Binding Specificity of a New Artificial ß-Glucan Recognition Protein and Its Application to ß-Glucan Detection in Mushroom Extracts.

ß-1,3-D-glucan (BG) activates innate immunity and enhances immune responses. Fungi, such as mushrooms, produce a relatively large amount of BG, the structure and molecular weight of which varies depending on the species of fungi. This study was conducted to develop a detection probe for quantifying or detecting BG from fungi using BG-binding proteins. The binding properties of a new ß-glucan recognition protein (BGRP) against various BGs were compared. With reference to the amino acid sequences of BGRP in insects, an artificial BGRP (supBGRP) was designed with higher production efficiency using gene recombination technology. SupBGRP was produced in Escherichia coli with high efficiency, and its reactivity with BG from fungi was the highest among the BG-binding proteins examined. SupBGRP exhibited high reactivity with 1,6-branched BG and will be useful for the quantification and detection of fungal BG.

Open-Label Study of the Influence of Food Containing the Royal Sun Mushroom, Agaricus brasiliensis KA21 (Agaricomycetes), on ß-Glucan-Specific Antibody Production in Healthy Human Volunteers.

The edible mushroom Agaricus brasiliensis contains a large amount ß-glucan, which is mainly composed of a ß-1,6-glucan structure. In this study, we investigated the effect of A. brasiliensis strain KA21 on the anti-ß-glucan antibody titer in healthy humans and the role of antibodies as an immunomodulator. Twenty-two healthy volunteers were fed the dried fruiting body of A. brasiliensis (900 or 1500 mg/day) for 12 weeks. The anti-ß-glucan antibody titer in the serum was determined by enzyme-linked immunosorbent assay. Immunoglobulin G (IgG) against ß-glucan was significantly upregulated after intake of A. brasiliensis. Murine experiments demonstrated improvement of anti-ß-glucan antibody production after intraperitoneal injection of Agaricus-derived ß-glucan. To understand the role of antibody against ß-glucan in exclusion of pathogenic fungi, we examined the interaction between HL-60 cells and antibody-treated heat-killed Candida albicans. Flow cytometry analysis indicated the upregulation of Candida-positive HL-60 cells after treatment with human IgG, whereas the competitive assay demonstrated that the main epitope of Candida-reacted IgG was the ß-1,6-glucan structure. Binding between HL-60 and IgG-opsonized C. albicans was suppressed by anti-Fcγ receptor 1 (FcγRI) neutralizing antibody. Finally, using FcγRI-expressed cells with the nuclear factor of activated T-cell reporter assay, we demonstrated that higher titers of anti-ß-glucan IgG can induce stronger Fc receptor-mediated cell activation through the formation of an antibody-ß-glucan complex. In conclusion, oral ingestion of A. brasiliensis KA21 promotes anti-ß-glucan antibody production and may contribute to preventing fungal infection through the activation of immune cells by forming antibody-ß-glucan complexes via an FcγR-dependent pathway.

Immunoreactivity of the Cold Water Extract of Royal Sun Culinary-Medicinal Mushroom, Agaricus brasiliensis Strain KA21 (Agaricomycetes), Assessed by Immunoglobulin Preparations for Intravenous Injection.

The royal sun medicinal mushroom, Agaricus brasiliensis, is a health food material that helps to improve quality of life. A. brasiliensis has long been used as a tea by extraction with cold and hot water. Our group has been investigating the immunopharmacological activities of the A. brasiliensis KA21 strain, which is cultivated outdoors. We prepared cold water (AgCWE) and hot water (AgHWE) extracts of this strain. AgCWE contained a larger proportion of proteins, including enzymes, and showed a brownish color during the extraction process. By contrast, chemical and immunochemical analyses revealed that AgHWE contained large amounts of ß-1,3-/1,6-glucans. In an attempt to elucidate the immunochemical characteristics of AgCWE, reactivities to immunoglobulin (Ig) preparations for intravenous injection were analyzed and compared with standard materials. To characterize brownish high-molecular weight components, standard phenol compounds such as caffeic acid (CA), trans-ferulic acid (FA), and coumaric acid (CouA) were polymerized to brownish polymerized polyphenols (PPPs) (i.e., polymerized CA, polymerized FA, and polymerized CouA) by laccase or peroxidase. The results obtained revealed that intravenous Ig reacted with all PPPs and PPPs cross-reacted with AgCWE and AgHWE. The isotype of the anti-PPP antibody was found to be IgG1, in contrast to that of the ß-glucan antibody, which was mainly IgG2. These results strongly suggest that A. brasiliensis extracts contain immunoreactive components against various classes of Igs.

Open-Label Study of the Influence of Food Containing the Royal Sun Mushroom, Agaricus brasiliensis KA21 (Higher Basidiomycetes), on the Quality of Life of Healthy Human Volunteers.

We conducted an open-label study in which food containing Agaricus brasiliensis KA21 was consumed continuously for 12 weeks. A questionnaire for subjective evaluation of the efficacy of this food (hereafter, subjective evaluation questionnaire) revealed significant improvements compared with before its intake; there were improvements in the scores of the amounts of hair loss and gray hair, fatigue and general malaise, eye strain, shoulder stiffness, coldness of extremities, difficulty staying awake during the day, and ease of getting out of bed. These findings suggest that intake of food containing A. brasiliensis KA21 results in the above-mentioned subjectively evaluated improvements, and the possibility that A. brasiliensis KA21 improves the body's immunity. Moreover, no issues regarding the safety of the test food were found.

Differences in antioxidant activities of outdoor- and indoor-cultivated Agaricus brasiliensis, and protective effects against carbon tetrachloride-induced acute hepatic injury in mice.

BACKGROUND: Agaricus brasiliensis (A. brasiliensis) is a medicinal mushroom that exerts various pharmacological actions. We previously demonstrated that different cultivation conditions altered the activity of the polyphenol-related enzymes from this mushroom. However, the influence of cultivation conditions on the antioxidant activity of the fruiting bodies remains unclear. Therefore, in this study we compared the antioxidative effects of fruiting bodies of A. brasiliensis cultivated outdoors and indoors. In addition, we assessed whether different cultivation methods affected the hepatoprotective effects against CCl4-induced liver injury. METHODS: We assessed the antioxidative effects of mushrooms cultivated in open-air or indoors using the DPPH radical-scavenging assay. Furthermore, we prepared experimental feeds containing outdoor- or indoor-cultivated A. brasiliensis. Acute liver injury was induced by CCl4 injection in mice that consumed feed containing outdoor- or indoor-cultivated A. brasiliensis. The hepatoprotective effects of these mushrooms were then evaluated by monitoring the reduction in the circulating levels of alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase. The significance of the differences between the means was assessed using Student's t-test. Finally, histopathological analysis of liver was performed. RESULTS: In the DPPH assay, the antioxidant activity of outdoor-cultivated A. brasiliensis was higher than that of indoor-cultivated mushroom. Moreover, in the mouse model of CCl4-induced hepatitis, the oral administration of outdoor-cultivated A. brasiliensis reduced liver damage significantly, but indoor-cultivated mushrooms failed to inhibit hepatitis. The hepatoprotective effects of outdoor-cultivated A. brasiliensis were observed even when ingestion commenced only 1 day before CCl4 injection, and these effects were not affected by excessive heat treatment. CONCLUSIONS: Outdoor cultivation significantly enhanced the antioxidative activity of A. brasiliensis fruiting bodies. In addition, outdoor-cultivated A. brasiliensis was more effective at protecting against CCl4-induced liver injury in mice than mushrooms grown in a greenhouse.

Cloning and characterization of laccase DNA from the Royal Sun medicinal mushroom, Agaricus brasiliensis (higher Basidiomycetes).

Laccase isozymes have been identified in several fungi. We report the cloning of 4 laccase genes from the medicinal mushroom Agaricus brasiliensis. The lac1 gene contained a 1560-base pair (bp) open reading frame (ORF) encoding 520 amino acids that was interrupted with 14 introns in genomic DNA. The deduced amino acid sequence indicated a multicopper oxidase signature 1 and 2 multicopper oxidase signature 2. The lac2 gene contained a 1566-bp ORF encoding 522 amino acids that was interrupted with 13 introns in genomic DNA. A number of different nucleotides were observed in whole regions containing the substitution of amino acid residues (lac2a and lac2b). The partial DNA fragments of lac3 and lac4 genes were subcloned using the semi-random two-step polymerase chain reaction method. The lac3 and lac4 genes contained coding sequences with a 1575-bp ORF encoding 525 amino acids and a 1584-bp ORF encoding 528 amino acids, respectively. However, the whole complementary DNA fragment of both laccases could not be amplified with polymerase chain reaction against the complementary DNA library; therefore, introns were deduced based on the GT-AG rule and multiple alignment of laccases from other fungi, which showed high identity. All laccases from A. brasiliensis conserved the fungal laccase signature sequence and suggest 2 subfamilies according to the location of introns and phylogenetic analysis. The genes lac2 and lac4 had a high degree of identity, and the lac2a gene was located upstream of the lac4 gene.

Agaricus brasiliensis KA21 improves circulatory functions in spontaneously hypertensive rats.

The present study aimed to clarify the effects of Agaricus brasiliensis KA21 (i.e., Agaricus blazei) mushroom on circulatory function. Spontaneously hypertensive rats (SHRs) were fed 10% A. blazei-containing pellets (agaricus group) or normal pellets (control group) for 5 weeks from 6 to 11 weeks of age. For Experiment 1, tail blood pressure and heart rate were measured in the conscious SHRs. For Experiment 2, echocardiographic and blood biochemical measurements were performed in the anesthetized SHRs. In Experiment 1, blood pressure and heart rate were significantly lower in the agaricus group compared with the control group throughout the observation period. In Experiment 2, the agaricus group also showed a significant decrease in cardiac output accompanied by a decrease in heart rate and an increase in early and late ventricular filling velocity (E/A ratio). Moreover, levels of escape enzymes such as creatine kinase (CK), CK-BB, CK-MB, asparate aminotransferase, lactate dehydrogenase, and aldolase were significantly lower than in the control group. We concluded that the ingestion of feed containing A. brasiliensis KA21 can improve hypertensive cardiovascular hemodynamics by decreasing the working load of the heart, presumably by lowering the sympathetic nervous tone in SHRs.

Royal sun medicinal mushroom, Agaricus brasiliensis Ka21 (higher Basidiomycetes), as a functional food in humans.

The Royal Sun medicinal mushroom, Agaricus brasiliensis, is used as a natural health product. In Japan, however, the quality control of some of these mushroom products has been viewed as a safety problem. Focusing on the quality control of A. brasiliensis KA21, we have performed several safety studies. To date, we have established evidence that this mushroom can be used safely as an immunostimulant and to mediate biochemical parameters associated with obesity or diabetes. Furthermore, to improve the manufacturing process of this mushroom, we have studied the relationship between its pharmaceutical actions and the conditions of its cultivation and thermal management. The purpose of this review is to report the findings of basic and clinical studies of the fruit body of A. brasiliensis KA21.

Analysis of the titer and reactivity of antibody/ies against fungal cell wall ß-glucans in human sera.

The aim of this work was to characterize an antibody response to ß-glucan (BG), a major component of the fungal cell wall, at each isotype in human sera. The titer and reactivity of the anti-human BG antibody were examined using enzyme-linked immunosorbant assay plates coated with Candida soluble cell wall ß-glucan as a standard antigen. The antibody was detected using anti-human immunoglobulin (Ig) G, IgM, and IgA. Its major class was IgG in all subjects. The antibody titer varied significantly. The anti-BG antibody showed greater reactivity to ß-glucans derived from pathogenic fungi than monoglycosyl-branched ß-glucans derived from mycelia culture medium. In addition, it was suggested that the anti-BG IgM antibody was bound relatively strongly to the ß1,3-glucan backbone and the anti-BG IgG antibody to ß1,6-glucan. The anti-BG antibody plays a variety of roles, according to class, in the host's response to fungi. We propose a new index of human response to ß-glucan that effects the understanding of the response to ß-glucan in humans.

Safety study of culinary-medicinal Royal Sun Agaricus, Agaricus brasiliensis S. Wasser et al. KA21 (higher Basidiomycetes) assessed by prokaryotic as well as eukaryotic systems.

As part of the safety evaluation of culinary-medicinal Royal Sun Agaricus, Agaricus brasiliensis KA21, for human consumption, we performed the bacterial reverse mutation test, the mouse micronucleus test, and mouse lymphoma test using A. brasiliensis KA21 as the test substance. The reverse mutation test utilized five bacterial strains, including Salmonella typhimurium TA100, TA1535, TA98, and TA1537, and Escherichia coli WP2 uvrAO. For the micronucleus test we used mice. For the mouse lymphoma test, we used one of the most commonly used mammalian cell mutagenesis systems; the L5178YTK +/- mouse lymphoma-TK assay detects the mutations at the thymidine kinase locus caused by base-pair changes, frameshift, and small deletions. All the tests were conducted according to the guidelines for genotoxicity testing of drugs by the Ministry of Health, Labor, and Welfare, Japan. In the bacterial reverse mutation test, no toxicity was observed up to a dose of 5,000 µg/plate. In the mouse micronucleus test, no toxicity was noted up to a dose of 1 g/kg body weight. In the mouse lymphoma test, frequency of the mutation was equal both in the presence or absence of KA21. Supporting the long history of human consumption of A. brasiliensis, the data shown in this study strongly indicate the safety of this mushroom.

Partial purification and characterization of polyphenoloxidase from culinary-medicinal Royal Sun mushroom (the Himematsutake), Agaricus brasiliensis S. Wasser et al. (Agaricomycetideae).

The Royal Sun mushroom, the Himematsutake culinary-medicinal mushroom, Agaricus brasiliensis has several polyphenoloxidase activities in a broad sense. Here we report the partial purification of tyrosinase-type polyphenoloxidase (PPO). PPO is purified from A. brasiliensis without browning using a two-phase partitioning with Triton X-114 and ammonium sulfate fractionation. Partially denaturing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide electrophoresis) staining with L-3,4-dihydroxyphenylalanine was performed and the indicated molecular sizes were approximately 70 kDa and 45 kDa. The purified enzyme is in its latent state and can be activated maximally in the presence of 1.6 mM sodium dodecyl sulfate (SDS). This enzyme catalyzes two distinct reactions, monophenolase and diphenolase activity, and the monophenolase activity showed a lag time typical of polyphenoloxidase. The K(m) value for 4-tert-butylcatechol was quite similar in the presence and absence of SDS, but the apparent V(max) value was increased 2.0-fold by SDS. Mimosine was a typical competitive inhibitor with K(i) values of 138.2 microM and 281.0 microM n the presence and absence of SDS, respectively.

Immunomodulating Activity of Agaricus brasiliensis KA21 in Mice and in Human Volunteers.

We performed studies on murine models and human volunteers to examine the immunoenhancing effects of the naturally outdoor-cultivated fruit body of Agaricus brasiliensis KA21 (i.e. Agaricus blazei). Antitumor, leukocyte-enhancing, hepatopathy-alleviating and endotoxin shock-alleviating effects were found in mice. In the human study, percentage body fat, percentage visceral fat, blood cholesterol level and blood glucose level were decreased, and natural killer cell activity was increased. Taken together, the results strongly suggest that the A. brasiliensis fruit body is useful as a health-promoting food.