Soichiro Nakamura

PROTECTIVE EFFECT OF LYSOZYME-GALACTOMANNAN OR LYSOZYME-PALMITIC ACID CONJUGATES AGAINST EDWARDSIELLA TARDA INFECTION IN CARP CYPRINUS CARPIO L.

The protective effect of lysozyme‐galactomannan or lysozyme‐palmitic acid conjugates orally administered to carp, Cyprinus carpio L. was investigated using a virulent strain of Gram‐negative Edwardsiella tarda isolated from an infected fish. Lysozyme‐galactomannan conjugate was prepared through controlled Maillard reaction. Lysozyme‐palmitic acid conjugate was prepared through base‐catalyzed ester exchange using N‐hydroxysuccinimide ester of palmitic acid. The conjugates provided substantial protection to carp infected with a Gram‐negative bacteria fish pathogen E. tarda NG 8104. Lytic activities of lysozyme conjugates with galactomannan and palmitic acid were about 80 and 71% of native lysozyme using Micrococcus lysodeikticus as a substrate. Feeding with lysozyme conjugates, for 8 days, significantly enhanced fish protection against E. tarda infection. The survival rate was 30% for lysozyme‐galactomannan conjugate treated fish and 20% for lysozyme‐palmitic acid conjugate treated fish after 6 days cultivation while all control fish died within 3 days. On the other hand, a recovery rate of 40% after 6 days was observed in the fish group that were fed lysozyme‐palmitic acid conjugate 3 and 2 h before and after E. tarda challenge, respectively, and for 6 consecutive days. The results of this work show the possibility of utilizing lysozyme conjugates with galactomannan or palmitic acid as a therapeutic for infection in fish.

Effects of ovalbumin glycoconjugates on alleviation of orally induced egg allergy in mice via dendritic‐cell maturation and T‐cell activation

SCOPE Glycation of allergens via Maillard reaction or chemical conjugation has been shown to influence susceptibility to food-induced allergies. It is hypothesized that mucosal immune response bias can be favorably altered by orally administering various forms of glycated ovalbumin (OVA). METHODS AND RESULTS Groups of Balb/c mice (n = 10) were orally sensitized to OVA and administered various forms of glycated OVA (glucose, mannose, glucomannan, galactomannan, and a mixture containing OVA and glucomannan). Outcomes post oral challenge were measured as clinical allergic signs, serum histamine, mouse mast cell protease 1 (MMCP-1), antibody activity, type-1/2 cytokines, percentage of T-regulatory cells (T-regs) and in vitro dendritic cell, and T-cell-related mechanisms. Clinical signs and specific IgE were decreased (p ≤ 0.05), and T-reg cell percentage was increased in the mannose and glucomannan treated groups. The OVA-mannose group also had less histamine, MMCP-1, specific IgG, IL-4 and IL-17, and more IL-12p70 (p ≤ 0.05). Other parameters measured did not differ significantly among groups. Also, OVA-glycated mannose reduced maturation and uptake by dendritic cells. Less activation of T cells and type-2 cytokine response in DC-T-cell cocultures were observed with OVA-glycated mannose stimulation. CONCLUSION This study validates, for the first time, the use of OVA-glycated mannose and glucomannan for potential beneficial dietary interventions for allergy.

Apricot carotenoids possess potent anti-amyloidogenic activity in vitro.

Alzheimers disease (AD) is the most common form of dementia and is characterized by the progressive accumulation of amyloid β protein (Aβ) in areas of the brain. There has been an increased interest in screening for food-grade anti-amyloidogenic compounds in foodstuffs. The purpose of this study was to screen and identify bioactive compounds with anti-amyloidogenicity in apricot fruits using synthetic Aβ(1-42). The anti-amyloidogenicity was investigated using thioflavin T fluorescence assay, electron microscopy, and dot blotting analysis. The carotenoid fraction from apricot showed strong inhibitory effects against oligomer and fibril formation of Aβ and fibril-destabilizing effects. Among the peaks in the HPLC chromatogram, lutein showed the strongest inhibitory effect on Aβ fibril formation. The inhibitory effect was dependent on the number and portion of hydroxyl groups on both sides of carotenoids. These findings suggest that lutein in fruits may be useful as a preventive agent for amyloid-associated diseases.

Macromolecular antimicrobial glycoprotein, achacin, expressed in a methylotrophic yeast Pichia pastoris

A cDNA encoding achacin, an antimicrobial glycoprotein from the body surface mucus of giant African snail Achacina fulica Férussac, was expressed in a methylotrophic yeast, Pichia pastoris, and recombinant achacin (rAch) was secreted in yeast minimal medium in a polyglycosylated form with 80 kDa. Carbohydrate analysis revealed that the glycosylated moiety of rAch was composed of 50 mol mannose and 2 mol N‐acetylglucosamine residues. Antimicrobial activity using Escherichia coli and Staphylococcus aureus showed that the rAch had a behavior similar to its native counterpart. The rAch showed so wide an antimicrobial spectrum that 0.1 mg/ml rAch inhibited the growth of Pseudomonas fluorescens, Staphylococcus epidermidis, and Streptococcus faecalis in addition to E. coli and S. aureus, whereas it did not appreciably affect the growth of Proteus mirabilis, Bacillus cereus and Micrococcus luteus. The rAch was also effective in preventing growth of Vibrio anguillarum and Vibrio parahaemolyticus. The results suggested that the rAch had great potential of using as an antimicrobial agent.

Genetically glycosylated ovomucoid third domain can modulate Immunoglobulin E antibody production and cytokine response in BALB/c mice

Background Food allergies are on the rise and it is estimated that in North America, 8% of the children and 4% of the adults have food allergies. Food allergies tend to occur more often in children than in adults due to their immature digestive and immune systems. Hens egg is among the most common cause of food‐induced allergic reactions in North America.

Antimicrobial effects of chlorogenic acid and related compounds

Chlorogenic acid (CGA) is a natural chemical ester composed of caffeic acid and (-)-quinic acid, and is further metabolized into active compounds in the living body. Here, we aimed to provide fundamental information on the antimicrobial action of CGA and related compounds against the Gram-negative bacterium Escherichia coli IFO 3301. Bacteriostatic effects were assessed by spectrophotometry, and bactericidal effects were determined by enumerating viable cells on MacConkey agar plates. CGA and related compounds exhibited specific antimicrobial activity and corresponding reduction in log survival ratio, in which ferulic, isoferulic, benzoic, and hydroxybenzoic acids exhibited obvious antimicrobial activity against E. coli. In a time-kill assay, it was observed that bactericidal effects were associated with treatment time, temperature, and dose. A reduction in log survival ratio was observed at low pH as well as under thermal stress condition. Thus, we demonstrated that CGA and related compounds have not only bacteriostatic effects but also bactericidal effects.

Attenuation of allergic immune response phenotype by mannosylated egg white in orally induced allergy in BALB/c mice.

Food allergies are attributed to an imbalance in immune response to ubiquitous antigens. A previous study demonstrated that mannose glycation (mannosylation) of ovalbumin decreased allergenicity in vivo. The proposed research targets mannosylation of various common allergens that may help prevent food allergy. Balb/c mice (n = 8) were sensitized toxin egg white, peanut, and whey and treated with mannosylated forms of the test antigens. Glucosylated peanut and cholera toxins were used as controls. Allergic status was assessed as clinical signs, serum histamine, mouse mast cell protease (MMCP), antibody activity, cytokines, and T regulatory cells (T-regs). Significant preventative effects were observed with mannosylated egg white treatment such as reduced clinical signs, histamine, MMCP, specific G, G1, and E antibody activities, and IL-4 and increased IL-10 and CD25(+) Foxp3(+) cells. Other groups did not differ significantly. It was concluded that mannosylated egg white provides a powerful tool to prevent allergic phenotypes with possible relevance to control human egg allergy.

Application of polymannosylated cystatin to surimi from roe-herring to prevent gel weakening.

A recombinant glycosylated cystatin with a polymannosyl chain was added to roe‐herring surimi for preventing gel weakening due to autolysis during cooking. Proteolysis of myosin heavy chain in the surimi was effectively suppressed while cooking at 90°C for 20 min after preincubation at 40°C for 30 min. The glycosylation of cystatin improved the stability against heating as well as proteolysis by cathepsin D. This process markedly improved the texture of the cooked surimi gel with gel strength 2.5 times that of unglycosylated control cystatin.

Enzymatic synthesis of novel phenol acid rutinosides using rutinase and their antiviral activity in vitro.

Novel rutinosides of vanillic acid, sinapic acid, ferulic acid, and caffeic acid were prepared via a rutinase-catalyzed transglycosylation reaction. Reaction mixtures containing rutin, phenolic acid, and rutinase derived from tartary buckwheat ( Fagopyrum tataricum ) seeds were incubated in 20 mM acetate buffer (pH 5.0) at 40 °C. The resulting rutinoside of each phenolic acid was purified by HPLC, and the structure was determined by NMR and FAB-MS analysis. Antiviral activity was determined using feline calicivirus (FCV) strain F9, which is a typical norovirus surrogate. It was found that rutinosylation of the phenolic acids increased their antiviral activity against FCV, with the sinapic acid rutinoside being the most effective. These results will contribute to the development of antiviral agents against noroviruses.

Apple polyphenols suppress antigen presentation of ovalbumin by THP-1-derived dendritic cells

Apple polyphenol extract (AP) and procyanidin contained in AP were investigated for their immunomodulatory effects using THP-1-derived human dendritic cells (TDDCs). The expression levels of HLA-DR (MHC class II) and CD86 (costimulatory molecule) were measured as an indicator of antigen presentation in TDDCs. A significant decrease in HLA-DR expression was observed in the AP and fractionated procyanidin-treated cells in the presence of ovalbumin (OVA), but no effect on CD86 expression was observed. The uptake of OVA was not inhibited by AP treatment, and the gene expression of membrane-associated RING-CH ubiquitin E3 ligase, MARCH1, was up-regulated by AP treatment. It can therefore be presumed that AP suppresses HLA-DR expression via the ubiquitin-proteasome pathway. Furthermore, the up-regulation of IL-12 and TNF-α was found in the procyanidin trimers-treated cells in the presence of OVA. These results suggest that apple polyphenols would be an effective factor for the development of immunomodulatory agents with suppressive effects of antigen presentation.