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[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.

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.

α-L-fucosyl residues attached at the non-reducing ends of glycoconjugates constitute histo-blood group antigens Lewis (Le) and ABO and play fundamental roles in various biological processes. Therefore, establishing a method for synthesizing the antigens is important for functional glycomics studies. However, regiospecific synthesis of glycosyl linkages, especially α-L-fucosyl linkages, is quite difficult to control both by chemists and enzymologists. Here, we generated an α-L-fucosynthase that specifically introduces Le(a) and Le(x) antigens into the type-1 and type-2 chains, respectively; i.e. the enzyme specifically accepts the disaccharide structures (Galβ1-3/4GlcNAc) at the non-reducing ends and attaches a Fuc residue via an α-(1,4/3)-linkage to the GlcNAc. X-ray crystallographic studies revealed the structural basis of this strict regio- and acceptor specificity, which includes the induced fit movement of the catalytically important residues, and the difference between the active site structures of 1,3-1,4-α-L-fucosidase (EC 3.2.1.111) and α-L-fucosidase (EC 3.2.1.51) in glycoside hydrolase family 29. The glycosynthase developed in this study should serve as a potentially powerful tool to specifically introduce the Le(a/x) epitopes onto labile glycoconjugates including glycoproteins. Mining glycosidases with strict specificity may represent the most efficient route to the specific synthesis of glycosidic bonds.

The reducing-end xylose-releasing exo-oligoxylanase (Rex) from Bacillus halodurans C-125, a novel family GH8 glycoside hydrolase, was crystallized by the hanging-drop vapour-diffusion method using 13.6 mg ml(-1) purified Rex, 5.6%(v/v) polyethylene glycol 4000, 70 mM sodium acetate pH 4.6 and 30%(v/v) glycerol. Suitable crystals grew after incubation for 5 d at 293 K. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 52.69, b = 86.02, c = 87.92 A. X-ray diffraction data were collected at a resolution of 1.35 A.

An intercellular interaction between mouse Ehrlich ascites tumor and non-malignant Chinese hamster cells occurred when these were co-cultured. That the intercellular processes which formed had emanated from the EA cells was revealed by immunofluoroscopy using anti-EA antiserum, and by direct microscopic examination. A passage of DNA from the EA to the CH cells was also observed. On long-term co-culture, new cell forms arose which were isolated, cloned, and propagated. They showed a CH karyotype and had acquired oncogenic potential and the ability to synthesize murine-specific antigens. These same heritable properties were also acquired by CH cells following their exposure to DNA isolated from EA cells.

Fucosyloligosaccharides have great therapeutic potential. Here we present a new route for synthesizing a Fucalpha1,2Gal linkage by introducing glycosynthase technology into 1,2-alpha-l-fucosidase. The enzyme adopts a unique reaction mechanism, in which asparagine-423 activated by aspartic acid-766 acts as a base while asparagine-421 fixes both a catalytic water and glutamic acid-566 (an acid) in the proper orientations. Glycosynthase activity of N421G, N423G, and D766G mutants was examined using beta-fucosyl fluoride and lactose, and among them, the D766G mutant most effectively synthesized 2'-fucosyllactose. 1,2-alpha-l-Fucosynthase is the first glycosynthase derived from an inverting alpha-glycosidase and from a glycosidase with an unusual reaction mechanism.

The hydrolytic reaction of family 18 chitinase has been considered to occur via substrate assisted catalysis. To kinetically investigate the enzyme reaction mechanism, we synthesized compounds designed to reduce the polarization of the carbonyl in N-acetyl group, GlcNAc-GlcN(TFA)-UMB (2) and GlcNAc-GlcN(TAc)-UMB (3). Kinetic parameters in the hydrolysis of these compounds by chitinase A from Serratia marcescens (ChiA) were compared with those from the hydrolysis of (GlcNAc)2-UMB (1). The kcat of 2 was 3.4% of 1, but the Km of 2 was 10-fold that of 1. In contrast, the kcat of 3 was only 0.3% of that of 1, and the two reactions had an identical Km. The drastic decreases in kcat were probably due to the weak nucleophilic activity of the C2-N-trifluoroacetamide and N-thioacetamide groups at reducing ends of compounds 2 and 3, respectively. These results indicate that the anchimeric assistance of the C2 N-acetamide group at GlcNAc plays a key role in the hydrolytic reactions catalyzed by family 18 chitinases.