homogeneity from a Ustilago esculenta culture filtrate by weak-cation-exchange, strong-cation-exchange, and size-exclusion chromatography. Three proteins in enzy-matically active fractions were digested with chymotrypsin followed by liquid chromatography-tandem mass spectrometry (LC/ MS/MS) analysis, resulting in the identification of three peptide sequences that shared significant similarity to a putative β-1,3-glucanase, a member of glucoside hydrolase family 16 (GH16) from Sporisorium reilianum SRZ2. A gene encoding a laminarin-degrading enzyme from 17. esculenta, lam16A, was isolated by PCR using degenerate primers designed based on the S. reilianum SRZ2 β-1,3-glucanase gene. Lam16A possesses a GH16 catalytic domain with an N-terminal signal peptide and a C-terminal gly-cosylphosphatidylinositol (GPI) anchor peptide. Recombinant Lam16A fused to an N-terminal FLAG peptide (Lam16A-FLAG) overexpressed in Aspergillus oryzae exhibited hydrolytic activity toward β-1,3-glucan specifically and was localized both in the extracellular and in the membrane fractions but not in the cell wall fraction. Lam16A without a GPI anchor signal peptide was secreted extracellularly and was not detected in the membrane fraction. Membrane-anchored Lam16A-FLAG was released completely by treatment with phosphatidylinositol-specific phospholipase C. These results suggest that Lam16A is anchored in the plasma membrane in order to modify β-1,3-glucan associated with the inner cell wall and that Lam16A is also used for the catab-olism of β-1,3-glucan after its release in the extracellular medium. [ABSTRACT FROM AUTHOR]

Osteoarthritis (OA) is a common disease with a genetic component for its etiology. Recently, a genetic association of a single nucleotide polymorphism (SNP), rs17039192 in HIF-2α with knee OA has been reported in a Japanese population; however, controversy exits for its replication and a role of HIF-2α in OA. This study aimed to evaluate the association of the SNP by a large-scale replication study. A total of 8,457 subjects (3,129 OA cases and 5,328 controls) from seven independent cohorts from six countries (Japan, China, Taiwan, Korea, Greece, and Australia) were recruited and genotyped. The association of rs17039192 with knee OA was evaluated by meta-analyses. The association of the HIF-2α SNP was not replicated in any of the populations. Contrary to the previous report, the odds ratios (ORs) of the risk allele frequency were all less than 1. A combined analysis for the seven populations also showed no replication of the association (OR = 0.91, 95% confidence interval = 0.81-1.03). Our large-scale meta-analysis showed that the association of rs17039192 in HIF-2α with knee OA is negative. The significance of HIF-2α in human OA (idiopathic OA as a common disease) should be further evaluated carefully. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1244-1248, 2012 [ABSTRACT FROM AUTHOR]

Hydrolytic enzymes responsible for laminarin degradation were found to be secreted during growth of Ustilago esculenta on laminarin. An enzyme involved in laminarin degradation was purified by assaying release of glucose from laminaribiose. Ion-exchange chromatography of the culture filtrate followed by size-exclusion chromatography yielded a 110-kDa protein associated with laminaribiose hydrolysis. LC/MS/MS analysis of the 110-kDa protein identified three peptide sequences that shared significant similarity with a putative glucoside hydrolase family (GH) 3 β-glucosidase in Ustilago maydis. Based on the DNA sequence of the U. maydis GH3 β-glucosidase, a gene encoding a putative GH3 β-glucosidase in U. esculenta ( Uebgl3A) was cloned by PCR. Based on the deduced amino acid sequence, the protein encoded by Uebgl3A has a molecular mass of 91 kDa and shares 90% identity with U. maydis GH3 β-glucosidase. Recombinant UeBgl3A expressed in Aspergillus oryzae released glucose from β-1,3-, β-1,4-, and β-1,6-linked oligosaccharides, and from 1,3-1,4-β-glucan and laminarin polysaccharides, indicating that UeBgl3A is a β-glucosidase. Kinetic analysis showed that UeBgl3A preferentially hydrolyzed laminaritriose and laminaritetraose. These results suggest that UeBgl3A is a key enzyme that produces glucose from laminarioligosaccharides during growth of U. esculenta on laminarin. [ABSTRACT FROM AUTHOR]

Abstract: A novel method for measuring an adhesion force of single yeast cell is proposed based on a nanorobotic manipulation system inside an environmental scanning electron microscope (ESEM). The effect of ambient humidity on a single yeast cell adhesion force was studied. Ambient humidity was controlled by adjusting the chamber pressure and temperature inside the ESEM. It has been demonstrated that a thicker water film was formed at a higher humidity condition. The adhesion force between an atomic force microscopy (AFM) cantilever and a tungsten probe which later on known as a substrate was evaluated at various humidity conditions. A micro-puller was fabricated from an AFM cantilever by use of focused ion beam (FIB) etching. The adhesion force of a single yeast cell (W303) to the substrate was measured using the micro-puller at the three humidity conditions: 100%, 70%, and 40%. The results showed that the adhesion force between the single yeast cell and the substrate is much smaller at higher humidity condition. The yeast cells were still alive after being observed and manipulated inside ESEM based on the result obtained from the re-culturing of the single yeast cell. The results from this work would help us to understand the ESEM system better and its potential benefit to the single cell analysis research. [Copyright &y& Elsevier]

Abstract: Cell’s adhesion is important to cell’s interaction and activates. In this paper, a novel method for cell–cell adhesion force measurement was proposed by using a nano-picker. The effect of the contact time on the cell–cell adhesion force was studied. The nano-picker was fabricated from an atomic force microscopy (AFM) cantilever by nano fabrication technique. The cell–cell adhesion force was measured based on the deflection of the nano-picker beam. The result suggests that the adhesion force between cells increased with the increasing of contact time at the first few minutes. After that, the force became constant. This measurement methodology was based on the nanorobotic manipulation system inside an environmental scanning electron microscope. It can realize both the observation and manipulation of a single cell at nanoscale. The quantitative and precise cell–cell adhesion force result can be obtained by this method. It would help us to understand the single cell interaction with time and would benefit the research in medical and biological fields potentially. [Copyright &y& Elsevier]

Osteoarthritis (OA) is one of the most prevalent skeletal diseases. Recently, we identified a novel gene on chromosome 3p24.3, named DVWA (double von Willebrand factor A domains), and its functional variants, which are associated with susceptibility to knee OA. Here we report the cloning and characterization of the DVWA gene. DVWA consisted of seven exons and had four alternative splicing variants, which encoded long (385 amino acid) and short (276 amino acid) proteins (L-DVWA and S-DVWA, respectively). S-DVWA was an N-terminal truncated form of L-DVWA and lacked a signal peptide and a part of a VWA domain. L-DVWA and S-DVWA transcripts were mainly expressed in articular cartilage. Immunoblot analysis using epitope-tagged proteins showed L-DVWA in the conditioned media and S-DVWA only in the cell, consistent with the in silico prediction. We also cloned the murine counterpart of DVWA, which was found to be identical to Col6a4, which has recently been reported. L-DVWA had 73% identity to the N-terminal sequence of the 2,309-amino acid Col6a4 protein. The mouse Dvwa/Col6a4 mRNA was present mainly in the small intestine in embryos and adults, but not in cartilage. The amino acid sequence of L-DVWA was conserved in higher species than chicken, but that of S-DVWA was unique in human. Knockdown of DVWA by siRNAs increased expression of chondrocyte matrix genes. Our study indicates that DVWA is evolutionally very unique, which, together with its specific expression in articular cartilage, suggests its specific role in human cartilage metabolism. [ABSTRACT FROM AUTHOR]

The article presents a study concerning practical preparation of D-Galactosyl-β→4-L-rhamnose (GalRha). It mentions D-Galactosyl has been produced enzymatically from 1.1m sucrose and 1.0m L-rhamnose by the concomitant actions of several enzymes including sucrose phosphorylase, UDP-glucose 4-epimerase and 1-phosphate uridylytransferase. It notes that the gathering of GalRha in one liter of the reaction mixture has reached 239 grams.

Asporin is an extracellular matrix (ECM) protein that regulates cartilage matrix gene expression and cartilage formation by modulating the transforming growth factor-beta (TGF-beta) signaling pathway. Our previous studies have indicated that asporin binds to TGF-beta1 directly and inhibits TGF-beta1-mediated expression of cartilage matrix genes. However, it is still unknown how asporin interacts with TGF-beta1 and influences its activity. Using competition assays, we determined that amino acids 159-205 of asporin mediate its interaction with TGF-beta1 and effectively repress TGF-beta1-induced cartilage matrix gene expression. Asporin also has a binding ability to type II collagen in vitro, but its binding pattern is different from that of TGF-beta1. In contrast with previous in vivo findings, asporin did not affect the interaction between TGF-beta1 and the TGF-beta type II receptor (TbetaRII) by itself or in the presence of type II collagen in vitro. However, in the presence of heparin/heparan sulfate, asporin inhibits the interaction between TGF-beta and TbetaRII in vitro. These findings suggest that asporin is one of the important cartilage matrix proteins that binds to the ECM and TGF-beta1 and thereby modulates interactions between TGF-beta and its signaling receptors. [ABSTRACT FROM AUTHOR]

Abstract: We characterized a glycoside hydrolase family 112 protein from Opitutus terrae (Oter_1377 protein). The enzyme phosphorolyzed d-galactosyl-β1→4-l-rhamnose (GalRha) and also showed phosphorolytic activity on d-galactosyl-β1→3-d-glucose as a minor substrate. In the reverse reaction, the enzyme showed higher activity on l-rhamnose derivatives than on d-glucose derivatives. The enzyme was stable up to 45°C and at pH 6.0–7.0. The values of k cat and K m of the phosphorolytic activity of the enzyme on GalRha were 60s−1 and 2.1mM, respectively. Thus, Oter_1377 protein was identified as d-galactosyl-β1→4-l-rhamnose phosphorylase (GalRhaP). The presence of GalRhaP in O. terrae suggests that genes encoding GalRhaP are widely distributed in different organisms. [Copyright &y& Elsevier]

Osteoarthritis (OA) is a common disease that has a definite genetic component. Only a few OA susceptibility genes that have definite functional evidence and replication of association have been reported, however. Through a genome-wide association study and a replication using a total of ~4,800 Japanese subjects, we identified two single nucleotide polymorphisms (SNPs) (rs7775228 and rs10947262) associated with susceptibility to knee OA. The two SNPs were in a region containing HLA class II/III genes and their association reached genome-wide significance (combined P = 2.43 × 10-8 for rs7775228 and 6.73 × 10-8 for rs10947262). Our results suggest that immunologic mechanism is implicated in the etiology of OA. [ABSTRACT FROM AUTHOR]

Between 2004 and 2007 we examined foods from Japanese retail shops for contamination with ochratoxin A (OTA) and fumonisins B1, B2, and B3. A total of 1,358 samples of 27 different products were examined for OTA, and 831 samples of 16 different products were examined for fumonisins. The limits of quantification ranged from 0.01 to 0.5 µg/kg for OTA and 2 to 10 µg/kg for the fumonisins. OTA was detected in amounts higher than limits of quantification in wheat flour, pasta, oatmeal, rye, buckwheat flour and dried buckwheat noodles, raisins, wine, beer, coffee beans and coffee products, chocolate, cocoa, and coriander. OTA was found in more than 90% of the samples of instant coffee and cocoa, and the highest concentration of OTA, 12.5 µg/kg, was detected in raisins. The concentration of OTA in oatmeal, rye, raisins, wine, and roasted coffee beans varied remarkably from year to year. Fumonisins were detected in frozen and canned corn, popcorn grain, corn grits, cornflakes, com soups, com snacks, beer, soybeans, millet, and asparagus. The highest concentrations of fumonisins B1, B2, and B3 were detected in corn grits (1,670, 597, and 281 µg/kg, respectively). All of the samples of corn grits were contaminated with fumonisins, and more than 80% of the samples of popcorn grain and corn snacks contained fumonisins. OTA and fumonisins were detected in several food products in Japan; however, although Japan has not set regulatory levels for these mycotoxins, their concentrations were relatively low. [ABSTRACT FROM AUTHOR]

We characterized three D-galactosyl-β1→3-N-acety1-D-hexosamine phosphorylase (EC 2.4.1.211) homologs from Clostridium phytofermentans (Cphy0577, Cphyl920, and Cphy3030 proteins). Cphy0577 and Cphy3030 proteins exhibited similar activity on galacto-N-biose (GNB; 1)-Gal-β1→3-D-GalNAc) and lacto-N-biose I (LNB; o-Gal-β1→3-DG1cNAc), thus indicating that they are D-galactosyl-β1→3-Nacetyl-D-hexosamine phosphorylases, subclassified as GNB/ LNB phosphorylase. In contrast, Cphyl920 protein phosphorolyzed neither GNB nor LNB. It showed the highest activity with L-rhamnose as the acceptor in the reverse reaction using α-D-galactose 1-phosphate as the donor, The reaction product was D-galactosyl-β1→4-L-rhamnose. The enzyme also showed activity on L-mannose, L-lyxose, D-glucose, 2-deoxy-o-glucose, and D-galactose in this order. When D-glucose derivatives were used as acceptors, reaction products were β-1,3-galactosides. Kinetic parameters of phosphorolytic activity on D-galactosyl-β1→4-L-rhamnose were kcat = 45 s-1 and Km = 7.9 mM, thus indicating that these values are common among other phosphorylases. We propose D-galactosyl-β1→4-L-rhamnose phosphorylase as the name for Cphy1920 protein. [ABSTRACT FROM AUTHOR]

Abstract: 2-Pyrone-4,6-dicarboxylic acid (PDC) is a catabolic intermediate in Sphingobium sp. SYK-6 (previously characterized as Sphingomonas paucimobilis SYK-6), which is a degrader of lignin-derived aromatic compounds. Recently, PDC has been also characterized as a novel starting material for several potentially useful synthetic polymers. In a previous study, we constructed a biosynthetic system in which PDC was generated efficiently from a chemically synthesized compound, protocatechuate. In order to develop an alternative system for production of PDC, we tried to generate it from glucose, which is a low-cost sugar that can be obtained from abundant cellulosic wastes and biomass crops. We designed a metabolic bypass to PDC from the shikimate pathway in recombinant Escherichia coli cells. PDC accumulated in the medium of recombinant E. coli cells that had been transformed with genes isolated from Emericella niger, E. coli, Pseudomonas putida, and Sphingobium sp. SYK-6. The yield of PDC depended on the combination of genes that we introduced into the cells and on the specific of host strain. Under optimal conditions, the yield and titer of PDC were, respectively, 17.3% and 0.35mg/l when the concentration of glucose was 2g/l and the culture volume was 50ml. Our results open up the possibility of novel utilization of biomass as the source of a useful chemical building block. [Copyright &y& Elsevier]

Homologs of the β-1,3-galactosyl- N-acetylhexosamine phosphorylase (GalHexNAcP) gene ( gnpA) were cloned from the genomic DNA of Propionibacterium acnes JCM6425 and P. acnes JCM6473, showing 99.9% and 97.9% nucleotide sequence identity, respectively, with the ppa0083 gene from the genome-sequenced P. acnes KPA171202. No gnpA gene was detected in the genomic DNA of type strain P. acnes ATCC25746. The recombinant enzyme from P. acnes JCM6425 (GnpA) showed approximately 70 times higher specific activity of phosphorolysis on galacto- N-biose (Galβ1→3GalNAc, GNB) than that on lacto- N-biose I (Galβ1→3GlcNAc). Km value for GnpA on GNB was high, but GnpA did not exhibit activity on any derivatives of GNB examined. These results indicate that GnpA is GalHexNAcP which should be classified as galacto- N-biose phosphorylase. The large kcat value of GnpA on GalNAc suggests that GnpA would be a useful catalyst for the synthesis of GNB. [ABSTRACT FROM AUTHOR]

A nanolaboratory is proposed to realize the capabilities of nanofabrication, nanoinstrumentation and nanoassembly to make three-dimensional structures, devices and systems in the nanoworld. A nanorobotic manipulation system is introduced to realize these capabilities inside a scanning electron microscope or a transmission electron microscope. Some precursors are introduced into the small working space in the nanoworld, so that cutting, bending, peeling and fixing operations in the nanoworld could be realized using nanomanipulators, electron-bean-induced deposition and other methodologies as in the macroworld. After making the three-dimensional structures, the nanodevices such as nanosensors and nanoactuators are fabricated using carbon nanotubes and a focused ion beam chemical vapor deposition process. Some nanodevices for bioapplications are also presented. [ABSTRACT FROM AUTHOR]

A β-1,3-galactosyl-N-acetyIhexosamine phosphorylase (GaIGIyNAcP) homolog gene was cloned from Vibrio vulnificus CMCP6. In synthetic reactions, the recombinant enzyme acted only with GIcNAc and GalNAc as acceptors in the presence of α-o-galactose-l-phosphate as a donor to form lacto-N-biose I (LNB) (Galβ1 → 3GIcNAc) and galacto-N-biose (GNB) (GaIβ1 → 3GaINAc), respectively. GIcNAc was a much better acceptor than GaINAc. The enzyme also phosphorolysed LNB faster than it phosphorolysed GNB, and the for LNB was approximately 60 times higher than the for GNB. This result indicated that the enzyme was remarkably different from GaIGIyNAcP from Bifidobacterium longum, which has similar activities with LNB and GNB, and GaIGIyNAcP from Clostridium perfringens, which is a GNB-specific enzyme. The enzyme is the first LNB-specific enzyme that has been found and was designated Iacto-N-biose I phosphorylase. The discovery of an LNB-specific GaIGIyNAcP resulted in recategorization of bifidobacterial GaIGIyNAcPs as galacto-N-biose/ lacto-N-biose I phosphorylases. [ABSTRACT FROM AUTHOR]

Lacto- N-biose phosphorylase (LNBP) from bifidobacteria is involved in the metabolism of lacto- N-biose I (Galβ1→3GlcNAc, LNB) and galacto- N-biose (Galβ1→3GalNAc, GNB). A homologous gene of LNBP (CPF0553 protein) was identified in the genome of Clostridium perfringens ATCC13124, which is a gram-positive anaerobic intestinal bacterium. In the present study, we cloned the gene and compared the substrate specificity of the CPF0553 protein with LNBP from Bifidobacterium longum JCM1217 (LNBPBl). In the presence of α-galactose 1-phosphate (Gal 1-P) as a donor, the CPF0553 protein acted only on GlcNAc and GalNAc, and GalNAc was a more effective acceptor than GlcNAc. The reaction product from GlcNAc/GalNAc and Gal 1-P was identified as LNB or GNB. The CPF0553 protein also phosphorolyzed GNB much faster than LNB, which suggests that the protein should be named galacto- N-biose phosphorylase (GNBP). GNBP showed a k cat/ K m value for GNB that was approximately 50 times higher than that for LNB, whereas LNBPBl showed similar k cat/ K m values for both GNB and LNB. Because C. perfringens possesses a gene coding endo-α- N-acetylgalactosaminidase, GNBP may play a role in the intestinal residence by metabolizing GNB that is available as a mucin core sugar. [ABSTRACT FROM AUTHOR]

Asporin (ASPN) is a small leucine-rich proteoglycan that is involved in pathological processes of osteoarthritis. Previously, we showed that asporin can inhibit transforming growth factor-β1 (TGF-β1)-mediated expression of cartilage matrix genes and chondrogenesis in vitro (Kizawa, H., Kou, I., Lida, A., Sudo, A., Miyamoto, Y., Fukuda, A., Mabuchi, A., Kotani, A., Kawakami, A., Yamamoto, S., Uchida, A., Nakamura, K., Notoya, K., Nakamura, Y., and Ikegawa, S. (2005) Nat. Genet. 37, 138-144). However, details about regulation of asporin itself are not yet known. Here, we examined ASPN expression in skeletal tissue and potential regulation of ASPN by TGF-β. In situ hybridization revealed the presence of ASPN mRNA in the perichondrium/periosteum of long bones, but its absence in articular cartilage and growth plates. Immunohistochemical analysis also showed ASPN protein expression predominantly in the perichondrium/periosteum. TGF-β1 induced endogenous ASPN mRNA expression over time in vitro, and this induction was suppressed by the TGF-β type I receptor kinase inhibitor SB431542. Inhibition of Smad3 significantly reduced TGF-β1- induced ASPN expression, whereas overexpression of Smad3 augmented the induction. Characterization of the human ASPN promoter region revealed a region from –126 to –82 that is sufficient for full promoter activity; however, TGF-β1 failed to increase activity through the ASPN promoter. Our findings indicate that TGF-β1 induces ASPN through Smad3 but that this induction is indirect. [ABSTRACT FROM AUTHOR]

Osteoarthritis (OA), the most prevalent form of skeletal disease, represents a leading cause of disability following middle age. OA is characterized by the loss of articular cartilage; however, the details of its etiology and pathogenesis remain unclear. Recently, we demonstrated a genetic association between the cartilage extracellular matrix protein asporin and OA (Kizawa, H., Kou, I., lida, A., Sudo, A., Miyamoto, Y., Fukuda, A., Mabuchi, A., Kotani, A., Kawakami, A., Yamamoto, S., Uchida, A., Nakamura, K., Notoya, K., Nakamura, Y., and Ikegawa, S. (2005) Nat. Genet. 37, 138-144). Furthermore, we showed that asporin binds to transforming growth factor-β (TGF-β), a key cytokine in OA pathogenesis, and inhibits TGF-β-induced chondrogenesis. To date, functional data for asporin have come primarily from mouse cell culture models of developing cartilage rather than from human articular cartilage cells, in which OA occurs. Here, we describe mechanisms for asporin function and regulation in human articular cartilage. Asporin blocks chondrogenesis and inhibits TGF-β1-induced expression of matrix genes and the resulting chondrocyte phenotypes. Small interfering RNA-mediated knockdown of asporin increases the expression of cartilage marker genes and TGF-β1; in turn, TGF-β1 stimulates asporin expression in articular cartilage cells, suggesting that asporin and TGF-β1 form a regulatory feedback loop. Asporin inhibits TGF-β/Smad signaling upstream of TGF-β type I receptor activation in vivo by co-localizing with TGF-β1 on the cell surface and blocking its interaction with the TGF-β type II receptor. Our results provide a basis for elucidating the role of asporin in the molecular pathogenesis of OA. [ABSTRACT FROM AUTHOR]

We conducted a survey of aflatoxin B1, B2, G1, and G2, ochratoxin A, and fumonisin B1, B2, and B3 contamination in various foods on the retail market in Japan in 2004 and 2005. The mycotoxins were analyzed by high-performance liquid chromatography, liquid chromatography-mass spectrometry, or high-performance thin-layer chromatography. Aflatoxins were detected in 10 of 21 peanut butter samples; the highest concentration of aflatoxin B1 was 2.59 µg/kg. Aflatoxin contamination was not found in corn products, corn, peanuts, buckwheat flour, dried buckwheat noodles, rice, or sesame oil. Ochratoxin A was detected in oatmeal, wheat flour, rye, buckwheat flour, green coffee beans, roasted coffee beans, raisins, beer, and wine but not in rice or corn products. Ochratoxin A concentrations in contaminated samples were below 0.8 µg/kg. Fumonisins were detected in popcorn, frozen corn, corn flakes, and corn grits. The highest concentrations of fumonisins B1, B2, and B3 in these samples were 354.0, 94.0, and 64.0 µg/kg, respectively. [ABSTRACT FROM AUTHOR]

Abstract: Cartilage intermediate layer protein (CILP) is an extracellular matrix protein abundant in cartilaginous tissues. CILP is implicated in common musculoskeletal disorders, including osteoarthritis and lumbar disc disease. Regulation of the CILP gene is largely unknown, however. We have found that CILP mRNA expression is induced by TGF-β1 and dependent upon signaling via TGF-β receptors. TGF-β1 induction of CILP is mediated by Smad3, which acts directly through cis-elements in the CILP promoter region. Pathways other than Smad3 also are involved in TGF-β1 induction of CILP. These observations, together with the finding that CILP protein binds and inhibits TGF-β1, suggest that CILP and TGF-β1 may form a functional feedback loop that controls chondrocyte metabolism. [Copyright &y& Elsevier]

Presents a study on transglycossylation activity of Dictyoglomus thermophilum amylase A. Characteristics of amylase A from Dictyoglamus thermophilum; Examination of the substrate specificity and reaction products of amylase A from D. thermophilum; Results that suggest the enzyme was able to use maltooligosaccharides and long chain amylose as substrate and has transglycosylation activity.

The embryonal carcinoma-derived cell line, ATDC5, differentiates into chondrocytes in response to insulin or insulin-like growth factor-I stimulation. In this study, we investigated the roles of mitogen-activated protein (MAP) kinases in insulin-induced chondrogenic differentiation of ATDC5 cells. Insulin-induced accumulation of glycosaminoglycan and expression of chondrogenic differentiation markers, type II collagen, type X collagen, and aggrecan mRNA were inhibited by the MEK1/2 inhibitor (U0126) and the p38 MAP kinase inhibitor (SB203580). Conversely, the JNK inhibitor (SP600125) enhanced the synthesis of glycosaminoglycan and expression of chondrogenic differentiation markers. Insulin-induced phosphorylation of ERK1/2 and JNK but not that of p38 MAP kinase. We have previously clarified that the induction of the cyclin-dependent kinase inhibitor, p21Cip-1/SDI-1/WAF-1, is essential for chondrogenic differentiation of ATDC5 cells. To assess the relationship between the induction of p21 and MAP kinase activity, we investigated the effect of these inhibitors on insulin-induced p21 expression in ATDC5 cells. Insulin-induced accumulation of p21 mRNA and protein was inhibited by the addition of U0126 and SB203580. In contrast, SP600125 enhanced it. Inhibitory effects of U0126 or stimulatory effects of SP600125 on insulin-induced chondrogenic differentiation were observed when these inhibitors exist in the early phase of differentiation, suggesting that MEK/ERK and JNK act on early phase differentiation. SB202580, however, is necessary not only for early phase but also for late phase differentiation, indicating that p38 MAP kinase stimulates differentiation by acting during the entire period of cultivation. These results for the first time demonstrate that up-regulation of p21 expression by ERK1/2 and p38 MAP kinase is required for chondrogenesis, and that JNK acts as a suppressor of chondrogenesis by down-regulating p21 expression. [Copyright &y& Elsevier]

A putative cytosolic α-mannosidase gene from a hyperthermophilic marine bacterium Thermotoga maritima was cloned and expressed in Escherichia coli. The purified recombinant enzyme appeared to be a homodimer of a 110-kDa subunit. The enzyme showed metal-dependent ability to hydrolyze p-nitrophenyl-α-d-mannopyranoside. In the absence of a metal, the enzyme was inactive. Cobalt and cadmium supported high activity (60 U/mg at 70 °C), while the activity with zinc and chromium was poor. Cobalt (0.8 mol) bound to 1 mol monomer with a Kd of 70 μM. The optimum pH and temperature were 6.0 and 80 °C, respectively. The activity was inhibited by swainsonine, but not by 1-deoxymannojirimycin, which is in agreement with the features of cytosolic α-mannosidase. [Copyright &y& Elsevier]

Background: Human eosinophils contain two eosinophil ribonucleases, eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN). In rats, 8 homologues of human ECP and EDN have been identified. To clarify the biological activity of rat eosinophil ribonucleases, we cloned rat eosinophil-associated ribonuclease (EAR)-1/rat ribonuclease 7 and rat EAR-2/rat ribonuclease 4, and produced recombinant rat pre-EAR-1 and pre-EAR-2 in a bacterial expression system. Methods: As we have already cloned the complete nucleotide sequence for rat EAR-1, we determined that for rat EAR-2 cDNA by the rapid amplification of cDNA ends procedure. Recombinant rat pre-EAR-1 and pre-EAR-2 were expressed in Escherichia coli as N-terminal 6 × histidine-tagged proteins, isolated from the insoluble fraction of the cell lysate and purified by a single-step method using an Ni-NTA resin column after solubilization with a 6 M guanidine solution. Results: The deduced amino acid sequence revealed that the molecular weight of EAR-2 containing the signal peptide is 17.3 kD and the isoelectric point is 8.59. The homology in amino acid sequence between rat pre-EAR-2, and human pre-ECP and human pre-EDN is 51 and 53%, respectively. The purified and refolded recombinant rat pre-EAR-1 and pre-EAR-2 showed bactericidal activity against E. coli and Staphylococcus aureus. Conclusions: These findings suggest that rat EAR-1 and EAR-2 act as host defense factors against bacterial infection in rats.Copyright © 2001 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder of skeletal malformations and presents progressive extra-skeletal ossification. The 617G>A (R206H) mutation in the activin receptor type IA ( ACVR1) gene has been identified in all examined individuals with FOP of various ethnic groups, including Caucasian and Chinese descents. Here, we examined three Japanese patients with FOP for ACVR1 mutations. We identified the 617G>A mutation in all three patients. Our results suggest that the mutation in the ACVR1 gene is common and recurrent in the global population. [ABSTRACT FROM AUTHOR]

Cyclic β-1,2-glucan synthase (CGS) is a key enzyme in production of cyclic β-1,2-glucans (CβGs) which are involved in bacterial infection or symbiosis to host organisms. Nevertheless, a mechanism of cyclization, the final step in the CGS reaction, has not been fully understood. Here we performed functional and structural analyses of the cyclization domain of CGS alone from Thermoanaerobacter italicus (TiCGSCy). We first found that β-glucosidase-resistant compounds are produced by TiCGSCy with linear β-1,2-glucans as substrates. The 1H-NMR analysis revealed that these products are CβGs. Next, action pattern analyses using β-1,2-glucooligosaccharides revealed a unique reaction pattern: exclusive transglycosylation without hydrolysis and a hexasaccharide being the minimum length of the substrate. These analyses also showed that longer substrate β-1,2-glucooligosaccharides are preferred, being consistent with the fact that CGSs generally produce CβGs with degrees of polymerization of around 20. Finally, the overall structure of the cyclization domain of TiCGSCy was found to be similar to those of β-1,2-glucanases in phylogenetically different groups. Meanwhile, the identified catalytic residues indicated clear differences in the reaction pathways between these enzymes. Overall, we propose a novel reaction mechanism of TiCGSCy. Thus, the present group of CGSs defines a new glycoside hydrolase family, GH189. Key points: • It was clearly evidenced that cyclization domain alone produces cyclic β-1,2-glucans. • The domain exclusively catalyzes transglycosylation without hydrolysis. • The present catalytic domain defines as a new glycoside hydrolase family 189. [ABSTRACT FROM AUTHOR]

Cyclic β-1,2-glucan synthase (CGS) is a key enzyme in production of cyclic β-1,2-glucans (CβGs) which are involved in bacterial infection or symbiosis to host organisms. Nevertheless, a mechanism of cyclization, the final step in the CGS reaction, has not been fully understood. Here we performed functional and structural analyses of the cyclization domain of CGS alone from Thermoanaerobacter italicus (TiCGSCy). We first found that β-glucosidase-resistant compounds are produced by TiCGSCy with linear β-1,2-glucans as substrates. The 1H-NMR analysis revealed that these products are CβGs. Next, action pattern analyses using β-1,2-glucooligosaccharides revealed a unique reaction pattern: exclusive transglycosylation without hydrolysis and a hexasaccharide being the minimum length of the substrate. These analyses also showed that longer substrate β-1,2-glucooligosaccharides are preferred, being consistent with the fact that CGSs generally produce CβGs with degrees of polymerization of around 20. Finally, the overall structure of the cyclization domain of TiCGSCy was found to be similar to those of β-1,2-glucanases in phylogenetically different groups. Meanwhile, the identified catalytic residues indicated clear differences in the reaction pathways between these enzymes. Overall, we propose a novel reaction mechanism of TiCGSCy. Thus, the present group of CGSs defines a new glycoside hydrolase family, GH189. Key points: • It was clearly evidenced that cyclization domain alone produces cyclic β-1,2-glucans. • The domain exclusively catalyzes transglycosylation without hydrolysis. • The present catalytic domain defines as a new glycoside hydrolase family 189. [ABSTRACT FROM AUTHOR]

Most Gram-negative bacteria synthesize osmo-regulated periplasmic glucans (OPG) in the periplasm or extracellular space. Pathogenicity of many pathogens is lost by knocking out opgG, an OPG-related gene indispensable for OPG synthesis. However, the biochemical functions of OpgG and OpgD, a paralog of OpgG, have not been elucidated. In this study, structural and functional analyses of OpgG and OpgD from Escherichia coli revealed that these proteins are β-1,2-glucanases with remarkably different activity from each other, establishing a new glycoside hydrolase family, GH186. Furthermore, a reaction mechanism with an unprecedentedly long proton transfer pathway among glycoside hydrolase families is proposed for OpgD. The conformation of the region that forms the reaction pathway differs noticeably between OpgG and OpgD, which explains the observed low activity of OpgG. The findings enhance our understanding of OPG biosynthesis and provide insights into functional diversity for this novel enzyme family. Two β-1,2-glucanases involved in periplasmic glucan synthesis, OpgG and OpgD from E. coli, belong to an uncharacterized glycoside hydrolase family. Biochemical and structural characterization show that OpgD employs a long range proton transfer. [ABSTRACT FROM AUTHOR]

Glycoside phosphorylases catalyze the phosphorolysis of oligosaccharides into sugar phosphates. Recently, we found a novel phosphorylase acting on β-1,2-glucooligosaccharides with degrees of polymerization of 3 or more (1,2-β-oligoglucan phosphorylase, SOGP) in glycoside hydrolase family (GH) 94. Here, we characterized SOGP from Lachnoclostridium phytofermentans (LpSOGP) and determined its crystal structure. LpSOGP is a monomeric enzyme that contains a unique β-sandwich domain (Ndom1) at its N-terminus. Unlike the dimeric GH94 enzymes possessing catalytic pockets at their dimer interface, LpSOGP has a catalytic pocket between Ndom1 and the catalytic domain. In the complex structure of LpSOGP with sophorose, sophorose binds at subsites +1 to +2. Notably, the Glc moiety at subsite +1 is flipped compared with the corresponding ligands in other GH94 enzymes. This inversion suggests the great distortion of the glycosidic bond between subsites −1 and +1, which is likely unfavorable for substrate binding. Compensation for this disadvantage at subsite +2 can be accounted for by the small distortion of the glycosidic bond in the sophorose molecule. Therefore, the binding mode at subsites +1 and +2 defines the substrate specificity of LpSOGP, which provides mechanistic insights into the substrate specificity of a phosphorylase acting on β-1,2-glucooligosaccharides. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented in response to the article on the research of T. Saito and colleagues on the role of hypoxia-inducible factor-2α in the development of osteoarthritis and skeletal growth.