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Find video protocols related to scientific articles indexed in Pubmed.
Spectroscopic investigation for the interaction of mycophenolate mofetil with ferrous ions.
Chem. Pharm. Bull.
PUBLISHED: 11-05-2014
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The interaction of mycophenolate mofetil (MMF) with ferrous ions (Fe(2+)) in the solid state, in water, and in polar organic solvents was investigated using (1)H-NMR, (13)C-NMR, IR, and UV-visible (Vis) spectroscopies. A red-purple colored substance was formed after grinding solid MMF and FeSO4·7H2O in a mortar. The IR spectrum of taken as a KBr tablet of the colored substance showed a new absorption band at 1651?cm(-1). Although the color disappeared when the sample was dissolved in water, it persisted in organic solvents such as MeOH or dimethyl sulfoxide (DMSO). The UV-Vis spectrum of a 0.25?mM MeOH solution of MMF showed a new absorption maximum at 507?nm in the presence of Fe(2+) ions, while an aqueous solution of the same mixture showed no significant change from the MMF solution. All the signals in the (13)C-NMR spectrum in DMSO-d6 solution were unambiguously assigned. Upon the addition of 0.5?eq. of Fe(2+) ions, all the carbon signals except those of the 2-morpholinoethyl group almost disappeared, which clearly indicated that the Fe(2+) ions were located far away from the 2-morpholinoethyl groups in the MMF molecules. On the basis of these results, we have concluded that the MMF-Fe(2+) complex is actually formed in the solid state as well as in polar organic solvents such as MeOH or DMSO.
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TGF- ? Signaling Cooperates with AT Motif-Binding Factor-1 for Repression of the ? -Fetoprotein Promoter.
J Signal Transduct
PUBLISHED: 07-03-2014
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?-Fetoprotein (AFP) is known to be highly produced in fetal liver despite its barely detectable level in normal adult liver. On the other hand, hepatocellular carcinoma often shows high expression of AFP. Thus, AFP seems to be an oncogenic marker. In our present study, we investigated how TGF-? signaling cooperates with AT motif-binding factor-1 (ATBF1) to inhibit AFP transcription. Indeed, the expression of AFP mRNA in HuH-7 cells was negatively regulated by TGF-? signaling. To further understand how TGF-? suppresses the transcription of the AFP gene, we analyzed the activity of the AFP promoter in the presence of TGF-?. We found that the TGF-? signaling and ATBF1 suppressed AFP transcription through two ATBF1 binding elements (AT-motifs). Using a heterologous reporter system, both AT-motifs were required for transcriptional repression upon TGF-? stimulation. Furthermore, Smads were found to interact with ATBF1 at both its N-terminal and C-terminal regions. Since the N-terminal (ATBF1N) and C-terminal regions of ATBF1 (ATBF1C) lack the ability of DNA binding, both truncated mutants rescued the cooperative inhibitory action by the TGF-? signaling and ATBF1 in a dose-dependent manner. Taken together, these findings indicate that TGF-? signaling can act in concert with ATBF1 to suppress the activity of the AFP promoter through direct interaction of ATBF1 with Smads.
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Serological surveillance development for tropical infectious diseases using simultaneous microsphere-based multiplex assays and finite mixture models.
PLoS Negl Trop Dis
PUBLISHED: 07-01-2014
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A strategy to combat infectious diseases, including neglected tropical diseases (NTDs), will depend on the development of reliable epidemiological surveillance methods. To establish a simple and practical seroprevalence detection system, we developed a microsphere-based multiplex immunoassay system and evaluated utility using samples obtained in Kenya.
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Risk factors and spatial distribution of Schistosoma mansoni infection among primary school children in Mbita District, Western Kenya.
PLoS Negl Trop Dis
PUBLISHED: 07-01-2014
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An increasing risk of Schistosoma mansoni infection has been observed around Lake Victoria, western Kenya since the 1970s. Understanding local transmission dynamics of schistosomiasis is crucial in curtailing increased risk of infection.
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Rhinorrhea in Parkinson's disease: a consecutive multicenter study in Japan.
J. Neurol. Sci.
PUBLISHED: 05-14-2014
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Recent reports suggest that rhinorrhea, defined as the presence of a runny nose unrelated to respiratory infections, allergies, or sinus problems, occurs more frequently among patients with Parkinson's disease (PD) than among healthy controls. We conducted a questionnaire survey in a multicenter study throughout Japan and compared the frequency of rhinorrhea between 231 PD and 187 normal control (NC) subjects. After excluding patients with rhinitis or paranasal sinusitis, a total of 159 PD and 59 NC subjects were included in our analysis. Rhinorrhea occurred more frequently in PD patients than NC subjects (33.3% vs. 11.9%; P=0.01). Among PD patients, rhinorrhea was more common in men than women (P=0.005). Rhinorrhea was not correlated with disease duration, modified Hoehn and Yahr score, disease type (akinesia rigidity vs. tremor dominant), or cardiac sympathetic function (evaluated by (123)I-metaiodobenzylguanidine uptake). To our knowledge, this is the first multicenter study on the frequency of PD-related rhinorrhea in Asian countries.
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Galactosyltransferases from Arabidopsis thaliana in the biosynthesis of type II arabinogalactan: molecular interaction enhances enzyme activity.
BMC Plant Biol.
PUBLISHED: 03-25-2014
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Arabinogalactan proteins are abundant proteoglycans present on cell surfaces of plants and involved in many cellular processes, including somatic embryogenesis, cell-cell communication and cell elongation. Arabinogalactan proteins consist mainly of glycan, which is synthesized by post-translational modification of proteins in the secretory pathway. Importance of the variations in the glycan moiety of arabinogalactan proteins for their functions has been implicated, but its biosynthetic process is poorly understood.
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Heat treatment effect on the electronic and magnetic structures of nanographene sheets investigated through electron spectroscopy and conductance measurements.
Phys Chem Chem Phys
PUBLISHED: 03-13-2014
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The heat treatment effect on the electronic and magnetic structures of a disordered network of nanographene sheets has been investigated by in situ measurements of X-ray photoemission spectroscopy, near-edge X-ray absorption fine structure (NEXAFS), and electrical conductance, together with temperature-programmed desorption measurements. Oxygen-containing functional groups bonded to nanographene edges in the pristine sample are almost completely decomposed under heat treatment up to 1300-1500 K, resulting in the formation of edges primarily terminated by hydrogen. The removal of the oxygen-containing groups enhances the conductance owing to the decrease in the electron transport barriers between nanographene sheets. Heat treatment above 1500 K removes also the hydrogen atoms from the edges, promoting the successive fusion of nanographene sheets at the expense of edges. The decrease in the ?* peak width in NEXAFS indicates the progress of the fusion reaction, that is, the extension of the ?-conjugation, which agrees with the increase in the orbital susceptibility previously reported. The fusion leads to the formation of local ?/sp(2) bridges between nanographene sheets and brings about an insulator-to-metal transition at 1500-1600 K, at which the bridge network becomes infinite. As for the magnetism, the intensity of the edge state peak in NEXAFS, which corresponds to the number of the spin-polarized edge states, decreases above 1500 K, though the effective edge-state spin density per edge state starts decreasing at approximately 200 K lower than the temperature of the edge state peak change. This disagreement indicates the development of antiferromagnetic short range ordering as a precursor of a spin glass state near the insulator-metal transition, at which the random network of inter-nanographene-sheet exchange interactions strengthened with the formation of the ?/sp(2) bridges becomes infinite.
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Magnetic edge-states in nanographene, HNO3-doped nanographene and its residue compounds of nanographene-based nanoporous carbon.
Phys Chem Chem Phys
PUBLISHED: 02-27-2014
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We investigated the magnetic and electronic properties of nanographene and its charge transfer effect, using near edge X-ray absorption fine structure (NEXAFS), magnetic susceptibility and ESR measurements, and elemental analysis, with the employment of nanoporous carbon, which consists of a three dimensional disordered network of loosely stacked nanographene sheets, in relation to the host-guest interaction with HNO3 as the electron-accepting guest. The adsorption of electron acceptor HNO3 decreases the intensity of the edge state peak in NEXAFS as a result of the charge-transfer-induced Fermi energy downshift, in agreement with the decrease in the edge-state spin concentration, and it also induces the structural expansion, which makes the inter-nanographene sheet distance elongated, resulting in weakening of the inter-nanographene-sheet antiferromagnetic interaction as evidenced by the decrease in the Weiss temperature. In addition, the decomposition of HNO3, which takes place with the electron-rich edge state as an oxidation catalyst, results in the creation of oxygen/nitrogen-containing functional groups bonded to the periphery of the nanographene sheets. Heat-treatment of the HNO3-ACFs under evacuation desorbs the HNO3 molecules completely, though a part of the oxygen/nitrogen-containing species remains strongly bonded to the edge even at a high temperature of ?800 °C, according to NEXAFS and elemental analysis results. These remaining species participate in the charge transfer, modifying the electronic structure as observed with the decrease in the orbital susceptibility and the strengthening of the inter-nanographene-sheet antiferromagnetic interaction.
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Crystal structure and characterization of the glycoside hydrolase family 62 ?-L-arabinofuranosidase from Streptomyces coelicolor.
J. Biol. Chem.
PUBLISHED: 01-30-2014
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?-L-arabinofuranosidase, which belongs to the glycoside hydrolase family 62 (GH62), hydrolyzes arabinoxylan but not arabinan or arabinogalactan. The crystal structures of several ?-L-arabinofuranosidases have been determined, although the structures, catalytic mechanisms, and substrate specificities of GH62 enzymes remain unclear. To evaluate the substrate specificity of a GH62 enzyme, we determined the crystal structure of ?-L-arabinofuranosidase, which comprises a carbohydrate-binding module family 13 domain at its N terminus and a catalytic domain at its C terminus, from Streptomyces coelicolor. The catalytic domain was a five-bladed ?-propeller consisting of five radially oriented anti-parallel ?-sheets. Sugar complex structures with l-arabinose, xylotriose, and xylohexaose revealed five subsites in the catalytic cleft and an l-arabinose-binding pocket at the bottom of the cleft. The entire structure of this GH62 family enzyme was very similar to that of glycoside hydrolase 43 family enzymes, and the catalytically important acidic residues found in family 43 enzymes were conserved in GH62. Mutagenesis studies revealed that Asp(202) and Glu(361) were catalytic residues, and Trp(270), Tyr(461), and Asn(462) were involved in the substrate-binding site for discriminating the substrate structures. In particular, hydrogen bonding between Asn(462) and xylose at the nonreducing end subsite +2 was important for the higher activity of substituted arabinofuranosyl residues than that for terminal arabinofuranoses.
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Mechanically controllable bi-stable states in a highly conductive single pyrazine molecular junction.
Nanotechnology
PUBLISHED: 07-12-2013
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We report the fabrication of a highly conductive single pyrazine molecular junction with Pt leads. Mechanically controllable break-junction measurements at low temperatures show two distinct high and low conductance states. These conductance values are two orders of magnitude larger than those of a conventional single molecular junction with anchoring groups because of direct binding of the ? conjugated molecule to a metal electrode with large density of states at the Fermi energy. Inelastic electron tunneling spectroscopy combined with density functional theory calculations highlights the vibration modes of the system for the two regimes. Theory allows us to assign the high and low conductance states of the molecular junction to two configurations in which the pyrazine axis is tilted and parallel with respect to the junction axis, respectively. Finally, we show that the pyrazine junction can be reversibly switched between the two bi-stable conductance states by mechanically stretching and relaxing the junction.
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A galactosyltransferase acting on arabinogalactan protein glycans is essential for embryo development in Arabidopsis.
Plant J.
PUBLISHED: 05-31-2013
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Arabinogalactan proteins (AGPs) are a complex family of cell-wall proteoglycans that are thought to play major roles in plant growth and development. Genetic approaches to studying AGP function have met limited success so far, presumably due to redundancy within the large gene families encoding AGP backbones. Here we used an alternative approach for genetic dissection of the role of AGPs in development by modifying their glycan side chains. We have identified an Arabidopsis glycosyltransferase of CAZY family GT31 (AtGALT31A) that galactosylates AGP side chains. A mutation in the AtGALT31A gene caused the arrest of embryo development at the globular stage. The presence of the transcript in the suspensor of globular-stage embryos is consistent with a role for AtGALT31A in progression of embryo development beyond the globular stage. The first observable defect in the mutant is perturbation of the formative asymmetric division of the hypophysis, indicating an essential role for AGP proteoglycans in either specification of the hypophysis or orientation of the asymmetric division plane.
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Perceptions of caregivers about health and nutritional problems and feeding practices of infants: a qualitative study on exclusive breast-feeding in Kwale, Kenya.
BMC Public Health
PUBLISHED: 05-20-2013
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Despite the significant positive effect of exclusive breast-feeding on child health, only 32% of children under 6 months old were exclusively breast-fed in Kenya in 2008. The aim of this study was to explore perceptions and feeding practices of caregivers of children under 6 months old with special attention to the caregivers indigenous knowledge, perceptions about the health and nutritional problems of their infants, and care-seeking behaviors that affect feeding practices.
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Phase-diagram-guided method for growth of a large crystal of glycoside hydrolase family 45 inverting cellulase suitable for neutron structural analysis.
J Synchrotron Radiat
PUBLISHED: 05-10-2013
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Neutron protein crystallography (NPC) is a powerful tool for determining the hydrogen position and water orientation in proteins, but a much larger protein crystal is needed for NPC than for X-ray crystallography, and thus crystal preparation is a bottleneck. To obtain large protein crystals, it is necessary to know the properties of the target protein in the crystallization solution. Here, a crystal preparation method of fungal cellulase PcCel45A is reported, guided by the phase diagram. Nucleation and precipitation conditions were determined by sitting-drop vapor diffusion. Saturation and unsaturation conditions were evaluated by monitoring crystal dissolution, and a crystallization phase diagram was obtained. To obtain a large crystal, crystallization solution was prepared on a sitting bridge (diameter = 5 mm). Initial crystallization conditions were 40 µl of crystallization solution (40 mg ml(-1) protein with 30.5% 3-methyl-1,5-pentanediol in 50 mM tris-HCl pH 8.0) with a 1,000 µl reservoir (61% 3-methyl-1,5,-pentanediol in 50 mM tris-HCl pH 8.0) at 293 K. After the first crystal appeared, the concentration of precipitant in the reservoir solution was reduced to 60% to prevent formation of further crystals. Finally, we obtained a crystal of 6 mm(3) volume (3 mm × 2 mm × 1 mm), which was suitable for neutron diffraction.
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Comparison of six commercially-available DNA polymerases for direct PCR.
Rev. Inst. Med. Trop. Sao Paulo
PUBLISHED: 03-26-2013
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The use of a "direct PCR" DNA polymerase enables PCR amplification without any prior DNA purification from blood samples due to the enzymes resistance to inhibitors present in blood components. Such DNA polymerases are now commercially available. We compared the PCR performance of six direct PCR-type DNA polymerases (KOD FX, Mighty Amp, Hemo KlenTaq, Phusion Blood II, KAPA Blood, and BIOTAQ) in dried blood eluted from a filter paper with TE buffer. GoTaq Flexi was used as a standard DNA polymerase. PCR performance was evaluated by a nested PCR technique for detecting Plasmodium falciparum genomic DNA in the presence of the blood components. Although all six DNA polymerases showed resistance to blood components compared to the standard Taq polymerase, the KOD FX and BIOTAQ DNA polymerases were resistant to inhibitory blood components at concentrations of 40%, and their PCR performance was superior to that of other DNA polymerases. When the reaction mixture contained a mild detergent, only KOD FX DNA polymerase retained the original amount of amplified product. These results indicate that KOD FX DNA polymerase is the most resistant to inhibitory blood components and/or detergents. Thus, KOD FX DNA polymerase could be useful in serological studies to simultaneously detect antibodies and DNA in eluents for antibodies. KOD FX DNA polymerase is thus not limited to use in detecting malaria parasites, but could also be employed to detect other blood-borne pathogens.
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Expression of Arabidopsis thaliana xylose isomerase gene and its effect on ethanol production in Flammulina velutipes.
Fungal Biol
PUBLISHED: 03-18-2013
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To improve the pentose fermentation rate in Flammulina velutipes, the putative xylose isomerase (XI) gene from Arabidopsis thaliana was cloned and introduced into F. velutipes and the gene expression was evaluated in transformants. mRNA expression of the putative XI gene and XI activity were observed in two transformants, indicating that the putative gene from A. thaliana was successfully expressed in F. velutipes as a xylose isomerase. In addition, ethanol production from xylose was increased in the recombinant strains. This is the first report demonstrating the possibility of using plant genes as candidates for improving the characteristics of F. velutipes.
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The structure of a Streptomyces avermitilis ?-L-rhamnosidase reveals a novel carbohydrate-binding module CBM67 within the six-domain arrangement.
J. Biol. Chem.
PUBLISHED: 03-13-2013
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?-L-rhamnosidases hydrolyze ?-linked L-rhamnosides from oligosaccharides or polysaccharides. We determined the crystal structure of the glycoside hydrolase family 78 Streptomyces avermitilis ?-L-rhamnosidase (SaRha78A) in its free and L-rhamnose complexed forms, which revealed the presence of six domains N, D, E, F, A, and C. In the ligand complex, L-rhamnose was bound in the proposed active site of the catalytic module, revealing the likely catalytic mechanism of SaRha78A. Glu(636) is predicted to donate protons to the glycosidic oxygen, and Glu(895) is the likely catalytic general base, activating the nucleophilic water, indicating that the enzyme operates through an inverting mechanism. Replacement of Glu(636) and Glu(895) resulted in significant loss of ?-rhamnosidase activity. Domain D also bound L-rhamnose in a calcium-dependent manner, with a KD of 135 ?m. Domain D is thus a non-catalytic carbohydrate binding module (designated SaCBM67). Mutagenesis and structural data identified the amino acids in SaCBM67 that target the features of L-rhamnose that distinguishes it from the other major sugars present in plant cell walls. Inactivation of SaCBM67 caused a substantial reduction in the activity of SaRha78A against the polysaccharide composite gum arabic, but not against aryl rhamnosides, indicating that SaCBM67 contributes to enzyme function against insoluble substrates.
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Ethanol production from high cellulose concentration by the basidiomycete fungus Flammulina velutipes.
Fungal Biol
PUBLISHED: 02-04-2013
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Ethanol production by Flammulina velutipes from high substrate concentrations was evaluated. F. velutipes produces approximately 40-60 g l(-1) ethanol from 15% (w/v) D-glucose, D-fructose, D-mannose, sucrose, maltose, and cellobiose, with the highest conversion rate of 83% observed using cellobiose as a carbon source. We also attempted to assess direct ethanol fermentation from sugarcane bagasse cellulose (SCBC) by F. velutipes. The hydrolysis rate of 15% (w/v) SCBC with commercial cellulase was approximately 20%. In contrast, F. velutipes was able to produce a significant amount of ethanol from 15% SCBC with the production of ?-glucosidase, cellobohydrolase, and cellulase, although the addition of a small amount of commercial cellulase to the culture was required for the conversion. When 9 mg g(-1) biomass of commercial cellulase was added to cultures, 0.36 g of ethanol was produced from 1 g of cellulose, corresponding to an ethanol conversion rate of 69.6%. These results indicate that F. velutipes would be useful for consolidated bioprocessing of lignocellulosic biomass to bioethanol.
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An autopsy case of sporadic amyotrophic lateral sclerosis associated with the I113T?SOD1 mutation.
Neuropathology
PUBLISHED: 01-29-2013
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A 64-year-old man noticed weakness in his arms and dyspnea upon exertion. Four months later he was admitted to our hospital, where muscle atrophy and hyperactive deep tendon reflexes in the arms were observed upon examination. A needle electromyograph study revealed acute and chronic denervation in the extremities, and he was diagnosed as having amyotrophic lateral sclerosis (ALS). Seven months after onset of the disease, he died of respiratory failure. Neuropathologically, neuronal cell loss was observed in the motor cortex, hypoglossal nuclei, cervical and lumbar anterior horns and Clarkes nuclei. Some of the remaining neurons contained neurofilamentous conglomerate inclusions (CIs). A small number of Lewy body-like hyaline inclusions (LBHIs) were also observed. No the Bunina bodies, skein-like inclusions or basophilic inclusions were detectable. Tract degeneration was moderate in the dorsal and ventral spinocerebellar tracts, mild in the pyramidal tract, but not discerned in the posterior column. Immunohistochemical examinations revealed that the CIs were strongly positive for phosphorylated neurofilament and moderately positive for ubiquitin and Cu/Zn superoxide dismutase 1 (SOD1). Moreover, a number of phosphorylated tau protein-positive globose neurofibrillary tangles (NFTs) and threads were observed in the periaqueductal gray matter, oculomotor nuclei and trochlear nuclei. Although the family history was negative for neuromuscular diseases, the neuropathological findings indicated features of familial ALS with a SOD1 mutation. In fact, DNA analysis of frozen-brain tissue revealed the presence of the I113T?SOD1 mutation. This case represents the first one of this mutation in a patient who showed CIs as well as LBHIs in the motor neurons at the same time, in addition to the NFTs in the mesencephalic tegmentum.
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?-galactosyl Yariv reagent binds to the ?-1,3-galactan of arabinogalactan proteins.
Plant Physiol.
PUBLISHED: 01-07-2013
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Yariv phenylglycosides [1,3,5-tri(p-glycosyloxyphenylazo)-2,4,6-trihydroxybenzene] are a group of chemical compounds that selectively bind to arabinogalactan proteins (AGPs), a type of plant proteoglycan. Yariv phenylglycosides are widely used as cytochemical reagents to perturb the molecular functions of AGPs as well as for the detection, quantification, purification, and staining of AGPs. However, the target structure in AGPs to which Yariv phenylglycosides bind has not been determined. Here, we identify the structural element of AGPs required for the interaction with Yariv phenylglycosides by stepwise trimming of the arabinogalactan moieties using combinations of specific glycoside hydrolases. Whereas the precipitation with Yariv phenylglycosides (Yariv reactivity) of radish (Raphanus sativus) root AGP was not reduced after enzyme treatment to remove ?-l-arabinofuranosyl and ?-glucuronosyl residues and ?-1,6-galactan side chains, it was completely lost after degradation of the ?-1,3-galactan main chains. In addition, Yariv reactivity of gum arabic, a commercial product of acacia (Acacia senegal) AGPs, increased rather than decreased during the repeated degradation of ?-1,6-galactan side chains by Smith degradation. Among various oligosaccharides corresponding to partial structures of AGPs, ?-1,3-galactooligosaccharides longer than ?-1,3-galactoheptaose exhibited significant precipitation with Yariv in a radial diffusion assay on agar. A pull-down assay using oligosaccharides cross linked to hydrazine beads detected an interaction of ?-1,3-galactooligosaccharides longer than ?-1,3-galactopentaose with Yariv phenylglycoside. To the contrary, no interaction with Yariv was detected for ?-1,6-galactooligosaccharides of any length. Therefore, we conclude that Yariv phenylglycosides should be considered specific binding reagents for ?-1,3-galactan chains longer than five residues, and seven residues are sufficient for cross linking, leading to precipitation of the Yariv phenylglycosides.
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Characterization of an ?-L-Rhamnosidase from Streptomyces avermitilis.
Biosci. Biotechnol. Biochem.
PUBLISHED: 01-07-2013
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The putative ?-L-rhamnosidase gene from Streptomyces avermitilis was cloned and expressed. The recombinant enzyme released L-rhamnose from p-nitrophenyl ?-L-rhamnoside, Citrus flavonoids such as naringin, rutin, and hesperidin, and gum arabic which is an arabinogalactan-protein. Calcium ions increased L-rhamnose production by the enzyme from gum arabic, whereas enzyme activity was not affected by any metal ions.
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Analysis of effects of meteorological factors on dengue incidence in Sri Lanka using time series data.
PLoS ONE
PUBLISHED: 01-01-2013
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In tropical and subtropical regions of eastern and South-eastern Asia, dengue fever (DF) and dengue hemorrhagic fever (DHF) outbreaks occur frequently. Previous studies indicate an association between meteorological variables and dengue incidence using time series analyses. The impacts of meteorological changes can affect dengue outbreak. However, difficulties in collecting detailed time series data in developing countries have led to common use of monthly data in most previous studies. In addition, time series analyses are often limited to one area because of the difficulty in collecting meteorological and dengue incidence data in multiple areas. To gain better understanding, we examined the effects of meteorological factors on dengue incidence in three geographically distinct areas (Ratnapura, Colombo, and Anuradhapura) of Sri Lanka by time series analysis of weekly data. The weekly average maximum temperature and total rainfall and the total number of dengue cases from 2005 to 2011 (7 years) were used as time series data in this study. Subsequently, time series analyses were performed on the basis of ordinary least squares regression analysis followed by the vector autoregressive model (VAR). In conclusion, weekly average maximum temperatures and the weekly total rainfall did not significantly affect dengue incidence in three geographically different areas of Sri Lanka. However, the weekly total rainfall slightly influenced dengue incidence in the cities of Colombo and Anuradhapura.
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Effect of lime pretreatment of brown midrib sorghums.
Biosci. Biotechnol. Biochem.
PUBLISHED: 12-07-2011
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The effect of lime pretreatment of brown midrib sorghums on enzymatic saccharification was investigated. Under most of the pretreatment conditions, the saccharification yields of bmrs were higher than those of the normal counterparts. This result suggests that bmr is useful to reduce pretreatment costs, because the amount of lime necessary for the pretreatment of biomass can reduced by using bmr mutants.
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Protein disulfide isomerase-immunopositive inclusions in patients with amyotrophic lateral sclerosis.
Amyotroph Lateral Scler
PUBLISHED: 07-11-2011
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The major pathological hallmarks of amyotrophic lateral sclerosis (ALS) are neuronal cytoplasmic inclusions (NCIs) and swollen neurites. Superoxide dismutase (SOD)-1-immunopositive NCIs are observed in patients with familial ALS (FALS), and TAR DNA-binding protein 43kDa (TDP-43)-immunopositive NCIs are found in patients with sporadic ALS (SALS). Protein disulfide isomerase (PDI) is a member of the thioredoxin superfamily and is believed to accelerate the folding of disulfide-bonded proteins by catalyzing the disulfide interchange reaction, which is the rate-limiting step during protein folding in the luminal space of the endoplasmic reticulum. Post mortem spinal cord specimens from five patients with SALS and one with FALS (I113T), and five normal controls were utilized in this immunohistochemical study. We found PDI-immunopositive swollen neurites and NCIs in the patients with ALS. Furthermore, PDI was colocalized with TDP-43 and SOD1 in NCIs. The accumulation of misfolding proteins may disturb axon transport and make swollen neurites. As the motor neuron is the longest cell in the nervous system, the motor system may selectively be disturbed. In conclusion, we assume that PDI is S-nitrosylated in the affected neurons, which inhibits its enzymatic activity and thus allows protein misfolding to occur in ALS.
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Smad inhibition by the Ste20 kinase Misshapen.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 06-20-2011
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The level of TGF-?/bone morphogenetic protein (BMP) signaling through Smad is tightly regulated to ensure proper embryonic patterning and homeostasis. Here we show that Smad activation by TGF-?/BMP is blocked by a highly conserved phosphorylation event in the ?-helix 1 region of Smad [T312 in Drosophila Smad1 (MAD)]. ?-helix 1 phosphorylation reduces Smad interaction with TGF-?/BMP receptor kinase and affects all receptor-activated Smads except Smad3. Tissue culture and transgenic studies in Drosophila further demonstrate that the biological activity of MAD is repressed by T312 phosphorylation in vivo. Through RNAi screening of the kinome, we have identified Misshapen (Msn) and the mammalian orthologs TNIK, MINK1, and MAP4K4 as the kinases responsible for ?-helix 1 phosphorylation. Targeted expression of an active form of Msn in the wing imaginal disk disrupted activation of endogenous MAD by Dpp and expression of the Dpp/MAD target gene. Msn kinases belong to the Ste20 kinase family that has been shown to act as MAP kinase kinase kinase kinase (MAP4K). Our findings thus reveal a function of Msn independent of its impact on MAP kinase cascades. This Smad inhibition mechanism by Msn likely has important implications for development and disease.
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Endo-beta-1,3-galactanase from winter mushroom Flammulina velutipes.
J. Biol. Chem.
PUBLISHED: 06-08-2011
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Arabinogalactan proteins are proteoglycans found on the cell surface and in the cell walls of higher plants. The carbohydrate moieties of most arabinogalactan proteins are composed of ?-1,3-galactan main chains and ?-1,6-galactan side chains, to which other auxiliary sugars are attached. For the present study, an endo-?-1,3-galactanase, designated FvEn3GAL, was first purified and cloned from winter mushroom Flammulina velutipes. The enzyme specifically hydrolyzed ?-1,3-galactan, but did not act on ?-1,3-glucan, ?-1,3:1,4-glucan, xyloglucan, and agarose. It released various ?-1,3-galactooligosaccharides together with Gal from ?-1,3-galactohexaose in the early phase of the reaction, demonstrating that it acts on ?-1,3-galactan in an endo-fashion. Phylogenetic analysis revealed that FvEn3GAL is member of a novel subgroup distinct from known glycoside hydrolases such as endo-?-1,3-glucanase and endo-?-1,3:1,4-glucanase in glycoside hydrolase family 16. Point mutations replacing the putative catalytic Glu residues conserved for enzymes in this family with Asp abolished activity. These results indicate that FvEn3GAL is a highly specific glycoside hydrolase 16 endo-?-1,3-galactanase.
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Clinicopathologic study on an ALS family with a heterozygous E478G optineurin mutation.
Acta Neuropathol.
PUBLISHED: 04-10-2011
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We investigated a family manifesting amyotrophic lateral sclerosis (ALS) with a heterozygous E478G mutation in the optineurin (OPTN) gene. Clinically, slow deterioration of motor function, mood and personality changes, temporal lobe atrophy on neuroimaging, and bizarre finger deformity were noted. Neuropathologically, TAR DNA-binding protein 43 (TDP-43)-positive neuronal intracytoplasmic inclusions were observed in the spinal and medullary motor neurons. In these cells, the immunoreactivity of nuclear TDP-43 was reduced. Consecutive sections revealed that the inclusions were also reactive with anti-ubiquitin and anti-p62 antibodies, but noticeably negative for OPTN. In addition, TDP-43/p62-positive glial cytoplasmic inclusions (GCIs) were scattered throughout the spinal cord and the medullary motor nuclei. Furthermore, Golgi fragmentation was identified in 70% of the anterior horn cells (AHCs). The presence of AHCs with preserved nuclear TDP-43 and a fragmented Golgi apparatus, which are unrecognizable in sporadic ALS, indicates that patients with the E4787G OPTN mutation would manifest Golgi fragmentation before loss of nuclear TDP-43. In the neocortex, GCIs were sparsely scattered among the primary motor and temporal cortices, but no neuronal TDP-43-positive inclusions were detected. In the amygdala and the ambient gyrus, argyrophilic grains and ballooned neurons were seen. The thorough neuropathologic investigations performed in this work demonstrated that OPTN-positive inclusion bodies, if any, were not prominent. We postulate that optineurinopathy is closely linked with TDP-proteinopathy and speculate that this heterozygous E478G mutation would cause ALS by acting through a dominant-negative mechanism.
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The Relationship between Performance and Trunk Movement During Change of Direction.
J Sports Sci Med
PUBLISHED: 03-01-2011
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The purpose of this study was to obtain the trunk kinematics data during a change-of-direction task and to determine the relationship between trunk kinematics and the change-of-direction performance. The design of this investigation was a descriptive laboratory study. Twelve healthy male collegiate soccer players (age: 21.3 ± 1.0 yrs, body mass: 67. 7 ± 6.7 kg, and height: 1.75 ± 0.05 m) participated in this study. Participants performed a shuttle run cutting task with a 180 degree pivot as quickly as possible. The shuttle run cutting time, ground contact time during a change-of-direction, and trunk inclination angle were measured. The shuttle run cutting time tends to correlate positively with ground contact time. During the change- of-direction task, the trunk forward inclination angle gradually increased during the first 50% of the stance phase and decreased subsequently whereas the trunk flexed, maintaining a left inclination during the first 40% of the stance phase and changing exponentially in the opposite direction. Forward angular displacement of the trunk between foot-contact and maximum trunk inclination correlated positively with the shuttle run cutting time (r = 0.61, p < 0.05) and ground contact time (r = 0.65, p < 0.05). These findings suggest that the change-of-direction performance could be related to the small angular displacement of the trunk during a change of direction. Moreover, it was considered that there might be optimal inclination angles related to change-of-direction performance. Therefore, coaches in field sports should check body posture and trunk movements during changes of direction. Key pointsSmall forward angular displacement of the trunk during a direction change is related to the change-of-direction performance.Trunk stability during a change of direction is an important factor in the change-of-direction performance.There might be a range of optimal angles of trunk inclination during a change of direction.Coaches in field sports should check the body posture and trunk movement of players when they require a change of direction or when they participate in sport-specific change-of-direction training.
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The structure and function of an arabinan-specific alpha-1,2-arabinofuranosidase identified from screening the activities of bacterial GH43 glycoside hydrolases.
J. Biol. Chem.
PUBLISHED: 02-21-2011
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Reflecting the diverse chemistry of plant cell walls, microorganisms that degrade these composite structures synthesize an array of glycoside hydrolases. These enzymes are organized into sequence-, mechanism-, and structure-based families. Genomic data have shown that several organisms that degrade the plant cell wall contain a large number of genes encoding family 43 (GH43) glycoside hydrolases. Here we report the biochemical properties of the GH43 enzymes of a saprophytic soil bacterium, Cellvibrio japonicus, and a human colonic symbiont, Bacteroides thetaiotaomicron. The data show that C. japonicus uses predominantly exo-acting enzymes to degrade arabinan into arabinose, whereas B. thetaiotaomicron deploys a combination of endo- and side chain-cleaving glycoside hydrolases. Both organisms, however, utilize an arabinan-specific ?-1,2-arabinofuranosidase in the degradative process, an activity that has not previously been reported. The enzyme can cleave ?-1,2-arabinofuranose decorations in single or double substitutions, the latter being recalcitrant to the action of other arabinofuranosidases. The crystal structure of the C. japonicus arabinan-specific ?-1,2-arabinofuranosidase, CjAbf43A, displays a five-bladed ?-propeller fold. The specificity of the enzyme for arabinan is conferred by a surface cleft that is complementary to the helical backbone of the polysaccharide. The specificity of CjAbf43A for ?-1,2-l-arabinofuranose side chains is conferred by a polar residue that orientates the arabinan backbone such that O2 arabinose decorations are directed into the active site pocket. A shelflike structure adjacent to the active site pocket accommodates O3 arabinose side chains, explaining how the enzyme can target O2 linkages that are components of single or double substitutions.
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Structure of arabinogalactan oligosaccharides derived from arabinogalactan-protein of Coffea arabica instant coffee powder.
Carbohydr. Res.
PUBLISHED: 02-09-2011
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Arabinogalactan-protein, previously isolated from instant coffee powder of Coffea arabica, has been subjected to partial mild acidic and enzymatic hydrolyses. Separation of obtained mixtures by size exclusion and HPLC chromatographies afforded series of oligosaccharides, structure of which were studied by NMR spectroscopy. Mild acidic hydrolysis afforded oligosaccharides without any ?Araf substituent while after enzymatic hydrolysis ?Araf was found in di-, tri-, and tetrasaccharides. In all cases ?Araf was a terminal substituent linked separately to O3, O6, and to both, O3 and O6, of ?Gal residues. Identification of di-, tri-, and tetrasaccharides containing ?-Araf enabled to distinguish in the (1)H NMR spectra ?Araf signals linked to O6 and O3 of neighboring ?Gal unit. Composition of polymeric residues after enzymatic and mild acidic hydrolyses was also analyzed.
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Myonuclear breakdown in sporadic inclusion body myositis is accompanied by DNA double strand breaks.
Neuromuscul. Disord.
PUBLISHED: 01-18-2011
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Rimmed vacuoles in sporadic inclusion body myositis (s-IBM) contain nuclear remnants. We sought to determine if the nuclear degeneration seen in s-IBM is associated with DNA damage. In muscle biopsy specimens from ten patients with s-IBM and 50 controls, we immunolocalized 1) phosphorylated histone H2AX (?-H2AX), which is a sensitive immunocytochemical marker of DNA double-strand breaks and 2) DNA-PK, which is an enzyme involved in double-strand break repair. In s-IBM, vacuolar peripheries often showed strong immunoreactivity to ?-H2AX and the three components of DNA-PK (DNA-PKcs, Ku70, and Ku80). A triple fluorescence study of Ku70, emerin, and DNA displayed nuclear breakdown and it suggested impaired nuclear incorporation of Ku70. The percentage of positive nuclei for ?-H2AX was significantly higher in vacuolated fibers than non-vacuolated fibers in s-IBM, or fibers in polymyosits. We hypothesize that a dysfunction of nuclear envelope may cause nuclear fragility, double-strand breaks and impaired nuclear transport in s-IBM.
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Induction of regulatory T cells by infliximab in Behcets disease.
Invest. Ophthalmol. Vis. Sci.
PUBLISHED: 01-01-2011
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To determine whether infliximab induces the development of Foxp3+ T regulatory (Treg) cells in patients with Behçets disease.
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Improvement of the transformation efficiency of Flammulina velutipes Fv-1 using the glyceraldehyde-3-phosphate dehydrogenase gene promoter.
Biosci. Biotechnol. Biochem.
PUBLISHED: 12-07-2010
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To improve the expression level of heterologous genes in Flammulina velutipes Fv-1, we constructed new vectors having glyceraldehydes-3-phosphate dehydrogenase (gpd) gene promoter to control the expression of target genes. When the hygromycin B phosphotransferase (hph) gene from Escherichia coli was controlled by the gpd promoter, transformation efficiency was 3-fold higher than the case of that controlled by the tryptophan synthetase gene (trp1) promoter.
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Lissencephaly-1 controls germline stem cell self-renewal through modulating bone morphogenetic protein signaling and niche adhesion.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 11-01-2010
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In the Drosophila ovary, bone morphogenetic protein (BMP) signaling activated by the niche promotes germline stem cell (GSC) self-renewal and proliferation, whereas E-cadherin-mediated cell adhesion anchors GSCs in the niche for their continuous self-renewal. Here we show that Lissencephaly-1 (Lis1) regulates BMP signaling and E-cadherin-mediated adhesion between GSCs and their niche and thereby controls GSC self-renewal. Lis1 mutant GSCs are lost faster than control GSCs because of differentiation but not because of cell death, indicating that Lis1 controls GSC self-renewal. The Lis1 mutant GSCs exhibit reduced BMP signaling activity, and Lis1 interacts genetically with the BMP pathway components in the regulation of GSC maintenance. Mechanistically, Lis1 binds directly to and stabilizes the SMAD protein Mothers against decapentaplegic (Mad), facilitates its phosphorylation, and thereby regulates BMP signaling. Finally, the Lis1 mutant GSCs accumulate less E-cadherin in the stem cell-niche junction than do their wild-type counterparts. Germline-specific expression of an activated BMP receptor thickveins (Tkv) or E-cadherin can partially rescue the loss phenotype of Lis1 mutant GSCs. Therefore, this study has revealed a role of Lis1 in the control of Drosophila ovarian GSC self-renewal, at least partly by regulating niche signal transduction and niche adhesion. It has been known that Lis1 controls neural precursor/stem cell proliferation in the developing mammalian brain; this study further suggests that Lis1, which is widely expressed in adult mammalian tissues, could regulate adult tissue stem cells through modulating niche signaling and adhesion.
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Recognition of the helical structure of beta-1,4-galactan by a new family of carbohydrate-binding modules.
J. Biol. Chem.
PUBLISHED: 09-08-2010
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The microbial enzymes that depolymerize plant cell wall polysaccharides, ultimately promoting energy liberation and carbon recycling, are typically complex in their modularity and often contain carbohydrate-binding modules (CBMs). Here, through analysis of an unknown module from a Thermotoga maritima endo-?-1,4-galactanase, we identify a new family of CBMs that are most frequently found appended to proteins with ?-1,4-galactanase activity. Polysaccharide microarray screening, immunofluorescence microscopy, and biochemical analysis of the isolated module demonstrate the specificity of the module, here called TmCBM61, for ?-1,4-linked galactose-containing ligands, making it the founding member of family CBM61. The ultra-high resolution X-ray crystal structures of TmCBM61 (0.95 and 1.4 ? resolution) in complex with ?-1,4-galactotriose reveal the molecular basis of the specificity of the CBM for ?-1,4-galactan. Analysis of these structures provides insight into the recognition of an unexpected helical galactan conformation through a mode of binding that resembles the recognition of starch.
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miR-21 and miR-31 converge on TIAM1 to regulate migration and invasion of colon carcinoma cells.
J. Biol. Chem.
PUBLISHED: 09-07-2010
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TGF-? promotes cell migration and invasion, an attribute that is linked to the pro-metastasis function of this cytokine in late stage cancers. The LIM 1863 colon carcinoma organoid undergoes epithelial-mesenchymal transition (EMT) in response to TGF-?. This process is markedly accelerated by TNF-?, and we found that the levels of miR-21 and miR-31 were prominently elevated under the synergistic actions of TGF-?/TNF-?. Consistent with this, overexpression of either miR-21 or miR-31 significantly enhanced the effect of TGF-? alone on LIM 1863 morphological changes. More importantly, transwell assays demonstrated the positive effects of both miR-21 and miR-31 in TGF-? regulation of LIM 1863 motility and invasiveness. Elevated levels of miR-21 and miR-31 also enhanced motility and invasiveness of other colon carcinoma cell lines. We present compelling evidence that TIAM1, a guanidine exchange factor of the Rac GTPase, is a direct target of both miR-21 and miR-31. Indeed in LIM 1863 cells, suppression of TIAM1 is required for miR-21/miR-31 to enhance cell migration and invasion. Therefore, we have uncovered miR-21 and miR-31 as downstream effectors of TGF-? in facilitating invasion and metastasis of colon carcinoma cells.
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Crystallization and preliminary crystallographic analysis of the glycoside hydrolase family 115 ?-glucuronidase from Streptomyces pristinaespiralis.
Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun.
PUBLISHED: 08-30-2010
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?-Glucuronidase from Streptomyces pristinaespiralis (SpGlcA115A) is composed of a single-chain peptide containing a catalytic domain belonging to glycosyl hydrolase family 115, a novel family of hemicellulolytic ?-glucuronidases. The enzyme catalyzes the hydrolysis of ?-linked 4-O-methylglucuronosyl and glucuronosyl residues from both polymeric xylans and oligosaccharides. SpGlcA115A was crystallized at 293?K using the sitting-drop vapour-diffusion method. The crystals belonged to space group R3 and diffracted to a resolution of 1.9?Å.
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Crystal structure of an Exo-1,5-{alpha}-L-arabinofuranosidase from Streptomyces avermitilis provides insights into the mechanism of substrate discrimination between exo- and endo-type enzymes in glycoside hydrolase family 43.
J. Biol. Chem.
PUBLISHED: 08-25-2010
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Exo-1,5-?-L-arabinofuranosidases belonging to glycoside hydrolase family 43 have strict substrate specificity. These enzymes hydrolyze only the ?-1,5-linkages of linear arabinan and arabino-oligosaccharides in an exo-acting manner. The enzyme from Streptomyces avermitilis contains a core catalytic domain belonging to glycoside hydrolase family 43 and a C-terminal arabinan binding module belonging to carbohydrate binding module family 42. We determined the crystal structure of intact exo-1,5-?-L-arabinofuranosidase. The catalytic module is composed of a 5-bladed ?-propeller topologically identical to the other family 43 enzymes. The arabinan binding module had three similar subdomains assembled against one another around a pseudo-3-fold axis, forming a ?-trefoil-fold. A sugar complex structure with ?-1,5-L-arabinofuranotriose revealed three subsites in the catalytic domain, and a sugar complex structure with ?-L-arabinofuranosyl azide revealed three arabinose-binding sites in the carbohydrate binding module. A mutagenesis study revealed that substrate specificity was regulated by residues Asn-159, Tyr-192, and Leu-289 located at the aglycon side of the substrate-binding pocket. The exo-acting manner of the enzyme was attributed to the strict pocket structure of subsite -1, formed by the flexible loop region Tyr-281-Arg-294 and the side chain of Tyr-40, which occupied the positions corresponding to the catalytic glycon cleft of GH43 endo-acting enzymes.
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Extracellular carbohydrate esterase from the basidiomycete Coprinopsis cinerea released ferulic and acetic acids from xylan.
Biosci. Biotechnol. Biochem.
PUBLISHED: 08-07-2010
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cDNA encoding an extracellular carbohydrate esterase (CcEst1) was cloned from the basidiomycete Coprinopsis cinerea. The recombinant CcEst1 expressed in Pichia pastoris acted on p-nitrophenyl acetate, alpha-naphthyl acetate, and methyl hydroxycinnamic acids, except for methyl sinapic acid. The enzyme released ferulic and acetic acids from wheat arabinoxylan and acetylated xylan respectively. Activity increased on the addition of endo-beta-1,4-xylanase.
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Degradation of carbohydrate moieties of arabinogalactan-proteins by glycoside hydrolases from Neurospora crassa.
Carbohydr. Res.
PUBLISHED: 07-01-2010
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Arabinogalactan-proteins (AGPs) are a family of plant proteoglycans having large carbohydrate moieties attached to core-proteins. The carbohydrate moieties of AGPs commonly have ?-(1?3)(1?6)-galactan as the backbone, to which other auxiliary sugars such as l-Ara and GlcA are attached. For the present study, an ?-L-arabinofuranosidase belonging to glycoside hydrolase family (GHF) 54, NcAraf1, and an endo-?-(1?6)-galactanase of GHF 5, Nc6GAL, were identified in Neurospora crassa. Recombinant NcAraf1 (rNcAraf1) expressed in Pichia pastoris hydrolyzed radish AGPs as well as arabinan and arabinoxylan, showing relatively broad substrate specificity toward polysaccharides containing ?-L-arabinofuranosyl residues. Recombinant Nc6GAL (rNc6GAL) expressed in P. pastoris specifically acted on ?-(1?6)-galactosyl residues. Whereas AGP from radish roots was hardly hydrolyzed by rNc6GAL alone, ?-(1?6)-galactan side chains were reduced to one or two galactan residues by a combination of rNcAraf1 and rNc6GAL. These results suggest that the carbohydrate moieties of AGPs are degraded by the concerted action of NcAraf1 and Nc6GAL secreted from N. crassa.
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Carbohydrate structural analysis of wheat flour arabinogalactan protein.
Carbohydr. Res.
PUBLISHED: 05-28-2010
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The water-extractable arabinogalactan protein (AGP) was isolated from bread wheat flour (Triticum aestivum L. variety Cadenza) and the structure of the arabinogalactan (AG) carbohydrate component was studied. Oligosaccharides, released by hydrolysis of the AG with a range of AGP-specific enzymes, were characterised by Matrix Assisted Laser Desorption Ionisation (MALDI)-Time of Flight (ToF)-Mass Spectrometry (MS), MALDI-ToF/ToF high energy collision induced dissociation (CID) and Polysaccharide Analysis by Carbohydrate gel Electrophoresis (PACE). The AG is composed of a ?-(1?3)-D-galactan backbone with ?-(1?6)-D-galactan side chains. These side chains are highly variable in length, from one to at least 20 Gal residues and are highly substituted with ?-L-Araf. Single GlcA residues are also present at the non-reducing termini of some short ?-(1?6)-galactan side chains. In addition, the ?-(1?6)-galactan side chains are also substituted with ?-L-Arap. We propose a polysaccharide structure of the wheat flour AGP that is substantially revised from earlier models.
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Development of a gene transfer system for the mycelia of Flammulina velutipes Fv-1 strain.
Biosci. Biotechnol. Biochem.
PUBLISHED: 05-07-2010
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To develop a gene transformation method for Flammulina velutipes, we constructed a vector with hph gene under control of the trp1 gene promoter. The vector was integrated into protoplast derived from mycelia by the calcium-polyethylene glycol method, as it has not been reported for F. velutipes. Transformation efficiency was much improved when transformation was performed by the restriction enzyme mediated integration method.
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Sleeping arrangement and house structure affect bed net use in villages along Lake Victoria.
Malar. J.
PUBLISHED: 03-14-2010
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Although insecticide-treated bed nets are effective tools, use often does not follow ownership. House structure and space arrangements may make the attempt to use bed nets difficult, especially for school age children. The objectives of this study were to explore whether an individuals sleeping arrangements and house structure affect bed net use in villages along Lake Victoria in western Kenya.
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Regulation of proapoptotic mammalian ste20-like kinase MST2 by the IGF1-Akt pathway.
PLoS ONE
PUBLISHED: 02-18-2010
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Hippo, a Drosophila serine/threonine kinase, promotes apoptosis and restricts cell growth and proliferation. Its mammalian homolog MST2 has been shown to play similar role and be regulated by Raf-1 via a kinase-independent mechanism and by RASSF family proteins through forming complex with MST2. However, regulation of MST2 by cell survival signal remains largely unknown.
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Phosphoinositide 3-kinase/Akt inhibits MST1-mediated pro-apoptotic signaling through phosphorylation of threonine 120.
J. Biol. Chem.
PUBLISHED: 11-24-2009
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The protein kinase mammalian sterile 20-like kinase 1 (MST1) is a mammalian homologue of the Drosophila hippo and plays a critical role in regulation of programmed cell death. MST1 exerts pro-apoptotic function through cleavage, autophosphorylation-Thr(183) and subsequent translocation to the nucleus where it phosphorylates a number of molecules, including LATS1/2, FOXO, JNK, and histone H2B. Here, we show that the cleavage of MST1 is inhibited by the phosphatidylinositol 3-kinase/Akt pathway. Akt interacts with MST1 and phosphorylates a highly conserved residue threonine 120 of MST1, which leads to inhibition of its kinase activity and nuclear translocation as well as the autophosphorylation of Thr(183). Phospho-MST1-Thr(120) failed to activate downstream targets FOXO3a and JNK. Further, inverse correlation between pMST1-Thr(120) and pMST1-Thr(183) was observed in human ovarian tumors. These findings indicate that the phosphorylation of MST1-Thr(120) by Akt could be a major mechanism of regulation of the Hippo/MST1 pathway by cell survival signaling.
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Nuclear contour irregularity and abnormal transporter protein distribution in anterior horn cells in amyotrophic lateral sclerosis.
J. Neuropathol. Exp. Neurol.
PUBLISHED: 10-10-2009
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The nucleocytoplasmic transport system is essential for maintaining cell viability; transport of proteins and nucleic acids between the nucleus and the cytoplasm occurs through nuclear pore complexes (NPCs). In this study, we examined the immunohistochemical distribution of the major protein components of NPCs, Nup62, Nup88, and Nup153, in spinal cords from controls and patients with sporadic or familial amyotrophic lateral sclerosis (SALS or FALS) and its mouse model. In control subjects, immunolabeling on the nuclear envelopes of anterior horn cells (AHCs) was invariably smooth and continuous, whereas in SALS and FALS patients, the AHCs predominantly showed irregular nuclear contours. Double immunofluorescence staining demonstrated that in SALS patients, importin-beta immunoreactivity was absent in the nuclei in a subset of AHCs; in these cells, Nup62 immunolabeling of nuclear membrane was invariably irregular, suggesting that there was dysfunctional nucleocytoplasmic transport in those AHCs. In the mouse model, Nup62-immunolabeled AHCs with irregular nuclear contours were predominant as early as the presymptomatic stage and the contours became progressively discontinuous along with disease development. Together, these observations suggest that dysfunctional nucleocytoplasmic transport may underlie the pathogenesis of ALS.
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Molecular cloning and expression in Pichia pastoris of a Irpex lacteus exo-beta-(1-->3)-galactanase gene.
Biosci. Biotechnol. Biochem.
PUBLISHED: 10-07-2009
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A gene encoding exo-beta-(1-->3)-galactanase from Irpex lacteus was cloned by reverse transcriptase-PCR. The deduced amino acid sequence showed high similarity with exo-beta-(1-->3)-galactanases from other sources. The molecular mass of the mature form was calculated to be 45,520 Da. The gene product expressed in Pichia pastoris specifically hydrolyzed beta-(1-->3)-galactooligosaccharides, as did other exo-beta-(1-->3)-galactanases. The recombinant enzyme showed high activity toward arabinogalactan-proteins (AGPs) from radish as well as beta-(1-->3)-galactan. Product analysis revealed that the enzyme released beta-(1-->6)-galactobiose, beta-(1-->6)-galactotriose, and alpha-L-arabinofuranosyl-(1-->3)-beta-galactosyl-(1-->6)-galactose together with Gal from beta-(1-->3)-galactans attached with and without beta-(1-->6)-galactosyl branches prepared from acacia gum. These results indicate that the exo-beta-(1-->3)-galactanase from I. lacteus efficiently hydrolyzes beta-(1-->3)-galactan main chains of AGPs by bypassing beta-(1-->6)-galactosyl side chains.
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Properties of ethanol fermentation by Flammulina velutipes.
Biosci. Biotechnol. Biochem.
PUBLISHED: 10-07-2009
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Basidiomycetes have the ability to degrade lignocellulosic biomass, and some basidiomycetes produce alcohol dehydrogenase. These characteristics may be useful in the direct production of ethanol from lignocellulose. Ethanol fermentation by basidiomycetes was investigated to examine the possibility of ethanol production by consolidated bioprocessing (CBP) using Flammulina velutipes. F. velutipes converted D-glucose to ethanol with a high efficiency (a theoretical ethanol recovery rate of 88%), but ethanol production from pentose was not observed. These properties of F. velutipes are similar to those of Saccharomyces cerevisiae, but the basidiomycete converted not only sucrose, but also maltose, cellobiose, cellotriose, and cellotetraose to ethanol, with almost the same efficiency as that for D-glucose. From these results, we concluded that F. velutipes possesses advantageous characteristics for use in CBP.
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The tetramer structure of the glycoside hydrolase family 27 alpha-galactosidase I from Umbelopsis vinacea.
Biosci. Biotechnol. Biochem.
PUBLISHED: 10-07-2009
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The crystal structure of Umbelopsis vinacea alpha-galactosidase I, which belongs to glycoside hydrolase family 27, was determined at 2.0 A resolution. The monomer structure was well conserved with those of glycoside hydrolase family 27 enzymes. The biological tetramer structure of this enzyme was constructed by the crystallographic 4-fold symmetry, and tetramerization appeared to be caused by three inserted peptides that were involved in the tetramer interface. The quaternary structure indicated that the substrate specificity of this enzyme might be related to the tetramer formation. Three N-glycosylated sugar chains were observed, and their structures were found to be of the high-mannose type.
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Bifunctional cytosolic UDP-glucose 4-epimerases catalyse the interconversion between UDP-D-xylose and UDP-L-arabinose in plants.
Biochem. J.
PUBLISHED: 09-17-2009
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UDP-sugars serve as substrates in the synthesis of cell wall polysaccharides and are themselves generated through sequential interconversion reactions from UDP-Glc (UDP-glucose) as the starting substrate in the cytosol and the Golgi apparatus. For the present study, a soluble enzyme with UDP-Xyl (UDP-xylose) 4-epimerase activity was purified approx. 300-fold from pea (Pisum sativum L.) sprouts by conventional chromatography. The N-terminal amino acid sequence of the enzyme revealed that it is encoded by a predicted UDP-Glc 4-epimerase gene, PsUGE1, and is distinct from the UDP-Xyl 4-epimerase localized in the Golgi apparatus. rPsUGE1 (recombinant P. sativum UGE1) expressed in Escherichia coli exhibited both UDP-Xyl 4-epimerase and UDP-Glc 4-epimerase activities with apparent Km values of 0.31, 0.29, 0.16 and 0.15 mM for UDP-Glc, UDP-Gal (UDP-galactose), UDP-Ara (UDP-L-arabinose) and UDP-Xyl respectively. The apparent equilibrium constant for UDP-Ara formation from UDP-Xyl was 0.89, whereas that for UDP-Gal formation from UDP-Glc was 0.24. Phylogenetic analysis revealed that PsUGE1 forms a group with Arabidopsis UDP-Glc 4-epimerases, AtUGE1 and AtUGE3, apart from a group including AtUGE2, AtUGE4 and AtUGE5. Similar to rPsUGE1, recombinant AtUGE1 and AtUGE3 expressed in E. coli showed high UDP-Xyl 4-epimerase activity in addition to their UDP-Glc 4-epimerase activity. Our results suggest that PsUGE1 and its close homologues catalyse the interconversion between UDP-Xyl and UDP-Ara as the last step in the cytosolic de novo pathway for UDP-Ara generation. Alternatively, the net flux of metabolites may be from UDP-Ara to UDP-Xyl as part of the salvage pathway for Ara.
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Crystallization of selenomethionyl exo-beta-1,3-galactanase from the basidiomycete Phanerochaete chrysosporium.
Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun.
PUBLISHED: 09-04-2009
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Exo-beta-1,3-galactanase from Phanerochaete chrysosporium (Pc1,3Gal43A) consists of a glycoside hydrolase family 43 catalytic domain and a substrate-binding domain that belongs to carbohydrate-binding module family 35. It catalyzes the hydrolysis of beta-1,3-galactan, which is the backbone of the arabinogalactan proteins; the C-terminal carbohydrate-binding module family 35 domain increases the local concentration of the enzyme around beta-1,3-galactan by its high affinity for the substrate. To enable phase determination using the multiwavelength anomalous dispersion method, selenomethionyl Pc1,3Gal43A was crystallized at 298 K using the hanging-drop vapour-diffusion method. The presence of selenium in the crystals was confirmed from the X-ray absorption spectrum. The crystals belonged to space group P2(1) and diffracted to 1.8 A resolution.
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A beta-l-Arabinopyranosidase from Streptomyces avermitilis is a novel member of glycoside hydrolase family 27.
J. Biol. Chem.
PUBLISHED: 07-16-2009
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Arabinogalactan proteins (AGPs) are a family of plant cell surface proteoglycans and are considered to be involved in plant growth and development. Because AGPs are very complex molecules, glycoside hydrolases capable of degrading AGPs are powerful tools for analyses of the AGPs. We previously reported such enzymes from Streptomyces avermitilis. Recently, a beta-l-arabinopyranosidase was purified from the culture supernatant of the bacterium, and its corresponding gene was identified. The primary structure of the protein revealed that the catalytic module was highly similar to that of glycoside hydrolase family 27 (GH27) alpha-d-galactosidases. The recombinant protein was successfully expressed as a secreted 64-kDa protein using a Streptomyces expression system. The specific activity toward p-nitrophenyl-beta-l-arabinopyranoside was 18 micromol of arabinose/min/mg, which was 67 times higher than that toward p- nitrophenyl-alpha-d-galactopyranoside. The enzyme could remove 0.1 and 45% l-arabinose from gum arabic or larch arabinogalactan, respectively. X-ray crystallographic analysis reveals that the protein had a GH27 catalytic domain, an antiparallel beta-domain containing Greek key motifs, another antiparallel beta-domain forming a jellyroll structure, and a carbohydrate-binding module family 13 domain. Comparison of the structure of this protein with that of alpha-d-galactosidase showed a single amino acid substitution (aspartic acid to glutamic acid) in the catalytic pocket of beta-l-arabinopyranosidase, and a space for the hydroxymethyl group on the C-5 carbon of d-galactose bound to alpha-galactosidase was changed in beta-l-arabinopyranosidase. Mutagenesis study revealed that the residue is critical for modulating the enzyme activity. This is the first report in which beta-l-arabinopyranosidase is classified as a new member of the GH27 family.
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Use of whole crop sorghums as a raw material in consolidated bioprocessing bioethanol production using Flammulina velutipes.
Biosci. Biotechnol. Biochem.
PUBLISHED: 07-07-2009
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The possibility of using two kinds of sorghum as raw materials in consolidated bioprocessing bioethanol production using Flammulina velutipes was investigated. Enzymatic saccharification of sweet sorghum was not as high as in brown mid-rib (bmr) mutated sorghum, but the amount of ethanol production was higher. Ethanol production from bmr mutated sorghum significantly increased when saccharification enzymes were added to the culture.
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Characterization of glycoside hydrolase family 6 enzymes from Coprinopsis cinerea.
Biosci. Biotechnol. Biochem.
PUBLISHED: 06-07-2009
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Recombinants, Cel6 from Coprinopsis cinerea (CcCel6)A, -B, and -C, were expressed by Escherichia coli. These enzymes produced cellobiose from phosphoric acid-swollen cellulose. When Avicel was used as the substrate, CcCel6A showed higher activity toward the substrate than CcCel6B or -C. In reaction with carboxymethylcellulose, CcCel6B and -C hydrolyzed the substrate, whereas CcCel6A failed to react.
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[A case of anti-AQP4 antibody-positive recurrent myelitis overlapped with autoimmune disorders including incomplete CREST syndrome revealed multiple discontinuous cord lesions].
Rinsho Shinkeigaku
PUBLISHED: 04-08-2009
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A 65-year-old woman presenting with multiple autoimmune disorders including incomplete CREST overlapping with aquaporin 4 (AQP4) antibody-positive recurrent myelitis was reported. She also clinically suffered from Sjogren syndrome and primary biliary cirrhosis (PBC). She had dysesthesia below C4 level, mild motor weakness and hyperreflexia without pathological reflexes on bilateral lower extremities. A T2-weighted MRI indicated multiple discontinuous spinal cord lesions at C1-5 and T7/8. A visual evoked potential study disclosed bilateral prolonged latency of P100. She clinically manifested not only incomplete CREST syndrome (facial teleangiectasia, sclerodactyly in bilateral fingers, and Raynauds phenomenon), but also Sjögren (sicca syndrome) and PBC (jaundice). Immunoserological study showed that she was positive for anti-nuclear, anti-centromere, and anti-AQP4 (= NMO-IgG) antibodies. A combination therapy with corticosteroid and plasmapheresis was effective for all clinical symptoms. Therefore, this case stresses on the relevance of anti-AQP 4 antibody to the other overlapping autoimmune disorders, such as CREST syndrome, when recurrent myelitis is clinically diagnosed.
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Crystallographic snapshots of an entire reaction cycle for a retaining xylanase from Streptomyces olivaceoviridis E-86.
J. Biochem.
PUBLISHED: 03-11-2009
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Retaining glycosyl hydrolases, which catalyse both glycosylation and deglycosylation in a concerted manner, are the most abundant hydrolases. To date, their visualization has tended to be focused on glycosylation because glycosylation reactions can be visualized by inactivating deglycosylation step and/or using substrate analogues to isolate covalent intermediates. Furthermore, during structural analyses of glycosyl hydrolases with hydrolytic reaction products by the conventional soaking method, mutarotation of an anomeric carbon in the reaction products promptly and certainly occurs. This undesirable structural alteration hinders visualization of the second step in the reaction. Here, we investigated X-ray crystallographic visualization as a possible method for visualizing the conformational itinerary of a retaining xylanase from Streptomyces olivaceoviridis E-86. To clearly define the stereochemistry at the anomeric carbon during the deglycosylation step, extraneous nucleophiles, such as azide, were adopted to substitute for the missing base catalyst in an appropriate mutant. The X-ray crystallographic visualization provided snapshots of the components of the entire reaction, including the E*S complex, the covalent intermediate, breakdown of the intermediate and the enzyme-product (E*P)complex.
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Crystallization and preliminary crystallographic analysis of beta-L-arabinopyranosidase from Streptomyces avermitilis NBRC14893.
Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun.
PUBLISHED: 03-02-2009
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Beta-L-arabinopyranosidase from Streptomyces avermitilis NBRC14893 is a monomeric protein consisting of a catalytic domain belonging to glycosyl hydrolase family 27, an unknown domain and a substrate-binding domain belonging to carbohydrate-binding module family 13. The complete enzyme (residues 45-658) has successfully been cloned and homologously expressed in the Streptomyces expression system. beta-L-Arabinopyranosidase was crystallized by the sitting-drop vapour-diffusion method. The crystals diffracted to 1.6 A resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 68.2, b = 98.9, c = 181.3 A. The Matthews coefficient was calculated to be 2.38 A(3) Da(-1).
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Identification of three mutant loci conferring carboxin-resistance and development of a novel transformation system in Aspergillus oryzae.
Fungal Genet. Biol.
PUBLISHED: 02-12-2009
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Mutants exhibiting resistance to the fungicide, carboxin, were isolated from Aspergillus oryzae, and the mutations in the three gene loci, which encode succinate dehydrogenase (SDH) B, C, and D subunits, were identified to be independently responsible for the resistance. A structural model of the SDH revealed the different mechanisms that confer carboxin-resistance in different mutations. The mutant AosdhB gene (AosdhB(cxr)) was further examined for possible use as a transformant selection marker. After transformation with AosdhB(cxr), carboxin-resistant colonies appeared within 4 days of culture, and all of the examined colonies carried the transgene. Insertion analyses revealed that the AosdhB(cxr) gene was integrated into AosdhB locus via homologous recombination at high efficiency. Furthermore, AosdhB(cxr) functioned as a successful selection marker in a transformation experiment in Aspergillus parasiticus, suggesting that this transformation system can be used for Aspergillus species.
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A novel family of hemicellulolytic alpha-glucuronidase.
FEBS Lett.
PUBLISHED: 02-09-2009
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Investigation of the xylanolytic enzyme system of the xylose-fermenting yeast Pichia stipitis resulted in the discovery of an extracellular alpha-glucuronidase efficiently debranching hardwood glucuronoxylan. This activity is not exhibited by more extensively investigated alpha-glucuronidases of glycoside hydrolase (GH) family 67, operating on substrates in which the uronic acid is linked to the non-reducing xylopyranosyl residues of main chain fragments. The N-terminus of the purified enzyme corresponded exactly to the P. stipitis gene ABN67901 coding for a protein of unknown function. BLAST search revealed the presence of similar genes in genomes of other microorganisms. These results lead to the emergence of a new family of alpha-glucuronidases.
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Heterologous expression, crystallization and preliminary X-ray characterization of CcCel6C, a glycoside hydrolase family 6 enzyme from the basidiomycete Coprinopsis cinerea.
Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun.
PUBLISHED: 01-31-2009
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CcCel6C is a gene that encodes a glycoside hydrolase family 6 (GH6) enzyme in the Coprinopsis cinerea genome. In the evolutionary tree of GH6 enzymes, the encoded enzyme was closely related to Cel6B from Humicola insolens, previously called endoglucanase VI, while its amino-acid sequence revealed a region corresponding to the C-terminal active-site-enclosing loop typical of cellobiohydrolase II. Here, the crystallization of CcCel6C produced in Escherichia coli is reported. The square prismatic crystal belonged to the triclinic space group P1, with unit-cell parameters a = 44.04, b = 45.11, c = 48.90 A, alpha = 77.81, beta = 87.34, gamma = 68.79 degrees. Diffraction data were collected to 1.6 A resolution.
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Mortality after exposure to polychlorinated biphenyls and polychlorinated dibenzofurans: a 40-year follow-up study of Yusho patients.
Am. J. Epidemiol.
PUBLISHED: 01-24-2009
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A 40-year follow-up study was conducted to examine mortality among 1,664 patients in Japan suffering from "Yusho," a disease caused by ingestion of rice oil contaminated with polychlorinated biphenyls and polychlorinated dibenzofurans. To evaluate the effects of exposure on mortality, the authors calculated standardized mortality ratios. National mortality rates for major causes of death were used as reference points. A total of 1,596 Yusho patients (95.9%) were followed until death or the end of the study (December 31, 2007). The standardized mortality ratios for most major causes of death were not significantly elevated, with the exceptions of all types of cancer (standardized mortality ratio (SMR) = 1.37, 95% confidence interval (CI): 1.11, 1.66), liver cancer (SMR = 1.82, 95% CI: 1.06, 2.91), and lung cancer (SMR = 1.75, 95% CI: 1.14, 2.57) in males. In addition, the standardized mortality ratios for all cancers, liver cancer, and lung cancer among males tended to decrease over time. Results from this study suggest that the carcinogenicity of polychlorinated biphenyls and polychlorinated dibenzofurans must be taken into account when evaluating mortality risk.
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Cloning and transcript analysis of multiple genes encoding the glycoside hydrolase family 6 enzyme from Coprinopsis cinerea.
Biosci. Biotechnol. Biochem.
PUBLISHED: 01-07-2009
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We searched the genome database of the basidiomycete Coprinopsis cinerea (Coprinus cinereus) and found five genes encoding the glycoside hydrolase family 6 (GH6) enzyme, CcCel6A, CcCel6B, CcCel6C, CcCel6D, and CcCel6E, designated in order of increasing locus number (CC1G_01107.1, CC1G_04166.1, CC1G_08276.1, CC1G_08277.1, and CC1G_10605.1). The amino acid sequence of CcCel6A suggests a two-domain structure consisting of an N-terminal family 1 carbohydrate-binding module (CBM1) and a GH6 catalytic domain, while the other genes lack CBM1. The transcripts of CcCel6A were observed at the active growth stage in cellulose culture, whereas they were absent from glucose culture. Cellobiose strongly induced transcription of CcCel6A. On the other hand, transcripts of CcCel6B, -D, and -E were detected in both glucose and cellulose cultures, and transcription of them was induced weakly by cellobiose. The transcript level of CcCel6C was not influenced by glucose or cellobiose.
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Rice seeds as sources of endophytic bacteria.
Microbes Environ.
PUBLISHED: 01-01-2009
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Endophytic bacteria are considered to originate from the external environment. To examine the hypothesis that rice (Oryza sativa, cultivar Kinuhikari) seeds are a source of endophytic bacteria, we isolated endophytic bacteria from the shoots, remains of the seeds, and roots of rice seedlings that were aseptically cultivated in vitro from surface-disinfected seeds. Of the various bacterial strains isolated, the closest relatives, identified by 16S rRNA gene sequencing, were: Bacillus firmus, B. fusiformis, B. pumilus, Caulobacter crescentus, Kocuria palustris, Micrococcus luteus, Methylobacterium fujisawaense, Me. radiotolerans, and Pantoea ananatis. The latter three species have been detected frequently inside both rice seedlings and mature rice plants. These results indicate that rice seeds are an important source of endophytic bacteria. The bacteria that colonize the seed interior appear to infect the subsequent generation via rice seeds and become the dominant endophytic species in the mature plant.
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Single molecule bridging between metal electrodes.
Phys Chem Chem Phys
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Single molecular junctions, in which a single molecule bridges between metal electrodes, have attracted wide attention as novel properties can appear due to their peculiar geometrical and electronic characters. The single molecular junction has also attracted attention due to its potential application in ultrasmall single molecular electronic devices, where single molecules are utilized as active electronic components. Thus, fabrication of single molecular junctions as well as understanding and controlling their properties (e.g. conductance, optical and magnetic properties) have become long-standing goals of scientists and engineers. This review article focuses on the experimental aspects of single molecular junctions, with primary focus on the electron transport mechanism.
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Prednisolone-sparing effect of cyclosporin A therapy for very elderly patients with myasthenia gravis.
Neuromuscul. Disord.
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Three very elderly (over 80years old) patients having generalized myasthenia gravis without thymoma were treated with cyclosporin A and followed for up to 24months. Cyclosporin A therapy quickly improved myasthenia gravis symptoms in all cases, which allowed a rapid reduction in the prednisolone dose and improvement of prednisolone-related hyperglycemia and hypertension. Combination therapy with prednisolone and low-dose cyclosporin A not only improved the clinical symptoms of the very elderly myasthenia gravis patients but also resulted in a rapid reduction in prednisolone dosage and prednisolone-related side effects. Attention should be paid to cyclosporin A-related renal dysfunction.
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Prevalence and risk factors of neurological impairment among children aged 6-9 years: from population based cross sectional study in western Kenya.
BMC Pediatr
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The burden of disability is more severe among children in low income countries. Moreover, the number of children with disabilities (CWDs) in sub-Saharan Africa is predicted to increase with reduction in child mortality. Although the issue on CWDs is important in sub-Saharan Africa, there are few researches on risk factors of disabilities. The purpose of this study was to evaluate the risk factors of neurological impairment (NI) among children in western Kenya.
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Are long-lasting insecticidal nets effective for preventing childhood deaths among non-net users? A community-based cohort study in western Kenya.
PLoS ONE
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Increasing the distribution and use of insecticide-treated nets (ITNs) in Sub-Saharan Africa has made controlling malaria with ITNs more practical. We evaluated community effects induced by ITNs, specifically long-lasting insecticidal nets (LLINs), under ordinary conditions in an endemic malaria area of Western Kenya.
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Characterization of an endo-processive-type xyloglucanase having a ?-1,4-glucan-binding module and an endo-type xyloglucanase from Streptomyces avermitilis.
Appl. Environ. Microbiol.
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We cloned two glycoside hydrolase family 74 genes, the sav_1856 gene and the sav_2574 gene, from Streptomyces avermitilis NBRC14893 and characterized the resultant recombinant proteins. The sav_1856 gene product (SaGH74A) consisted of a catalytic domain and a family 2 carbohydrate-binding module at the C terminus, while the sav_2574 gene product (SaGH74B) consisted of only a catalytic domain. SaGH74A and SaGH74B were expressed successfully and had molecular masses of 92 and 78 kDa, respectively. Both recombinant proteins were xyloglucanases. SaGH74A had optimal activity at 60°C and pH 5.5, while SaGH74B had optimal activity at 55°C and pH 6.0. SaGH74A was stable over a broad pH range (pH 4.5 to 9.0), whereas SaGH74B was stable over a relatively narrow pH range (pH 6.0 to 6.5). Analysis of the hydrolysis products of tamarind xyloglucan and xyloglucan-derived oligosaccharides indicated that SaGH74A was endo-processive, while SaGH74B was a typical endo-enzyme. The C terminus of SaGH74A, which was annotated as a carbohydrate-binding module, bound to ?-1,4-linked glucan-containing soluble polysaccharides such as hydroxyethyl cellulose, barley glucan, and xyloglucan.
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PHF20 is an effector protein of p53 double lysine methylation that stabilizes and activates p53.
Nat. Struct. Mol. Biol.
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PHF20 is a multidomain protein and subunit of a lysine acetyltransferase complex that acetylates histone H4 and p53 but whose function is unclear. Using biochemical, biophysical and cellular approaches, we determined that PHF20 is a direct regulator of p53. A Tudor domain in PHF20 recognized p53 dimethylated at Lys370 or Lys382 and a homodimeric form of this Tudor domain could associate with the two dimethylated sites on p53 with enhanced affinity, indicating a multivalent interaction. Association with PHF20 promotes stabilization and activation of p53 by diminishing Mdm2-mediated p53 ubiquitylation and degradation. PHF20 contributes to upregulation of p53 in response to DNA damage, and ectopic expression of PHF20 in different cell lines leads to phenotypic changes that are hallmarks of p53 activation. Overall our work establishes that PHF20 functions as an effector of p53 methylation that stabilizes and activates p53.
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Activation of transforming growth factor-?/Smad signaling reduces aggregate formation of mislocalized TAR DNA-binding protein-43.
Neurodegener Dis
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TAR DNA-binding protein of 43 kDa (TDP-43) is naturally located in the nucleus and has been identified as the major component of cytoplasmic ubiquitinated inclusions in patients with amyotrophic lateral sclerosis (ALS). We have reported that TDP-43 and phosphorylated Smad2 (pSmad2), an intracellular mediator protein of transforming growth factor-? (TGF?) signaling, are co-localized within cytoplasmic inclusions in the anterior horn cells of sporadic ALS patients.
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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.