Yasunori Nagamatsu

The cadherin‐like protein is essential to specificity determination and cytotoxic action of the Bacillus thuringiensis insecticidal CryIAa toxin

The Bacillus thuringiensis CryIAa toxin binds a cadherin‐like protein (BtR175) on the brush‐border membranes of the Bombyx mori midgut columnar cells, which are the targets. By introducing the BtR175 gene with a baculovirus, Spodoptera frugiperda Sf9 cells expressed BtR175 protein on the cell membrane and became susceptible to the CryIAa toxin. The toxin bound the cadherin repeat adjacent to the membrane and made a pore that passed inorganic ions, causing the cell to swell and burst. This was not observed with a BtR175 variant lacking the toxin‐binding site. This in vitro experiment mimicked the specific insecticidal action of the toxin in vivo well.

Analysis of Structural Components and Molecular Construction of Soybean Soluble Polysaccharides by Stepwise Enzymatic Degradation

Soybean soluble polysaccharides (SSPS) extracted from soybean cotyledons have a pectin-like structure. The core polysaccharides after treatments with four kinds of hemicellulases and a pectinase contained approximately equal numbers of L-rhamnouse and D-galacturonate residues, suggesting the presence of the rhamnogalacturonan (RG) I structure consisting of the diglycosyl repeating unit, -4)-α-D-GalpA-(1→2)-α-L-Rhap-(1-. The lengths of RG chains were calculated as approximately 15, 28, and 100 diglycosyl repeats. The RG components linked to each other by intervention of galacturonan (GN) chains, constituting the backbone of SSPS. All arabinose residues, which constitute 21% of total SSPS sugars, were found to be in side chains from RG regions, and this was also true for galactose residues, which constitute 50% of total sugars. Of arabinose residues, 94% are present as α-1,3- or α-1,5-arabinans, and 89% of galactose residues were present as β-1,4-galactans. Galactan chains are modified with arabinose, xylose, fucose, and glucose at the sites close to the RG regions.

Structural Studies by Stepwise Enzymatic Degradation of the Main Backbone of Soybean Soluble Polysaccharides Consisting of Galacturonan and Rhamnogalacturonan

Soybean soluble polysaccharides (SSPS) extracted from soybean cotyledons are acidic polysaccharides and have a pectin-like structure. The results of a structural analysis of SSPS by using polygalacturonase (PGase) and rhamnogalacturonase (RGase) clarified that the main backbone consisted of galacturonan (GN) and rhamnogalacturonan (RG), which were composed of the diglycosyl repeating unit, -4)-α-D-GalpA-(1→2)-α-L-Rhap-(1-. The side chains of β-1,4-galactans, branched with fucose and arabinose residues, were linked to the C-4 side of rhamnose residues in the RG regions. The degree of polymerization (dps) of GN, which linked the RG regions together, was estimated to be about 4-10 residues, and some were modified with xylose residues on the C-3 side of the galacturonates. The dps of GN at the reducing end of SSPS was estimated to be about 7-9 residues. Moreover, the fragment of the basic structure of the RG region, -[4)-α-D-GalpA-(1→2)-α-L-Rhap- (1-]2-, some of which had long-chain β-1,4-galactans branched on the C-4 side of rhamnose residues, were liberated from SSPS by the RGase treatment. The dps of the galactan side chain was estimated to be about 43-47 residues by an analysis of the digestion products from the β-galactosidase treatment.

Effect of soybean soluble polysaccharides on the stability of milk protein under acidic conditions

Abstract Soybean soluble polysaccharide (SSPS) extracted from soybean cotyledons has a pectin-like structure. SSPS has been used as a dispersion stabilizer for milk protein under acidic conditions, below the isoelectric point of milk protein, in a manner similar to high methoxyl pectin. SSPS at concentrations of less than 0.2% stabilized acidic beverages prepared with 8.0% of milk solid non-fat at pH 3.4–4.4, in contrast with pectin which needed at least 0.4% concentration. To elucidate the structure of the polysaccharide, which seemed likely to have a significant influence on its ability to stabilize acidic beverages, SSPS was digested with purified pectinases and hemicellulases, and the stabilizing effects of the digestion products on acidic beverages were analyzed. The stabilizing ability of SSPS was reduced by digestion of its neutral sugar side chains, arabinan or galactan, and was only slightly changed by digestion of its galacturonan main backbone with polygalacturonase or rhamnogalacturonase. This indicates that neutral sugar side chains, which mainly consist of arabinose and galactose residues, play a significant role in the ability of SSPS to stabilize acidic beverages. In contrast, the stabilizing effect of pectin was reduced by digestion of its galacturonan backbone, and was not changed by digestion of its neutral sugar side chains. This indicates that SSPS may differ from pectin in the mechanism by which it stabilizes milk proteins under acidic conditions.

Purification and characterization of Bombyx mori chitinases.

Two isozymes of chitinase (EC 3.2.1.14) were purified from the fifth-instar larvae of Bombyx mori by chromatography on DEAE-Cellulofine A-500, hydroxylapatite, Butyl-Toyopearl 650M, and Fractogel EMD DEAE 650 (M). These two isozymes were glycoproteins with different apparent molecular masses of 65 and 88 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pHs of the 65 and 88 kDa chitinases were 5.5 and 6.5, respectively, towards a short substrate, N-acetylchitopentaose (GlcNAc5), whereas their high activities were observed in a wide pH range between 4 and 10 towards a longer substrate, glycolchitin. Steady-state kinetic analysis of these chitinases was performed using a series of N-acetylchitooligosaccharides (GlcNAcn, n = 2-6) and glycolchitin as the substrates. Kinetic parameters for both chitinases could be obtained in the hydrolysis of glycolchitin, but not in that of N-acetylchitooligosaccharides because of strong substrate inhibition. Both chitinases similarly hydrolysed N-acetylchitooligosaccharides except for GlcNAc2 as follows: GlcNAc3 to GlcNAc plus GlcNAc2, GlcNAc4 to two molecules of GlcNAc2, GlcNAc5 to GlcNAc2 plus GlcNAc3, and GlcNAc6 to GlcNAc2 plus GlcNAc4 as well as two molecules of GlcNAc3. These results suggest that these chitinases are endo-type hydrolases, and preferred the longer-chain N-acetylchitooligosaccharides. With respect to activity, the 65 kDa chitinase was 1.7-fold more active than the 88 kDa chitinase with regard to the initial velocity in the reaction of 0.1 mM N-acetylchitooligosaccharides (GlcNAcn, n = 3-6), whereas in the overall reaction of glycolchitin (kcat/K(m)), the 88 kDa chitinase was four times more active than the 65 kDa chitinase. Regarding the affinity (1/K(m)) to glycolchitin, the affinity of the 88 kDa chitinase was 5.8-fold higher than that of the 65 kDa chitinase. The protein amino acid and gene nucleotide sequences were partly determined. Both N-terminal amino acid sequences of the 65 and 88 kDa chitinases were identical as ADSRARIVXYFSNWAVYRPG. The partial amino acid (113 amino acids) and nucleotide sequences (278 nucleotides) analysed from a mixture of 65 and 88 kDa chitinases included the two conserved regions of the family of 18 glycosyl hydrolases. All these results suggest that the B. mori chitinases are similar to Manduca sexta chitinase in primary structure and kinetic behaviour, and may be involved in the initial and intermediate stages of chitin degradation.

Analysis of the molecular construction of xylogalacturonan isolated from soluble soybean polysaccharides

Soluble soybean polysaccharides (SSPS) extracted from soybean cotyledons are acidic polysaccharides, and exhibited a pectin-like structure. After digesting galacturonan with polygalacturonase, two novel galacturonan (GN) fragments, which were directly linked to xylosyl oligosaccharides, were obtained. One consisted of (β-D-Xyl)7 branched at the C-3 site of 1,4-linked (α-D-GalA)4, and the other consisted of (β-D-Xyl)4 branched at the C-3 site of 1,4-linked (α-D-GalA)3.

Three Cry toxins in two types from Bacillus thuringiensis strain M019 preferentially kill human hepatocyte cancer and uterus cervix cancer cells.

Bacillus thuringiensis strain M019, non-pathogenic to lepidopteran and dipteran insects, produces a parasporal inclusion that consists of three 84-kDa Cry proteins (CPs). CP78A and CP78B, which exhibit 83.5% amino acid identity, were new variants of the previously reported HeLa cell-killing protein (parasporin-1). CP84 was a novel CP showing low-level homology, of 21.9% (56.4% similarity), with the insecticidal Cry2 toxin. In vitro solubilization with dithiothreitol at pH 10.2 and limited hydrolysis with trypsin resulted in the removal of N-terminal portions of the CPs and their activation. The 70-kDa proteins (15- and 55-kDa fragments) from CP78A and CP78B and the 73-kDa protein (14- and 59-kDa fragments) from CP84 exhibited varying degrees of cytocidal activity preferentially toward human hepatocyte cancer HepG2 cells and uterus cervix cancer HeLa cells causing cell swelling or the formation of vacuoles in the cytoplasm. These toxins appeared to attack an identical target on human cells.

High production of pyoluteorin and 2,4-diacetylphloroglucinol by Pseudomonas fluorescens S272 grown on ethanol as a sole carbon source

Pseudomonas fluorescens S272 newly isolated from a soil sample produced a considerable amount of pyoluteorin and 2,4-diacetylphloroglucinol when grown on ethanol as a single carbon source. The coproduction of approximately 150 μg/ml of pyoluteorin and approximately 500 μg/ml of 2,4-diacyl-phloroglucinol was achieved by flask cultutivation in a medium containing approximately 2% ethanol. A high CN ratio and inorganic phosphate limitation in the medium were also important factors to be considered for optimization of production of the antibiotics.

β-Galactosidase and Its Significance in Ripening of “Saijyo” Japanese Persimmon Fruit

The fruit extracts of ripening cv. Japanese Persimmon, “Saijyo”, contained a number of glycosidases and glycanases. Among them, β-galactosidase appeared to be the most significant, and the activity increased in parallel with tissue ripening. Persimmon β-galactosidase was presented in at least three isoforms, β-galactosidase-I (pI=4.88), β-galactosidase-II (pI=6.76), and β-galactosidase-III (pI=7.05). β-Galactosidase-III had exo-type galactanase activity, while the others did not. The activity of endo-type glycanases was a maximum in immature green or yellow fruits. The firmness of the pulp tissue decreased dramatically, and the amount of water-soluble polysaccharide (WSS) increased. The enzyme activities of exo-type glycosidases, especially β-galactosidase, appeared maximal in mature red fruits. The amount of extractable pectin remained unchanged, although the galactose content of the high-molecular-weight fraction in WSS decreased dramatically. These results suggest that the ripening of persimmon was caused by the solubilization of pectic polysaccharide by endo-type glycanases and digestion by exo-type glycosidases. β-Galactosidase, in particular, seemed to play a major role in ripening the fruit.

Protective effects of bacterial glyceroglycolipid M874B against cell death caused by exposure to heat and hydrogen peroxide

It was revealed by bioassay using sodA and katA mutants of Bacillus subtilis that the bacterial monogalactosyldiacylglycerol M874B, previously characterized as an alkyl peroxyl radical scavenger, was also capable of protecting cells from death caused by heating and exogenous H2O2. Chemical assays using the Fenton reaction and xanthine-xanthine oxidase revealed that M874B could quench hydroxyl radicals but not superoxide anions. Wheat monogalactosyldiacylglycerol, but neither digalactosyldiacylglycerol nor synthetic diacylglycerol, also had the same activities as those of M874B, although it was less efficient than M874B. These results suggest that monogalactosyldiacylglycerols such as M874B are a new type of oxygen radical scavengers capable of quenching some reactive oxygen species.