Enzyme-responsive molecular assembly system with amylose-primer surfactants.
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Amylose↗
Biomedical subjects
Shinichi Kitamura
Publications and source records attributed to Shinichi Kitamura.
Characterization of a GDP-D-mannose 3'',5''-epimerase from rice.
The enzymatic characterization of GDP-d-mannose 3'',5''-epimerase (GME), a key enzyme in the biosynthesis of vitamin C in plants is described. The GME gene (Genbank Accession No. AB193582) in rice was cloned, and expressed as a fusion protein in Escherichia coli. Reaction products from GDP-d-mannose, as produced by GME catalysis, were separated by recycling HPLC on an ODS column, and were determined to be GDP-l-galactose and GDP-l-gulose, based on their NMR spectra and sugar analysis. The reaction catalyzed by GME was inhibited by GDP, and was strongly accelerated by NAD(+) in contrast to the case of GME from Arabidopsis thaliana. This difference in the effect of NAD(+) on GME activity can be attributed to the NAD binding domain which is conserved in the rice gene, but not in the Arabidopsis thaliana gene. The apparent K(m) and k(cat) were determined to be 1.20x10(-5)M and 0.127s(-1), respectively, in the presence of 20microM NAD(+). The fractions of GDP-d-mannose, GDP-l-galactose and GDP-l-gulose, at equilibrium, were approximately 0.75, 0.20 and 0.05, respectively.
Arabidopsis↗
Characterization of soluble and putative membrane-bound UDP-glucuronic acid decarboxylase (OsUXS) isoforms in rice.
Arabinoxylans in crop plants are the major sugar components of the cell walls, and UDP-xylose is a key substrate in the biosynthesis of xylans. In this study, the six putative UDP-D-glucuronic acid decarboxylase genes from rice (Oryza sativa UDP-xylose synthase; OsUXS) were cloned. Except for the soluble form of OsUXS3 (GenBank Accession No. \AB079064), the remaining five OsUXS enzymes contain a putative membrane-bound region. The six OsUXS genes were classified into three types by phylogenetic analysis and were expressed during the development of rice seeds. The HPLC retention times of the enzyme products and NMR data, indicate that the recombinant OsUXS2 enzyme catalyzes the conversion of UDP-D-glucuronic acid to UDP-D-xylose. Interestingly, the reactions catalyzed by the recombinant OsUXS2 and OsUXS3 enzymes were inhibited by NADP+, and accelerated by NADPH. The catalytic activities of the recombinant OsUXS2 and OsUXS3 enzymes were strongly inhibited by UDP, UTP, TDP, and TTP. The expression levels of OsUXS genes changed in different manners during the development of rice seeds, suggesting that each corresponding OsUXS enzyme plays a significant role in rice seed development at a certain stage. In the present study, we report that the UXS2-type enzyme of rice is not only characterized for the first time but also show significant findings involved in the gene expression of OsUXSs.
Amino Acid Motifs↗
High-coverage profiling analysis of genes expressed during rice seed development, using an improved amplified fragment length polymorphism technique.
A novel method, based on quantitative PCR and amplified fragment length polymorphism was applied to the analysis of high-coverage gene expression profiles during the development of rice seeds. This represents the first report of the application of this method to plants, which permitted the detection and analysis of approximately 70% of all the genes that are expressed in rice. The method was used to compare gene expression at different developmental stages, subspecies or cultivars, and phyletic lines to identify genes of interest through differences in their level of expression. Using this approach, even novel anonymous genes could be detected. Examples of these include the soluble starch synthase (SS) II-I and the rice branching enzyme 4 (rbe4) genes in the starch synthesis pathway. A profiling database was compiled and the results compared with public data on full-length cDNA sequences of rice. The method enables candidate novel genes to be immediately identified among the large numbers of genes that are expressed during the development of rice seeds. Our results will contribute to a better understanding of comparative transcriptomics in all plant species.
Gene Expression Profiling↗
Structural characterization and biological activities of an exopolysaccharide kefiran produced by Lactobacillus kefiranofaciens WT-2B(T).
Lactobacillus kefiranofaciens, isolated from kefir grains, produces an extracellular polysaccharide when cultured, not only in PYG10 medium but also in a liquid medium containing a rice hydrolysate that had been previously degraded by treatment with a glucoamylase. The maximum yield of the polysaccharide, using the rice hydrolysate as the medium, was 2.5 g/L after a 7-day culture period at pH 5.0 and 33 degrees C. Compositional analysis, methylation analysis, specific rotation, and (1)H and (13)C NMR spectroscopy revealed that the structures of polysaccharides obtained from these two different culture media are essentially identical. The polysaccharide is composed of a hexasaccharide repeating unit and, thus, is known as kefiran. The weight-average molecular weight and the z-average radius of gyration of a sample, purified from the rice hydrolysate medium, were determined to be 7.6 x 10(5) g/mol and 39.9 nm, respectively, by gel permeation chromatography equipped with a multiangle laser-light-scattering photometer. Changes in blood pressure and serum components were examined in SHRSP/Hos rats, using doses of 100 and 300 mg of kefiran/kg of rat. A suppression in the increase in blood pressure was observed in these rats after 30 days. This activity is discussed in terms of the concentration of serum components of the rat, with emphasis on lipid components such as cholesterols, triglycerides, and free fatty acids.
Animals↗
Effects of an exopolysaccharide (kefiran) on lipids, blood pressure, blood glucose, and constipation.
Lactobacillus kefiranofaciens was reported to produce an exopolysaccharide named kefiran. In the present study, we developed a new medium, rice hydrolyzate (RH) medium, for the culture of L. kefiranofaciens. Structural analyses revealed that the exopolysaccharide produced by L. kefiranofaciens from RH medium was composed of a hexasaccharide repeating unit, and essentially identical to the kefiran reported in previous studies. A study on the effects of kefiran in animals demonstrated that kefiran significantly suppressed increase of blood pressure and reduced the serum cholesterol levels in SHRSP/Hos rats when subjects consumed excessive dietary cholesterol. Kefiran supplementation demonstrated the ability to significantly lower blood glucose in KKAy mice. In addition, the administration of kefiran in constipated SD rats caused an obvious improvement in the levels of fecal moisture and wet weights of feces. These results suggest that kefiran could be used as a functional food to prevent some commonly occurring diseases.
Animals↗
Cloning and expression of a UDP-glucuronic acid decarboxylase gene in rice.
A cDNA fragment was cloned from rice immature seeds by the RT-PCR method. The deduced amino acid sequence of the cDNA showed a high degree of identity with UDP-d-glucuronic acid decarboxylase (UXS) from other plants and was most similar to the soluble UXS from Arabidopsis. The recombinant protein, expressed in an Escherichia coli system, catalysed the conversion of UDP-d-glucuronic acid to UDP-d-xylose, confirming that the gene encoded UXS. The uxs gene was expressed in mature, harvested rice seeds as well as in immature seeds 14 d post-anthesis, suggesting that the uxs gene is necessary at the beginning of the germination period. This is the first report of the cloning of the uxs gene from monocots.
Amino Acid Sequence↗
Crystal structure of a catalytic site mutant of beta-amylase from Bacillus cereus var. mycoides cocrystallized with maltopentaose.
The X-ray crystal structure of a catalytic site mutant of beta-amylase, E172A (Glu172 --> Ala), from Bacillus cereus var. mycoides complexed with a substrate, maltopentaose (G5), and the wild-type enzyme complexed with maltose were determined at 2.1 and 2.0 A resolution, respectively. Clear and continuous density corresponding to G5 was observed in the active site of E172A, and thus, the substrate, G5, was not hydrolyzed. All glucose residues adopted a relaxed (4)C(1) conformation, and the conformation of the maltose unit for Glc2 and Glc3 was much different from those of other maltose units, where each glucose residue of G5 is named Glc1-Glc5 (Glc1 is at the nonreducing end). A water molecule was observed 3.3 A from the C1 atom of Glc2, and 3.0 A apart from the OE1 atom of Glu367 which acts as a general base. In the wild-type enzyme-maltose complex, two maltose molecules bind at subsites -2 and -1 and at subsites +1 and +2 in tandem. The conformation of the maltose molecules was similar to that of the condensation product of soybean beta-amylase, but differed from that of G5 in E172A. When the substrate flips between Glc2 and Glc3, the conformational energy of the maltose unit was calculated to be 20 kcal/mol higher than that of the cis conformation by MM3. We suggest that beta-amylase destabilizes the bond that is to be broken in the ES complex, decreasing the activation energy, DeltaG(++), which is the difference in free energy between this state and the transition state.
Bacillus cereus↗
Translational diffusion coefficient of cycloamylose in aqueous sodium hydroxide.
Seven cyclic (1 --> 4)-alpha-D-glucan (cycloamylose) samples ranging in weight-average molecular weight from 5 x 10(3) to 1.8 x 10(4) and gamma-cyclodextrin have been studied by sedimentation equilibrium in dimethylsulfoxide (at 25 degrees C) and by dynamic light scattering in 0.5 N aqueous sodium hydroxide (at 25 degrees C), a good solvent for linear amylose. The measured translational diffusion coefficients D in the aqueous NaOH agree fairly closely with previous Monte Carlo results for cyclic (1 --> 4)-alpha-D-glucan chains with excluded volume, when correction is made for the effects of bead diameter and fluctuating hydrodynamic interaction (HI) on the Kirkwood theory on which the computation of D was based. These D data are also explained almost quantitatively by Yamakawa and Fujii's expression for the associated KP ring (based on the Kratky-Porod wormlike chain) with the molecular parameters for linear amylose if the fluctuating HI and excluded-volume effects are taken into account. It is concluded that the translational diffusion behavior of cycloamylose in the aqueous NaOH is consistent with the conformational characteristics derived from the conformational energy of maltose and dilute-solution data for linear amylose.
Cyclodextrins↗
Fourth 3D structure of the chitosan molecule: conformation of chitosan in its salts with medical organic acids having a phenyl group.
Chitosan salts with two medical organic acids having phenyl groups (salicylic and gentisic acids) exhibited fiber diffraction patterns of a new type of crystal which does not compare with known types I and II. The crystals, called type III salts, showed a fiber repeat of 2.550 nm and a meridional reflection at the 5th layer line. These results coupled with a conformational analysis indicate the chain conformation of chitosan with the salts to be a 5/3 helix, this helix differing from those of type I (an extended two-fold helix) and type II (a relaxed two-fold helix or a 4/1 helix). The fiber patterns of all the type III salts were similar. This observation has also been found with type II salts and is an indication that the acid ions are not arranged in regular positions in the crystals. A comparison of solid-state 13C-NMR spectra of the gentisic acid salt and the aspirin salt, which could not be crystallized, suggests that, in the latter salt, the chitosan molecules also formed a 5/3 helix.
Acids↗
Dependence of the mechanical properties of a pullulan film on the preparation temperature.
The mechanical properties of pullulan films prepared at various temperatures were investigated. The films prepared at high temperatures (40 degrees C and 60 degrees C; H-films) did not show any clear plastic deformation in tensile test, indicating that they were brittle. In contrast, those prepared at low temperatures (4 degrees C, 13 degrees C, and 25 degrees C; L-films) showed such deformation. The latter films had higher values for both tensile strength and elastic modulus than the former, indicating that the L-films were stiffer and more flexible than the H-films. Stretching the L-films clearly showed a shear deformation band inclined at 45 degrees to the stretching direction, indicating that they were amorphous.
Elasticity↗
Monte Carlo study of cycloamylose: chain conformation, radius of gyration, and diffusion coefficient.
Cyclic (1 --> 4)-alpha-D-glucan chains with or without excluded volume have been collected from a huge number (about 10(7)) of linear amylosic chains generated by the Monte Carlo method with a conformational energy map for maltose, and their mean-square radii of gyration and translational diffusion coefficients D (based on the Kirkwood formula) have been computed as functions of x (the number of glucose residues in a range from 7 to 300) and the excluded-volume strength represented by the effective hard-core radius. Both /x and D in the unperturbed state weakly oscillate for x < 30 and the helical nature of amylose appears more pronouncedly in cyclic chains than in linear chains. As x increases, these properties approach the values expected for Gaussian rings. Though excluded-volume effects on them are always larger in cycloamylose than in the corresponding linear amylose, the ratios of and the hydrodynamic radius of the former to the respective properties of the latter in good solvents can be slightly lower than or comparable to the (asymptotic) Gaussian-chain values when x is not sufficiently large. An interpolation expression is constructed for the relation between the gyration-radius expansion factors for linear and cyclic chains from the present Monte Carlo data and the early proposed asymptotic relation with the aid of the first-order perturbation theories.
Carbohydrate Conformation↗
Immunohistochemical localization of hemicelluloses and pectins varies during tissue development in the bamboo culm.
The distribution of hemicelluloses and pectins in bamboo internodes was studied immunocytochemistrically at various stages of development. The ultra-structures of bamboo cell walls have been reported previously at various stages. The internodes were identically classified into three developmental phases: primary wall stage (phase I), unlignified secondary wall stage (phase II) and lignified wall stage (phase III), using the same bamboo culm. (1-->3, 1-->4)-Beta-glucans were distributed in nearly all tissues in an actively elongating stage. Limited amounts of beta-glucans were deposited in primary walls and the middle lamellae, but were limited to the phloem in secondary walls. This suggests that the function of beta-glucans might be different in phloem vis-à-vis other tissues. Highly-substituted xylans were located in nearly all tissues of early phase I, but had disappeared in all tissues immediately prior to lignification. In contrast, low-branched xylan epitopes were present only in the protoxylem in phase I, but were present in all tissues immediately prior to lignification in phase II. In phase III, the epitopes were densely localized in lignified walls, suggesting that the substitution of xylans is closely related to maturation. Methyl-esterified (but not unesterified) pectins were present in all tissues of early phase I. Just before and after lignification, both types of pectins were concentrated in the phloem and protoxylem. Xyloglucans were largely distributed in the phloem and in lignified tissues, suggesting that they might be closely correlated with maturation. This represents the first account of the distribution of hemicelluloses and pectins at the tissue and ultrastructural level in bamboo internodes at various stages of development.
Bambusa↗
Toward understanding the different function of two types of parenchyma cells in bamboo culms.
The bamboo, woody monocot, has two types of parenchyma cells in the ground tissues of its culm, in contrast to a single type of parenchyma cell in rice, maize and other major crop species. The distribution of cell wall components, including lignin, (1-->3), (1-->4)-beta-D-glucans (MGs), the highly-substituted glucuronoarabinoxylans (hsGAXs) and low-branched xylans (lbXs) in ground parenchyma tissue of Phyllostachys heterocycla var. pubescens culms was studied at various developmental stages using light microscopy (LM), UV-microscopy, transmission electron microscopy (TEM) and immunolabeling techniques. The short parenchyma cell walls were lignified in 2-month-old bamboo culms just as the long parenchyma cell walls were. The lignified regions were confined to the portions in contact with the long parenchyma cell walls, while the walls at the cell corner region never lignified, even in 7-year-old culms. Significant differences were also found in the hemicellulose distribution between the short and long parenchyma cell walls. In bamboo parenchyma tissue, MGs were localized in short parenchyma cell walls and few were found in long parenchyma cell walls in both young and 7-year-old culms. The distribution of hsGAXs was similar to that of MGs in young culms, but they only appeared in the cell corner region of short parenchyma cells in old culms. Low-branched xylans were distributed in the lignified, but not in unlignified parenchyma cell walls. Based on this evidence, the differences of function in both short and long parenchyma cells in a bamboo culm are discussed.
Cell Wall↗
Amphiphilic block copolymer with a molecular recognition site: induction of a novel binding characteristic of amylose by self-assembly of poly(ethylene oxide)-block-amylose in chloroform.
Solution properties of amphiphilic methoxy poly(ethylene oxide)-block-amyloses (MPEO-amyloses) in chloroform were investigated by SLS and DLS. The results indicated that MPEO-amyloses dissolved in chloroform containing 2 wt % DMSO by their self-associations. The complexation of MPEO-amylose with methyl orange (MO) was significantly enhanced in the amylose domain of the associate in chloroform. The blue shift of the maximum absorption and strong induced circular dichroism with exciton coupling were observed in the MPEO-amylose MO complex in chloroform. The self-assembly of MPEO-amylose in chloroform shows a unique feature for binding with MO. MPEO-block-amylose is a novel amphiphilic polymer with amylose as a molecular recognition site.
Amylose↗
Effect of addition of water-soluble chitin on amylose film.
Amylose films blended with chitosan, which were free from additives such as acid, salt, and plasticizer, were prepared by casting mixtures of an aqueous solution of an enzymatically synthesized amylose and that of water-soluble chitin (44.1% deacetylated). The presence of a small amount of chitin (less than 10%) increased significantly the permeability of gases (N2, O2, CO2, C2H4) and improved the mechanical parameters of amylose film; particularly, the elastic modulus and elongation of the blend films were larger than those of amylose or chitin films. No antibacterial activity was observed with either amylose or water-soluble chitin films. But amylose films having a small amount of chitin showed strong antibacterial action, suggesting a morphological change in water-soluble chitin on the film surface by blending with amylose molecule. These facts suggested the presence of a molecular complex of amylose and chitosan.
Acetylation↗