Hydration of vinyl ether groups by unsaturated glycoside hydrolases and their role in bacterial pathogenesis

Many pathogenic microorganisms invade mammalian and/or plant cells by producing polysaccharide-degrading enzymes (lyases and hydrolases). Mammalian glycosaminoglycans and plant pectins that form part of the cell surface matrix are typical targets for these microbial enzymes. Unsaturated glycoside hydrolase catalyzes the hydrolytic release of an unsaturated uronic acid from oligosaccharides, which are produced through the reaction of matrix-degrading polysaccharide lyase. This enzymatic ability suggests that unsaturated glycoside hydrolases function as virulence factors in microbial infection. This review focuses on the molecular identification, bacterial distribution, and structure/function relationships of these enzymes. In contrast to general glycoside hydrolases, in which the catalytic mechanism involves the retention or inversion of an anomeric configuration, unsaturated glycoside hydrolases uniquely trigger the hydrolysis of vinyl ether groups in unsaturated saccharides but not of their glycosidic bonds. [Int Microbiol 2007; 10(4):233-243

Combined analysis of microstructures within an annual ring of Douglas fir (Pseudotsuga menziesii) by dynamic mechanical analysis and small angle X-ray scattering

Dynamic mechanical analysis (DMA) and small angle X-ray scattering (SAXS) measurements of water-saturated wood of Douglas fir (Pseudotsuga menziesii) in the temperature range of 0 ℃ to 100 ℃ were focused to clarify microstructural changes within an annual ring. The following results were obtained. Thermal softening behavior caused by micro-Brownian motion of lignin was observed in both earlywood and latewood. The peaks of tanδ were found at around 95 ℃ for earlywood and at around 90 ℃ for latewood. These results suggested that the structures of lignin in the cell wall were different between earlywood and latewood. SAXS measurements of water-saturated earlywood and latewood in water were performed with precise temperature control. The scattering intensity increased with increasing temperature, indicating that the density of the matrix was reduced at higher temperature. One-dimensional SAXS intensity at the equator, which approximately represents cellulose microfibrils arrangement in the matrix, was intensively analyzed using the WoodSAS model. The result of this model fitting showed that the cellulose microfibril diameter of latewood was higher than that of earlywood. In addition, the value of interfibrillar distance decreased monotonically in the earlywood, while it decreased rapidly in the latewood from 60 ℃ to 90 ℃. The changes in the cellulose microfibril (CMF) diameter and the interfibrillar distance with increasing temperature between earlywood and latewood by SAXS measurement were different. The differences in CMF diameter and inter-fibril distance between earlywood and latewood measured by SAXS also support the hypothesis that lignin structure differs between earlywood and latewood based on the results of DMA measurements

Characterization of catalytic α-1,3-glucanase isozymes from Paenibacillus glycanilyticus FH11 by using Brevibacillus system; Essential for suppression of Streptococcus mutans biofilms

S. mutans has been implicated in the etiology of dental caries by facilizing the colonization of tooth surfaces and playing a key role in the development of the virulent dental plaque. α-1,3-Glucan, which is a key structural constituent of the biofilm matrix (dental plaque), synthesized by glucosyltransferase type B (gtfB) in the presence of ingested sucrose. α-1,3-Glucanases also called mutanases, which hydrolyze α-1,3-glucan, are classified into two families of glycoside hydrolases, fungal (type 71) and bacterial (type 87). Because of being considered to degrade α-1,3-glucan, α-1,3-glucanases have been purified and characterized from various microbial sources. However, there are few reports on S. mutans biofilm study. For the host cell expression, Brevibacillus system is an effective bacterial expression system for secretory proteins. B. choshinensis is a gram-positive bacterium and easy to handle non-sporulating bacterium, lacking extracellular protease, that has been already shown to provide a high level of recombinant protein expression. Recently, many proteins are produced from this expression system and use for medical treatment, research study (1). Therefore, in this study we attempted to use Brevibacillus expression system to express, purify, and characterize of α-1,3- glucanase. In addition, we aimed to investigate the effect of recombinant enzyme on α-1,3-glucan biofilm produced by S. mutans from the viewpoints of formation and the effect of toothpaste agent on enzyme activity. Two novel catalytic domains of α-1,3-glucanase isozyme genes were cloned from P. glycanilyticus strain FH11 and heterologously expressed in Brevibacillus system. The recombinant isozymes, in termed CatAgl-FH1 and CatAgl-FH2, were purified to homogeneity with specific activity 0.70 U/mg and 0.77 U/mg respectively. The molecular mass of catalytic domain was estimated 62 kDa by SDS-PAGE. Both recombinant enzymes exhibited the different properties. The optimal pH of CatAgl-FH1 and CatAgl-FH2 were 5.5 and 6.0, respectively. The pH stability of CatAgl-FH1 and CatAgl-FH2 were in a range of pH 4.0-11.0 and 4.5-9.0, respectively. The optimal temperature of CatAgl-FH1 and CatAgl-FH2 were 60°C and 55°C, respectively and they were stable until 60°C. Thin Layer chromatography revealed their mode of hydrolysis towards α-1,3-glucan was endo-cleavage pattern. The major products of CatAgl-FH1 were di- and trisaccharide but mainly trisaccharide was for CatAgl-FH2. Both enzymes showed high tolerance against high concentration of sodium fluoride. However, each enzyme activity on surfactants were stepped down when sodium dodecyl sulfate and benzethonium concentration were increased. Please click Additional Files below to see the full abstract

Author Correction:C151 in KEAP1 is the main cysteine sensor for the cyanoenone class of NRF2 activators, irrespective of molecular size or shape

Correction to: Scientific Reportshttps://doi.org/10.1038/s41598-018-26269-9, published online 23 May 2018 This Article contains an error in Figure 3.As a result of an error during the preparation of the figures for this Article, the western blots shown in Figure 3A and 3B contained an additional lane for the protein Tubulin. This is because an additional sample was loaded in the last lane of the gel to prevent potential stretching of the gel in this lane during electrophoresis if left empty. It was subsequently left uncropped from the tubulin blot shown in the published figure. The corrected Figure 3 and its accompanying legend appear below. C151 in KEAP1 is the primary sensor for MCE-23 and MCE-1 in MEF cells. Western blot analyses of total cell lysates of KEAP1-knockout MEF cells rescued with either wild-type (WT), single cysteine mutant C151S, double cysteine mutant C273W/C288E or triple cysteine mutant C151S/C273W/C288E of mouse N-terminally tagged HA-KEAP1. Cells (3 × 105 per well), growing in 6-well plates, were exposed to vehicle (0.1% DMSO) (A,B), MCE-23 (A) or MCE-1 (B) for 3 h, after which the cells were lysed. Immunoblotting was performed on cell lysates using antibodies raised against NRF2, HA and α-tubulin.</p

“Transmantle sign”を示す限局性皮質異形成における神経細胞の成熟と分化の未熟性：層特異的マーカー発現による解析

Transmantle dysplasia is a rare type of focal cortical dysplasia (FCD) characterized by expansion of the cortex from the deep white matter to the surface and in which there is a FCD IIA or IIB pathologic pattern. To characterize possible mechanisms underlying this regional disorder of radial migrating cells, we studied the expression patterns of neocortical layer-specific markers using immunohistochemistry in surgical specimens from 5 FCD IIA and 4 FCD IIB cases in children. All neuronal cells expressed the mature neuron marker MAP2/2B but not the microglia markers Iba-1 and CD68. Some layer-specific markers showed distinct expression patterns. TBR1-positive, SATB2-positive, and FOXP1-positive cells were diffusely distributed in the cortex and/or the white matter. TBR1-positive and FOXP1-positive cells were generally more numerous in FCD IIB than in FCD IIA and were mostly in the cortical molecular and upper layers. FOXP1-, FOXP2-, and CUTL1-positive cells also expressed the immature neuron marker, Nestin/PROX1, whereas TBR1-, CTIP2-, and SATB2-positive cells only expressed MAP2/2B. These data highlight differences between FCD IIB and FCD IIA with more cells having the immature marker in upper layer markers in the former. By analyzing layer-specific marker expression patterns, we identified apparent neuronal maturation differences between FCD IIA and FCD IIB in cases of transmantle dysplasia.博士（医学）・乙第1312号・平成25年5月29

FcRγ-dependent immune activation initiates astrogliosis during the asymptomatic phase of Sandhoff disease model mice

Sandhoff disease (SD) is caused by the loss of β-hexosaminidase (Hex) enzymatic activity in lysosomes resulting from Hexb mutations. In SD patients, the Hex substrate GM2 ganglioside accumulates abnormally in neuronal cells, resulting in neuronal loss, microglial activation, and astrogliosis. Hexb−/− mice, which manifest a phenotype similar to SD, serve as animal models for examining the pathophysiology of SD. Hexb−/− mice reach ~8 weeks without obvious neurological defects; however, trembling begins at 12 weeks and is accompanied by startle reactions and increased limb tone. These symptoms gradually become severe by 16–18 weeks. Immune reactions caused by autoantibodies have been recently associated with the pathology of SD. The inhibition of immune activation may represent a novel therapeutic target for SD. Herein, SD mice (Hexb−/−) were crossed to mice lacking an activating immune receptor (FcRγ−/−) to elucidate the potential relationship between immune responses activated through SD autoantibodies and astrogliosis. Microglial activation and astrogliosis were observed in cortices of Hexb−/− mice during the asymptomatic phase, and were inhibited in Hexb−/− FcRγ−/− mice. Moreover, early astrogliosis and impaired motor coordination in Hexb−/− mice could be ameliorated by immunosuppressants, such as FTY720. Our findings demonstrate the importance of early treatment and the therapeutic effectiveness of immunosuppression in SD