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Agaro-oligosaccharides (AGO), hydrolysis products of agarose, is known to have antioxidant and anti-inflammatory properties. Speculating that AGO is effective for preventing aging, we investigated the longevity-supporting effects of AGO and their mechanisms using Caenorhabditis elegans.Caenorhabditis elegans were fed AGO from young adulthood. The lifespan, locomotory activity, lipofuscin accumulation, and heat stress resistance of the worms were examined. To elucidate mechanisms of AGO-mediated longevity, we conducted comprehensive expression analysis using microarrays. Moreover, we used quantitative real-time PCR (qRT-PCR) to verify the genes showing differential expression levels. Furthermore, we measured the lifespan of loss-of-function mutants to determine the genes related to AGO-mediated longevity.AGO extended the lifespan of C. elegans, reduced lipofuscin accumulation, and maintained vigorous locomotion. The microarray analysis revealed that the endoplasmic reticulum-unfolded protein response (ER-UPR) and insulin/insulin-like growth factor-1-mediated signaling (IIS) pathway were activated in AGO-fed worms. The qRT-PCR analysis showed that AGO treatment suppressed sir-2.1 expression, which is a negative regulator of ER-UPR. In loss-of-function mutant of sir-2.1, AGO-induced longevity and heat stress resistance were decreased or cancelled completely. Furthermore, the pro-longevity effect of AGO was decreased in loss-of-function mutants of abnormal Dauer formation (daf) -2 and daf-16, which are IIS pathway-related genes.AGO delays the C. elegans aging process and extends their lifespan through the activations of ER-UPR and the IIS pathway.

[Purpose] Recently, a photo-based smartphone application for angle measurement-"Grid line imaging application Professional"-was developed to evaluate joint disease treatments. The aim of this study was to determine the accuracy and reliability of the application. [Participants and Methods] We measured the knee joint of a mannequin using an application and a universal goniometer. Twelve examiners measured eight knee joints of mannequins at different arbitrary angles using the application and a universal goniometer. Correlations between the application and universal goniometer measurements were examined using scatter plots and correlation coefficients. Systematic errors of the application were visually confirmed using the Bland-Altman method. Intra-class correlation coefficients were used to evaluate the inter-examiner reliability of the application. [Results] The application and universal goniometer measurements showed a good correlation (r=0.99) and no systematic error. The intra-class correlation coefficient for inter-examiner reliability was 0.999. Furthermore, to evaluate intra-examiner reliability, six examiners measured six different knee joints twice using the application on a 2-day interval. The intra-class correlation coefficient for intra-examiner reliability was 0.982. [Conclusion] The accuracy of the application was equivalent to that of a universal goniometer, and both the inter- and intra-examiner reliabilities of the application were almost perfect.

We investigated the physicochemical properties of starches extracted from Vigna angularis variants Noto-dainagon, Kyoto-dainagon, dainagon, and erimo-type adzuki beans. The particle size was larger in the Noto-dainagon starch than in the other starches, but all starches displayed a C-type crystal structure in the X-ray diffraction analysis. The onset and peak temperatures of gelatinization were approximately 7 °C lower in the Noto-dainagon and Kyoto-dainagon starches than in the dainagon and adzuki bean starches. The dainagon and adzuki bean starches also demonstrated lower swelling power at 60 °C than the other starches. The rapid visco-analyser curves of all starches showed no breakdown during the heating process, and the final viscosities of the starches increased with decreasing temperature. The apparent amylose and phosphorus contents were highest in the Noto-dainagon starch (36.6% and 95.6 ppm, respectively). The branched- chain length degree of polymerization 6–12 was lowest in the adzuki bean starch. In vitro and in vivo analyses of the digestive properties indicated that dainagon starch was more indigestible than the other starches. We considered that dainagon starch was a potentially viable health-promoting ingredient.

We investigated the physicochemical properties of starches extracted from 8 lotus (Nelumbo nucifera Gaertn.) rhizomes harvested in different months (September 2012 to May 2013). The physicochemical properties of the lotus starches depended on the harvest date. The peak viscosity (PV) in the Rapid Visco-Analyser analysis, and the viscosity at 65 °C (V65) in the rotational viscometer analysis were significantly lower in SEP starch (extracted from the September-harvested sample) than in the other lotus starches. The Spearman’s rank correlation coefficients of potassium ion (K) content vs. V65 and of K content vs. PV were 0.905 and 0.714, respectively, indicating that potassium ions are important for expressing the pasting properties of lotus starch. Principal component analysis suggested that the potassium, magnesium, calcium, and phosphorus contents are important for displaying both the pasting and gelatinization properties of the lotus starches. Meanwhile, the cluster analysis revealed that physicochemical properties of the SEP starch were different from those of the starches harvested in other months.

Protease has been shown to be an effective food additive for improving the quality of gluten-free rice bread. We found that rice protein hydrolysate by bacillolysin (Protin SD-NY10) and thermolysin (Thermoase) increased the specific volume of gluten-free rice starch bread. Many fine bubble cells were observed in the crumb of the rice starch bread containing rice protein hydrolysate by proteases using scanning electron microscopy. The temperature dependence between 30 and 60 °C of the tanδ values of the rice starch batter containing the protein hydrolysate was increased by comparing rice starch bread without the hydrolysate. SDS-PAGE of the rice protein hydrolysates by both proteases demonstrated that both proteases produced low molecular weight protein (ca 10 kDa) from α-glutelin in the rice protein. Judging from the protein hydrolysate by the proteases, the degradation of the α-glutelin and the hydrolysate are necessary to increase the specific volume of gluten-free rice starch bread.

A basidiomycetous yeast, Pseudozyma tsukubaensis , has been isolated from a variety of plant materials. Previous molecular phylogenetic analyses have suggested this yeast is an asexual stage of the smut fungus Macalpinomyces spermophorus (synonym Ustilago spermophora , Ustilaginales). To reveal the teleomorph–anamorph connection, yeast-like isolates from germinated teliospores of morphologically identified M. spermophorus on Eragrostis ferruginea from Japan were analyzed. The internal transcribed spacer region of rDNA of the yeast isolates was mostly identical with that of known isolates of P. tsukubaensis . Physiological analysis showed that the characteristics of the yeast isolates correlated well with P. tsukubaensis . These data revealed that P. tsukubaensis is the anamorph of M. spermophorus and should be treated as a synonym of M. spermophorus . In addition, the erythritol-producing ability of the yeast isolates derived from teliospores of M. spermophorus were confirmed by high-performance ion-exchange chromatography and 13 C NMR analyses.

We have recently generated a highly efficient 1,2-α-l-fucosynthase (BbAfcA N423H mutant) by protein engineering of 1,2-α-l-fucosidase from Bifidobacterium bifidum JCM 1254. This synthase could specifically introduce H-antigens (Fucα1-2Gal) into the non-reducing ends of oligosaccharides and in O-linked glycans in mucin glycoprotein. In the present study, we show an extended application of the engineered 1,2-α-l-fucosynthase by demonstrating its ability to insert Fuc residues into N- and O-glycans in fetuin glycoproteins, GM1 ganglioside, and a plant-derived xyloglucan nonasaccharide. This application study broadens the feasibility of this novel H-antigen synthesis technique in functional glycomics.

Fucα1-2 Gal linkages, or H-antigens, constitute histo-blood group antigens and are involved in various physiological processes. In addition, recent studies have shown that the H-antigen-containing glycans play an important role, not only in establishing harmonious relationship between gut microbes and the host, but also in preventing gut dysbiosis-related diseases. Therefore, development of an efficient method for introducing Fuc residue at Gal residue at the nonreducing end of glycans via α-(1→2) linkage is desired for research as well as medicinal purposes. In this study, we succeeded in derivatizing inverting 1,2-α-l-fucosidase (AfcA) into a highly efficient 1,2-α-l-fucosynthase. The synthase specifically synthesized H type 1-, type 2-, type 3- and type 4-chain-containing oligosaccharides with yields of 57-75% based on acceptor depletion. The synthase was also able to specifically introduce Fuc residues into Lewis a/x antigens to produce Lewis b/y antigens, with yields of 43% and 62%, respectively. In addition, the enzyme efficiently introduced H-antigens into sugar chains of porcine gastric mucins, as revealed by lectin blotting and mass spectroscopy analysis of the sugars. Detailed acceptor specificity analysis using various monosaccharides and oligosaccharides unraveled unique substrate recognition feature of this synthase at the subsite (+1), which can be explained by our previous X-ray crystallographic study of AfcA. These results show that the synthase developed in this study could serve as an alternative to other H-antigen synthesis methods involving α-1,2-fucosyltransferases and retaining α-fucosidase.

We investigated the physicochemical properties of starches from rhizomes of two lotus (Nelumbo nucifera Gaertn.) cultivars, Shinashirobana cultivar and Kanasumi-line No. 20, harvested at the same farm in Japan. A rapid visco analyser analysis indicated that pasting temperatures of the starches from Shinashirobana cultivar and Kanasumi-line No. 20 were 62.1 and 66.5 °C, respectively, and the peak viscosities of the starch of both lotuses were similar 244 and 240 RVU, respectively. The starch from the Kanasumi-line No. 20 at 60 °C had low viscosity, 16.2 mPa∙s, but the starch from Shinashirobana cultivar had higher viscosity, 2,720 mPa∙s, at 60 °C. The onset and peak temperatures of the differential scanning calorimetry (DSC) curve of the starch from Shinashirobana cultivar were 4–5 °C lower than those of the starch from Kanasumi-line No. 20, although both exhibited similar ΔH values (13.4 and 11.6 J/g). The distribution patterns of the branched chain lengths of amylopectin in the two starches indicated that the rate of low-degree of polymerization (DP 5–12) of all branched chains was higher for the starch from Shinashirobana cultivar than that for the starch from Kanasumi-line No. 20. These results suggest that the increased rate of DP 5−12 in the starch from Shinashirobana cultivar is related to the higher viscosity of the starch.

We created a glycosynthase from a GH19 chitinase from rye seeds (RSC-c), that has a long-extended binding cleft consisting of eight subsites; -4, -3, -2, -1, +1, +2, +3 and +4. When wild-type RSC-c was incubated with α-(GlcNAc)3-F [α-(GlcNAc)3 fluoride], (GlcNAc)3 and hydrogen fluoride were produced through the Hehre resynthesis-hydrolysis mechanism. Glu89, which acts as a catalytic base, and Ser120, which fixes a nucleophilic water molecule, were mutated to produce two single mutants, E89G and S120A, and a double mutant, E89G/S120A. E89G only produced a small amount of (GlcNAc)7 from α-(GlcNAc)3-F in the presence of (GlcNAc)4 S120A, with the highest F(-)-releasing activity, produced a larger amount of (GlcNAc)7, a fraction of which was decomposed by its own residual hydrolytic activity. However, the double mutant E89G/S120A, of which the hydrolytic activity was completely abolished while its F(-)-releasing activity was only moderately affected, produced the largest amount of (GlcNAc)7 from α-(GlcNAc)3-F and (GlcNAc)4 without decomposition. We concluded that E89G/S120A was an efficient glycosynthase, that enabled the addition of a three-sugar unit.

Exo-β-D-glucosaminidase (EC 3.2.1.165) from Photobacterium profundum (PpGlcNase) is an inverting GH (glycoside hydrolase) belonging to family 9. We have determined the three-dimensional structure of PpGlcNase to describe the first structure–function relationship of an exo-type GH9 glycosidase. PpGlcNase has a narrow and straight active-site pocket, in contrast with the long glycan-binding cleft of a GH9 endoglucanase. This is because PpGlcNase has a long loop, which blocks the position corresponding to subsites −4 to −2 of the endoglucanase. The pocket shape of PpGlcNase explains its substrate preference for a β1,4-linkage at the non-reducing terminus. Asp139, Asp143 and Glu555 in the active site were located near the β-O1 hydroxy group of GlcN (D-glucosamine), with Asp139 and Asp143 holding a nucleophilic water molecule for hydrolysis. The D139A, D143A and E555A mutants significantly decreased hydrolytic activity, indicating their essential role. Of these mutants, D139A exclusively exhibited glycosynthase activity using α-GlcN-F (α-D-glucosaminyl fluoride) and GlcN as substrates, to produce (GlcN)2. Using saturation mutagenesis at Asp139, we obtained D139E as the best glycosynthase. Compared with the wild-type, the hydrolytic activity of D139E was significantly suppressed (

Papain (cysteine protease), subtilisin (Protin SD-AY10, serine protease), and bacillolysin (Protin SD-NY10, metallo protease) increased the specific volume of gluten-free rice breads by 19–63% compared to untreated bread. In contrast, Newlase F (aspartyl protease) did not expand the volume of the rice bread. In a rheological analysis, the viscoelastic properties of the gluten-free rice batters also depended on the protease categories. Principal component analysis (PCA) analysis suggested that the storage and loss moduli (G′ and G″, respectively) at 35 °C, and the maximum values of G′ and G″, were important factors in the volume expansion. Judging from the PCA of the viscoelastic parameters of the rice batters, papain and Protin SD-AY10 improved the viscoelasticity for gluten-free rice bread making, and Protin SD-NY effectively expanded the gluten-free rice bread. The rheological properties differed between Protin SD-NY and the other protease treatments.

We describe the novel substrate specificities of two independently evolved lacto-N-biosidases (LnbX and LnbB) towards the sugar chains of globo- and ganglio-series glycosphingolipids. LnbX, a non-classified member of the glycoside hydrolase family, isolated from Bifidobacterium longum subsp. longum, was shown to liberate galacto-N-biose (GNB: Galβ1-3GalNAc) and 2'-fucosyl GNB (a type-4 trisaccharide) from Gb5 pentasaccharide and globo H hexasaccharide, respectively. LnbB, a member of the glycoside hydrolase family 20 isolated from Bifidobacterium bifidum, was shown to release GNB from Gb5 and GA1 oligosaccharides. This is the first report describing enzymatic release of β-linked GNB from natural substrates. These unique activities may play a role in modulating the microbial composition in the gut ecosystem, and may serve as new tools for elucidating the functions of sugar chains of glycosphingolipids.

In single-wafer cleaning of semiconductor chips and flat panel displays, various cleaning methods tailored to the process, such as two-fluid spray cleaning, high-pressure jet spray cleaning, ultrasonic cleaning spray cleaning, and scrub cleaning have been proposed and put to practical use. In this study, Polyethylene Latex (PSL) particles 0.2 µm in diameter and 1 µm in diameter were electro-sprayed on the SiO2 The soil samples, and then cleaning efficiency were measured using two types of ultrasonic sprayers with frequencies of 1 MHz and 3 MHz confirmed. The soil samples are controlled for adhesion by deforming the PSL particles by heating the silicon wafer from 300K to 425K. From the experimental results, it was found that the is determined by factors other than the conventionally reported radiation flow in PSL particles. Figure 1

In this study, polystyrene powder was subjected to CF4 plasma-treatment using a polygonal barrel-plasma-treatment system to enhance the hydrophobicity of the particles. The characterization of the treated samples showed that the orange-peel-like surface of the particles was changed into rugged structures by the CF4 plasma-treatment. The plasma-treatment using our system also resulted in the fluorination of the particle surfaces. The contact angle of a water droplet loaded on the sample treated at a radio frequency (RF) power of 150 W for 10 min (149.1°) was significantly larger than that on the untreated sample (123.6°): both the contact angle value and the fluorine content of sample depended on the RF power. These results indicate that the hydrophobicity of the polystyrene powder was improved by our system, allowing preparation of stable liquid marbles, including CuCl2 solutions, which can be used as NH3 gas sensors.

In this study, tetrafluoromethane (CF4) plasma-treatments of polymethylmethacrylate (PMMA) powder were performed using a polygonal barrel-plasma-treatment system to improve the PMMA's hydrophobicity. Characterization of the treated samples showed that the PMMA particle surfaces were fluorinated by the CF4 treatment. The smooth surfaces of the particles changed into nano-sized worm-like structures after the plasma-treatment. The hydrophobicity of the treated PMMA samples was superior to that of the untreated samples. It was noted that the hydrophobicity of the treated samples and the surface fluorination level depended on the plasma-treatment time and radiofrequency (RF) power; high RF power increased the sample temperature, which in turn decreased the hydrophobicity of the treated samples and the surface fluorination because of the thermal decomposition of PMMA. The water-repellent effects were evaluated by using paper towels to show the application of the plasma-treated PMMA particles, with the result that the paper towel coated with the treated sample was highly water-repellent.

Infant gut-associated bifidobacteria possess species-specific enzymatic sets to assimilate human milk oligosaccharides, and lacto-N-biosidase (LNBase) is a key enzyme that degrades lacto-N-tetraose (Galβ1–3GlcNAcβ1–3Galβ1–4Glc), the main component of human milk oligosaccharides, to lacto-N-biose I (Galβ1–3GlcNAc) and lactose. We have previously identified LNBase activity in Bifidobacterium bifidum and some strains of Bifidobacterium longum subsp. longum (B. longum). Subsequently, we isolated a glycoside hydrolase family 20 (GH20) LNBase from B. bifidum; however, the genome of the LNBase+ strain of B. longum contains no GH20 LNBase homolog. Here, we reveal that locus tags BLLJ_1505 and BLLJ_1506 constitute LNBase from B. longum JCM1217. The gene products, designated LnbX and LnbY, respectively, showed no sequence similarity to previously characterized proteins. The purified enzyme, which consisted of LnbX only, hydrolyzed via a retaining mechanism the GlcNAcβ1–3Gal linkage in lacto-N-tetraose, lacto-N-fucopentaose I (Fucα1–2Galβ1–3GlcNAcβ1–3Galβ1–4Glc), and sialyllacto-N-tetraose a (Neu5Acα2–3Galβ1–3GlcNAcβ1–3Galβ1–4Gal); the latter two are not hydrolyzed by GH20 LNBase. Among the chromogenic substrates examined, the enzyme acted on p-nitrophenyl (pNP)-β-lacto-N-bioside I (Galβ1–3GlcNAcβ-pNP) and GalNAcβ1–3GlcNAcβ-pNP. GalNAcβ1–3GlcNAcβ linkage has been found in O-mannosyl glycans of α-dystroglycan. Therefore, the enzyme may serve as a new tool for examining glycan structures. In vitro refolding experiments revealed that LnbY and metal ions (Ca2+ and Mg2+) are required for proper folding of LnbX. The LnbX and LnbY homologs have been found only in B. bifidum, B. longum, and a few gut microbes, suggesting that the proteins have evolved in specialized niches. Background: Phenotypically lacto-N-biosidase-positive Bifidobacterium longum JCM1217 does not possess a gene homologous to previously identified lacto-N-biosidase. Results: Hypothetical proteins BLLJ_1505 and BLLJ_1506 encode lacto-N-biosidase and its designated chaperone, respectively. Conclusion: The enzyme showed unique and unexpected substrate specificity. Significance: The enzyme is important for understanding how B. longum consumes human milk oligosaccharides and also may serve as a new tool in glycobiology.

The wide prevalence of gluten-related disorders has led to increase in the demand for gluten-free foods. Rice is a gluten-free and less allergenic cereal. However, bread made from rice flour, i.e., gluten-free rice bread, is generally of poor quality because rice flour cannot develop a network with gluten-like properties. In this study, we investigated the effects of protease treatment on gluten-free rice to improve the quality of its bread. Bread treated with a commercial protease from Bacillus stearothermophilus (thermoase) was of higher quality, i.e., good crumb appearance, high volume, and soft texture, depending on the amount of enzyme added. Rice proteins in the protease-treated bread were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which showed that glutelins and prolamins were hardly digested by thermoase in comparison with other proteins. Scanning electron microscopy revealed that many cellular structures were formed in the thermoase-treated bread; however, these structures were rare in the untreated control. Bread crumb color was not affected by the treatment. The staling rate was much lower for the thermoase-treated bread than for the control. These results indicate that thermoase treatment can be successfully used to improve the quality of gluten-free rice bread by partial digestion of rice proteins.

A new plasma-treatment system was developed for preparing a functional powdery material. The nitrogen (N)-doped titanium dioxide (TiO 2 ) powder was prepared to determine the validity of this system. The physical properties of the plasma-treated TiO 2 powder agreed with those of a sample heated at 550 °C in the presence of ammonia, revealing the formation of the N-doped TiO 2 . The absorbance in the visible region and the N contents of the plasma-treated samples depended on the plasma-treatment conditions that included the treatment time, RF power, and N 2 pressure. The specific surface areas of the samples were found to be 231–242 m 2 /g, which were similar to that of the untreated TiO 2 powder (236 m 2 /g).

A class IV chitinase belonging to the glycoside hydrolase 19 family from Nepenthes alata (NaCHIT1) was expressed in Escherichia coli. The enzyme exhibited weak activity toward polymeric substrates and significant activity toward (GlcNAc)(n) [β-1,4-linked oligosaccharide of GlcNAc with a polymerization degree of n (n = 4-6)]. The enzyme hydrolyzed the third and fourth glycosidic linkages from the non-reducing end of (GlcNAc)(6). The pH optimum of the enzymatic reaction was 5.5 at 37°C. The optimal temperature for activity was 60°C in 50 mM sodium acetate buffer (pH 5.5). The anomeric form of the products indicated that it was an inverting enzyme. The k(cat)/K(m) of the (GlcNAc)(n) hydrolysis increased with an increase in the degree of polymerization. Amino acid sequence alignment analysis between NaCHIT1 and a class IV chitinase from a Picea abies (Norway spruce) suggested that the deletion of four loops likely led the enzyme to optimize the (GlcNAc)(n) hydrolytic reaction rather than the hydrolysis of polymeric substrates.

Nanoparticle catalysts of PtRu/C for the direct methanol fuel cell anode were synthesized by a radiolytic process. Bimetallic substructures were controlled by varying irradiation dose rate and by addition of NH4OH or NaH2PO2. Material characterization was performed with the transmission electron microscopy, the X-ray diffraction, the X-ray fluorescence spectroscopy and the X-ray absorption fine structure techniques. Methanol oxidation activity was evaluated by the linear sweep voltammetry. We concluded that the structure of the radiolytically synthesized catalysts has a Pt-rich core/Ru-rich shell structure or incomplete alloy structure. A correlation between the substructures and catalytic activities was found by using a pairing factor defined from coordination numbers determined by the extend X-ray absorption fine structure analysis, which indicates the validity of the bifunctional mechanism in the PtRu nanoparticle system. This radiolytic process is promising for synthesizing advanced PtRu/C catalysts with well-mixed bimetallic substructures enhancing methanol oxidation.

To investigate the substrate recognition at the minus subsites of glycoside hydrolase family 10 xylanases, the kinetic parameters of four xylanases on all four p-nitrophenyl β-glycosides of β-1,4-gluco/xylo-disaccharides were determined. All four xylanases hydrolyzed all the four substrates examined. The Km values of all the enzymes on the four substrates lined up in the same order, indicating that both the subsites -1 and -2 of all the enzymes prefer xylose to glucose. The comparison of the parameters on the substrates gave detailed information on the substrate recognition at each subsite -1 and -2.

Glycosynthases are engineered glycoside hydrolases (GHs) that catalyse the synthesis of glycoside from glycosyl-fluoride donors and suitable acceptors. We have determined five crystal structures of the glycosynthase mutants reducing-end xylose-releasing exo-oligoxylanase, an inverting GH, that exhibit various levels of glycosynthetic activities. At the active site of the Y198F mutant, the most efficient glycosynthase, a water molecule is observed at the same position as nucleophilic water (NW) in the parent enzyme, and the loss of the fixation of the direction of the lone pair of water molecules in the mutant drastically decreases hydrolytic activity. Water molecules were also observed at each active site of the general base mutant, but they were shifted 1.0-3.0 A from the NW in the wild type. Their positions exhibited a strong correlation with the strength of glycosynthase activity. Here, we propose that a structural prerequisite for the sufficient glycosynthase reaction is the presence of a water molecule at the NW position, and mutation at the NW holder provides a general strategy for inverting GHs. The idea on the position of a water molecule may also be applicable to the design of efficient glycosynthases from retaining GHs.

還元末端キシロース遊離エキソオリゴキシラナーゼ（Rex, EC. 3.2.1.156）は，糖加水分解酵素ファミリー 8 に属する反転型キシラン分解酵素であり，キシロオリゴ糖から還元末端キシロース（X1）を遊離する反応を触媒する。Rex は X1 の存在下のみ，α キシロビオシルフルオリド（α-X2F）を加水分解する，Hehre-合成-加水分解反応を示した。一般塩基触媒残基（D263）変異酵素ライブラリーを作製し，グライコシンターゼ活性により α-X2F と X1 からキシロトリオース（X3）を最も良く蓄積する D263C 変異酵素を選抜した。しかしながら，D263C のフッ素イオン遊離活性は親酵素より低かった。次に，一般塩基触媒残基と共に求核試薬である水を保持している残基である Y198 をフェニルアラニンに変異させたところ，フッ素イオン遊離活性が低下することなく加水分解活性のみに大幅に低下が見られた。Rex の Y198F 変異酵素は D263C よりも X3 の蓄積が多かった。最近になり，糖加水分解酵素ファミリー 95 に属する α1,2 フコシダーゼのグライコシンターゼ化が報告された。この場合は，一般塩基触媒残基と相互作用している別の残基に変異を入れることが有効であった。両者ともに，一般塩基触媒残基は変異させない方が良かった。

Flake-shaped fine particles were modified with a thin TiN layer by a hexagonal-barrel-sputtering technique. To determine the optimum sputtering conditions, TiN films were deposited on a glass substrate by the reactive sputtering technique by varying the values of N2 percentage, total pressure, radio-frequency (RF) power, and substrate temperature. From the analysis of XRD patterns, it was determined that a N2 percentage of 25%, a total pressure of 1.2 Pa, a RF power of 200 W, and room temperature were suitable for the preparation of TiN films. Under these optimized conditions, Al flakes were modified with a TiN by the barrel-sputtering technique. The results of optical microscopy, X-ray diffraction measurements, scanning electron microscopy, and energy-dispersive X-ray spectroscopy measurements revealed that the surface of each Al flake was successfully coated uniformly with a TiN layer. [doi:10.2320/matertrans.MRA2008014]

We studied problems in the treatment policies from our treatment experience with 229 cases of asymptomatic saccular unruptured cerebral aneurysm. We used clipping or coiling as needed where the aneurysm was larger than 3 mm, or was located in the dura, and the patient was younger than 70 years, had no serious general disease and had given informed consent. Clipping was used in 170 cases. The mean age was 57.7 years. In 1 case of a high-positioned basilar tip aneurysm, clipping was considered difficult during operation, so it was changed to coiling. Surgical complications were found in 9.4% of cases (transient: 5.3%, permanent: 4.1%). Venous damage occurred in 5 cases, chronic subdural hematoma in 3 cases, memory loss in 3 cases, tight clipping in 2 cases, aggravation of existing cerebral infarction in 1 case, diminution of vision in 1 case and spasm in 1 case. There were no deaths. In the cases of multiple aneurysm, complications were found frequently when surgery was done in 1 session. Coiling was used in 16 cases. The mean age was 63.3 years. In 1 case of a relatively wide neck basilar tip aneurysm, the stability of the coil was poor, so coiling was changed to clipping. Two cases required blood transfusion or suturing of blood vessels because of hematoma at the puncture site. In 1 case, a cholesterol crystal embolism developed. Combined treatment by clipping and coiling was used in 2 cases. Neither case was problematic. Conservative treatment was used in 41 cases. The mean age was 64.3 years. Sixteen cases were treated conservatively because the patients were older than 70 years. The remaining 25 cases were younger than 70 years but were treated conservatively because the aneurysm was located extradurally (10 cases), serious medical complications were found (5 cases) or treatment was indicated but no informed consent was obtained (10 cases). The aneurysm ruptured and the patient died in 2 cases in which no informed consent was obtained. Many of the complications in clipping are preventable, so more careful operative procedures are needed. With coiling, complications attendant upon catheterization pose problems. In 2 cases in which the conservative treatment was not given because informed consent was not obtained, the aneurysm ruptured and the patient died. A more positive attitude about obtaining informed consent on the part of neurosurgeons may be needed.

Reducing end xylose-releasing exo-oligoxylanase from Bacillus halodurans C-125 (Rex) hydrolyzes xylooligosaccharides whose degree of polymerization is greater than or equal to 3, releasing the xylose unit at the reducing end. It is a unique exo-type glycoside hydrolase that recognizes the xylose unit at the reducing end in a very strict manner, even discriminating the beta-anomeric hydroxyl configuration from the alpha-anomer or 1-deoxyxylose. We have determined the crystal structures of Rex in unliganded and complex forms at 1.35-2.20-A resolution and revealed the structural aspects of its three subsites ranging from -2 to +1. The structure of Rex was compared with those of endo-type enzymes in glycoside hydrolase subfamily 8a (GH-8a). The catalytic machinery of Rex is basically conserved with other GH-8a enzymes. However, subsite +2 is blocked by a barrier formed by a kink in the loop before helix alpha10. His-319 in this loop forms a direct hydrogen bond with the beta-hydroxyl of xylose at subsite +1, contributing to the specific recognition of anomers at the reducing end.

肛門縁から18cm口側の直腸癌に対して高位前方切除術を行った10カ月後に,肛門管内に壁内転移を認めた稀な症例を経験したので報告する.症例は64歳,男性.平成15年2月13日,直腸癌の診断にて高位前方切除術を施行した(中分化腺癌, Rs, 2型, 50×60mm, 全周性, se, P0, H0, M(-), ly1, v0, n(-), D3, aw(-), ow(-), ew(-), CurA, Stage II).術後10カ月目の大腸内視鏡検査で,腫瘍下縁が歯状線上にある中心に潰瘍を形成した大きさ20mmの粘膜下腫瘍を認めた.生検検査結果は腺癌で,肛門管癌と診断した.平成16年1月14日,腹会陰式直腸切断術を施行した.切除標本において,肛門管後壁の腫瘍の大きさは25×20mm, 肛門縁より11cm口側の吻合部の腫瘍は15×7mmで,いずれの病変も中分化腺癌であった.この2つの病変には連続性がなく,原発直腸癌と同様の組織像であった.以上より吻合部近くの病変は局所再発,肛門管の病変は壁内転移と判断した.

The gene encoding family 8 glycoside hydrolases from Bacillus halodurans C-125 (BH2105), an alkalophilic bacterium with a known genomic sequence, was expressed in Escherichia coli. The protein was expressed with the intact N-terminal sequence, suggesting that it did not possess a signal peptide and that it was an intracellular enzyme. The recombinant enzyme showed no hydrolytic activity on xylan, whereas it had been annotated as xylanase Y. It hydrolyzed xylooligosaccharide whose degree of polymerization is greater than or equal to 3 in an exo-splitting manner with anomeric inversion, releasing the xylose unit at the reducing end. Judging from its substrate specificity and reaction mechanism, we named the enzyme reducing end xylose-releasing exo-oligoxylanase (Rex). Rex was found to utilize only the beta-anomer of the substrate to form beta-xylose and alpha-xylooligosaccharide. The optimum pH of the enzymatic reaction (6.2-7.3) was found in the neutral range, a range beneficial for intracellular enzymes. The genomic sequence suggests that B. halodurans secretes two endoxylanases and possesses two alpha-arabinofuranosidases, one alpha-glucuronidase, and three beta-xylosidases intracellularly in addition to Rex. The extracellular enzymes supposedly hydrolyze xylan into arabino/glucurono-xylooligosaccharides that are then transported into the cells. Rex may play a role as a key enzyme in intracellular xylan metabolism in B. halodurans by cleaving xylooligosaccharides that were produced by the action of other intracellular enzymes from the arabino/glucurono-xylooligosaccharides.

Vibrio proteolyticus chitobiose phosphorylase (ChBP) belongs to glycosyl transferase family 36 (GT-36), and catalyzes the reversible phosphorolysis of chitobiose into alpha-GlcNAc-1-phosphate and GlcNAc with inversion of the anomeric configuration. As the first known structures of a GT-36 enzyme, we determined the crystal structure of ChBP in a ternary complex with GlcNAc and SO(4). It is also the first structures of an inverting phosphorolytic enzyme in a complex with a sugar and a sulfate ion, and reveals a pseudo-ternary complex structure of enzyme-sugar-phosphate. ChBP comprises a beta sandwich domain and an (alpha/alpha)(6) barrel domain, constituting a distinctive structure among GT families. Instead, it shows significant structural similarity with glycoside hydrolase (GH) enzymes, glucoamylases (GH-15), and maltose phosphorylase (GH-65) in clan GH-L. The structural similarity reported here, together with distant sequence similarities between ChBP and GHs, led to the reclassification of family GT-36 into a novel GH family, namely GH-94.