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Featured researches published by Masakatsu Usui.


Food Research International | 2002

Emulsifying properties and bactericidal action of chitosan–lysozyme conjugates

Youtao Song; Elfadil E. Babiker; Masakatsu Usui; Akira Saito; Akio Kato

Abstract The solubility of chitosan was greatly improved by conjugation with lysozyme through Maillard-type protein–polysaccharide reaction. The emulsifying properties, especially emulsion stability, were improved by lysozyme–chitosan conjugation. The improvement in emulsifying properties was better in the conjugates of high molecular weight-type chitosan (HMC) than low molecular-type chitosan (LMC). The lysozyme–HMC conjugate has greatly improved emulsion stability at acidic pH. The conjugates have greatly enhanced bactericidal action against Escherichia coil K-12, a typical Gram-negative bacterium. Although the bactericidal activity of LMC was much lower than that of HMC, it was improved by conjugation with lysozyme. Thus, chitosan–lysozyme conjugate can be used as a new functional ingredient having excellent emulsifying properties and bactericidal action.


Bioscience, Biotechnology, and Biochemistry | 2013

Contribution of structural reversibility to the heat stability of the tropomyosin shrimp allergen.

Masakatsu Usui; Akihito Harada; Takayuki Ishimaru; Emiri Sakumichi; Fumihiko Saratani; Chiho Sato-Minami; Hiroyuki Azakami; Taiko Miyasaki; Ken-ichi Hanaoka

Tropomyosins are common heat-stable crustacean allergens. However, their heat stability and their effects on antigenicity have not been clarified. We purified tropomyosin in this study from raw kuruma prawns (Marsupenaeus japonicus) without heat processing. SDS-PAGE of the purified protein showed a band at approximately 35 kDa that cross-reacted with IgE from the serum of a shrimp-allergic patient, identifying it as Pen j 1. The circular dichroism spectrum of native Pen j 1 revealed the common α-helical structure of tropomyosins which easily collapsed upon heating to 80 °C. However, there were no insoluble aggregates after heating, and the protein regained its native CD spectral pattern after cooling to 25 °C. There was no significant difference in total IgG production between mice sensitized with native and heated Pen j 1. These results suggest that heat-denatured Pen j 1 refolded upon cooling and maintained its antigenicity following the heat treatment.


Bioscience, Biotechnology, and Biochemistry | 2003

Functional Properties of Glycosylated Lysozyme Secreted in Pichia pastoris

Akira Saito; Yukikazu Sako; Masakatsu Usui; Hiroyuki Azakami; Akio Kato

Various mutant lysozymes having the N-glycosylation signal sequence, R21T (Asn19-Tyr20-Thr21), G49N (Asn49- Ser50-Thr51), R21T/G49N (Asn19-Tyr20-Thr21/Asn49-Ser50-Thr51), were secreted in the Pichia pastoris expression system. The secreted amounts of these mutant glycosylated lysozymes were almost the same as those of wild-type lysozyme (about 30 mg/liter). Glycosylation of the mutant lysozymes was confirmed by SDS-PAGE patterns, Endo-H treatment, TOF-MS analysis and chemical analysis. The composition of the carbohydrate chain attached to the single glycosylated lysozymes, R21T and G49N, was GlcNAc2Man9-11, while that of the double glycosylated lysozyme, R21T/G49N, was GlcNAc4Man27-32. The results of a CD analysis and lytic activity suggested that the conformation of the single glycosylated lysozymes had been conserved, while that of the double glycosylated lysozyme was less stable. The emulsifying properties of the lysozyme when glycosylated were greatly improved, being especially noteworthy in the double glycosylated lysozyme.


Bioscience, Biotechnology, and Biochemistry | 2003

Reduction of Antigenicity of Cry j I, Major Allergen of Japanese Cedar Pollen, by the Attachment of Polysaccharides

Masakatsu Usui; Akira Saito; Naohiro Taniguchi; Noriaki Nishijima; Hiroyuki Azakami; Akio Kato

An attempt was made to mask the allergenic structure of a major allergen protein, Cry j I (CJI), in Japanese cedar pollen using the Maillard-type polysaccharide conjugation. The SDS-PAGE pattern of the CJI-galactomannan conjugate prepared by the Maillard reaction showed broad bands widely distributed from 50 kDa to more than 100 kDa, suggesting the attachment of galactomannan. The competitive enzyme-linked immunosorbent assay showed that the IgE antibody in the sera of ceder pollen-sensitive patients reacted strongly with CJI, while it did not react with the CJI-galactomannan conjugate. This result suggests that the antigenicity of CJI is greatly reduced by the conjugation with galactomannan.


Nahrung-food | 2002

Relationship between the stability of lysozymes mutated at the inside hydrophobic core and secretion in Saccharomyces cerevisiae

Youtao Song; J. Sakai; Akira Saito; Masakatsu Usui; Hiroyuki Azakami; Akio Kato

The relationship between the stability of lysozymes mutated at the inside hydrophobic core and secretion was investigated to understand the optimal secretion of mutant lysozymes in the Saccharomyces cerevisiae. S91T mutant lysozyme increased in the methyl residue inside the core greatly increased the conformational stability. The secretion amount of S91T in S. cerevisiae increased greatly compared with wild-type lysozyme. On the other hand, I55V and T40S/I55V mutant lysozymes decreased in methyl residue inside the core brought about their unstable conformation. The secretion amounts of these unstable mutant lysozymes significantly decreased. In addition, the effect of glycosylation on the secretion of these mutants was investigated. The secretion amounts of glycosylated lysozyme S91T/G49N with stable hydrophobic core greatly increased compared with that of glycosylated lysozyme G49N, while those of mutant I55V/G49N and T40S/I55V/G49N with unstable hydrophobic core greatly decreased. These results indicate that the secretion amounts of mutant lysozymes increase in proportion to the hydrophobic core stabilities and that a similar good correlation was obtained with glycosylated lysozymes.


Journal of Agricultural and Food Chemistry | 2009

Reduction of antigenicity of Cry j 1, a major allergen of Japanese cedar pollen, by thermal denaturation.

Rieko Aoki; Akira Saito; Masakatsu Usui; Hiroyuki Azakami; Akio Kato

The soluble aggregates of Cry j 1, a major allergen of Japanese cedar pollen, were formed without any coagulates during heat treatment at acidic pH 5, as shown in HPLC and SDS-PAGE patterns. A remarkable change in the CD spectrum was observed between native and heat-denatured Cry j 1 at a linear rate of 1 degrees C/min from 40 to 90 degrees C. The negative peak of native Cry j 1 at 222 nm was moved to 218 nm, suggesting the transition of an alpha-helix to beta-structure during heat denaturation. The increase in beta-structure was also observed during heat denaturation by monitoring the fluorescence with Thioflavin T. These results suggest that Cry j 1 forms intermolecular cross-beta-structure between denatured proteins during heating at 90 degrees C. The antigenicity of Cry j 1 detected by dot-blotting was greatly diminished during heating at a linear rate of 1 degrees C/min from 40 to 90 degrees C without any coagulates. These results suggest that IgE epitopes exposed on the molecular surface of Cry j 1 was buried inside soluble aggregates through intermolecular beta-structure formed by heating.


Bioscience, Biotechnology, and Biochemistry | 2015

Relationship between the risk for a shrimp allergy and freshness or cooking

Masakatsu Usui; Akihito Harada; Shinya Yasumoto; Yoshimasa Sugiura; Anri Nishidai; Maria Ikarashi; Honami Takaba; Taiko Miyasaki; Hiroyuki Azakami; Masakazu Kondo

Tropomyosins are defined as risk factors for shrimp allergy. However, their concentration in different preparations has not been clarified. We quantified the tropomyosin concentration in shrimp meat, which was cooked using several methods or was stored under various conditions. The results demonstrated that shrimp meat from various preparations and storage conditions maintained tropomyosin concentrations that were sufficient to cause food allergies.


Bioscience, Biotechnology, and Biochemistry | 2014

Unstable mutant lysozymes are degraded through the interaction with calnexin homolog Cne1p in Saccharomyces cerevisiae

Hiroyuki Azakami; Masayoshi Uehara; Ryohei Matsuo; Yuta Tsurunaga; Yuichiro Yamashita; Masakatsu Usui; Akio Kato

Cne1p is a yeast homolog of calnexin, which is a constituent of endoplasmic reticulum (ER)-associated protein quality control system in mammals. Cne1p may be involved in the degradation of misfolded lysozymes in Saccharomyces cerevisiae. To test this, c-Myc-tagged lysozymes were expressed in CNE1-deficient S. cerevisiae. The expression and secretion of an unstable lysozyme mutant G49N/D66H were enhanced and its intracellular localization was changed in the CNE1-deficient strain. Furthermore, when Cne1p was co-expressed with unstable lysozyme mutants (G49N/D66H, G49N/C76A, and K13D/G49N), its affinity to the misfolded mutant proteins was revealed by co-immunoprecipitation. The interaction with Cne1p was abrogated by the addition of tunicamycin, an inhibitor of N-glycosylation, indicating that N-linked carbohydrates might be necessary for protein binding to Cne1p. These results suggest that in yeasts, Cne1p interacts with misfolded lysozyme proteins possibly causing their retention in the ER and subsequent elimination via ER-associated degradation. Graphical Abstract In yeasts, Cne1p interacts with misfolded lysozymes possibly causing their retention in the ER and subsequent elimination via ER-associated degradation.


FEBS Letters | 2004

Effective reduction of antigenicity of hen egg lysozyme by site‐specific glycosylation

Masakatsu Usui; Toshiaki Shimizu; Yasuko Goto; Akira Saito; Akio Kato

Various mutant lysozymes were constructed by genetic modification and secreted in yeast expression system to evaluate the changes in the antigenicity of hen egg lysozyme (HEL). Although Arg68, the most critical residue to antigenicity of HEL, was substituted with Gln, the binding of monoclonal antibodies (mAbs) with the mutant lysozyme did not critically reduce, remaining 60% of the binding with mAb. In contrast, glycosylated mutant lysozyme G49N whose glycine was substituted with asparagine dramatically reduced the binding with mAb. The oligomannosyl type of G49N lysozyme reduced binding with mAb to one‐fifth, while the polymannosyl type of G49N lysozyme completely diminished the binding with mAb. This suggests that the site‐specific glycosylation of lysozyme in the interfacial region of lysozyme–antibody complex is more effective to reduce the antigenicity than the mutation of single amino acid substitution in the interfacial region.


Marine Drugs | 2018

Orally Administered Phlorotannins from Eisenia arborea Suppress Chemical Mediator Release and Cyclooxygenase-2 Signaling to Alleviate Mouse Ear Swelling

Yoshimasa Sugiura; Masakatsu Usui; Kunio Imai; Makoto Kakinuma; Hideomi Amano; Masaaki Miyata

Phlorotannin is the collective term for polyphenols derived from brown algae belonging to the genera Ascopyllum, Ecklonia, Eisenia, Fucus and Sargassum etc. Since the incidence of allergies is currently increasing in the world, there is a focus on phlorotannins having anti-allergic and anti-inflammatory effects. In this study, six purified phlorotannins (eckol; 6,6′-bieckol; 6,8′-bieckol; 8,8′-bieckol; phlorofucofuroeckol (PFF)-A and PFF-B) from Eisenia arborea, orally administered to mice, were examined for their suppression effects on ear swelling. In considering the suppression, we also examined whether the phlorotannins suppressed release of chemical mediators (histamine, leukotriene B4 and prostaglandin E2), and mRNA expression and/or the activity of cyclooxygenase-2 (COX-2), using RBL-2H3 cells, a cultured mast cell model. Results showed that the phlorotnannins exhibited suppression effects in all experiments, with 6,8′-bieckol, 8,8′-bieckol and PFF-A showing the strongest of these effects. In conclusion, orally administered phlorotannins suppress mouse ear swelling, and this mechanism apparently involves suppression of chemical mediator release and COX-2 mRNA expression or activity. This is the first report of the anti-allergic effects of the orally administered purified phlorotannins in vivo. Phlorotannins show potential for use in functional foods or drugs.

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