RESUMO
Various physiologically active effects of polymerized polyphenols have been reported. In this study, we synthesized a polymerized polyphenol (mL2a-pCA) by polymerizing caffeic acid using mutant Agaricus brasiliensis laccase and analyzed its physiological activity and mechanism of action. We found that mL2a-pCA induced morphological changes and the production of cytokines and chemokines in C3H/HeN mouse-derived resident peritoneal macrophages in vitro. The mechanisms of action of polymerized polyphenols on in vitro mouse resident peritoneal cells have not been characterized in detail previously. Herein, we report that the mL2a-pCA-induced production of interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) in C3H/HeN mouse-derived resident peritoneal cells was inhibited by treatment with the Rac1 inhibitor NSC23766 trihydrochloride. In addition, we found that mL2a-pCA activated the phosphorylation Rac1. Taken together, the results show that mL2a-pCA induced macrophage activation via Rac1 phosphorylation-dependent pathways.
Assuntos
Lacase/química , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Macrófagos/patologia , Neuropeptídeos/imunologia , Polifenóis/administração & dosagem , Polifenóis/química , Proteínas rac1 de Ligação ao GTP/imunologia , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Ativação Enzimática , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Fosforilação/efeitos dos fármacosRESUMO
Brazil-grown outdoor-cultivated Agaricus brasiliensis KA21 fruiting body (KA21) significantly increases the production of serum anti-beta-glucan antibody. Therefore, KA21 ingestion may be useful for the prevention and alleviation of fungal infections. This study aimed to determine the effects of KA21 in fungal infections in animals. KA21 was administered to nine dogs infected with Malassezia. Notably, the anti-beta-glucan antibody titer remained unchanged or tended to decrease in the oral steroid arm, whereas in the non-steroid arm, antibody titer increased in almost all animals after KA21 ingestion. Dogs showing improved clinical symptoms exhibited increased anti-beta-glucan antibody titers. The results of this study suggest that KA21 ingestion may alleviate the symptoms of Malassezia and other fungal infections and that continuous ingestion may help prolong recurrence-free intervals. Additionally, the ingestion of KA21 during oral steroid dosage reduction or discontinuation may enable smoother steroid withdrawal.
Assuntos
Agaricus , Doenças do Cão , Carpóforos , Malassezia , Animais , Cães , Agaricus/química , Carpóforos/química , Malassezia/efeitos dos fármacos , Doenças do Cão/microbiologia , Doenças do Cão/tratamento farmacológico , Dermatomicoses/veterinária , Dermatomicoses/prevenção & controle , Dermatomicoses/tratamento farmacológico , Dermatomicoses/microbiologia , beta-Glucanas/administração & dosagem , beta-Glucanas/farmacologia , Masculino , Brasil , Dermatite/tratamento farmacológico , Dermatite/veterinária , Dermatite/microbiologia , Dermatite/prevenção & controle , Feminino , Anticorpos Antifúngicos/sangueRESUMO
We investigated whether outdoor-cultivated Agaricus brasiliensis (KA21) could reduce the side effects caused by the anticancer medicine 5-fluorouracil (5-FU). The adverse effects of 5-FU were analyzed in mice by orally administering the drug every day for 5 days. Leukopenia, diarrhea, body weight loss, anorexia, kidney injury, gastrointestinal tract injury, and hair loss were evaluated as side effects. We determined whether these side effects were reduced by the intake of outdoor-cultivated A. brasiliensis. The side effects were reduced in mice receiving the outdoor-cultivated A. brasiliensis but not in those receiving the indoor-cultivated A. brasiliensis. These results suggest that outdoor-cultivated A. brasiliensis is beneficial in reducing the side effects of the anticancer medicine and might, therefore, be useful in improving the quality of life of patients with cancer. Furthermore, because antioxidants have been reported to contribute to the suppression of the side effects of anticancer medicines, the antioxidant activities of different mushrooms were determined. The antioxidant activity of the outdoor-cultivated A. brasiliensis was the highest among all the tested mushrooms. These findings make it clear that the cultivation conditions of A. brasiliensis are important in suppressing the side effects of anticancer medicines. Strong antioxidant activity might be one of the mechanisms through which this pharmacological activity is mediated.
Assuntos
Agaricus/química , Antineoplásicos/efeitos adversos , Antioxidantes/química , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/prevenção & controle , Agricultura , Animais , Feminino , Fluoruracila/efeitos adversos , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Melanin-producing Cryptococcus and Aspergillus are highly invasive and can suppress or escape the immune system of the host. Since non-melanin-producing strains do not affect the immune system, melanin may play a role in immune system suppression. Artificial melanin synthesized using conventional methods is insoluble, making structural and functional analysis of this chemical difficult. In this study, we describe a melanin solubilization method based on polymerization of homogentisic acid (solubilizing component) and an equivalent amount of L-DOPA in the presence of laccase. In addition, we investigated the effect of melanin on the immune system. Homogentisic acid and L-DOPA mixed melanin (HALD), the synthetic solubilized melanin, did not exert a cytotoxic effect on mouse macrophages. HALD suppressed cytokine and reactive oxygen species production by macrophages when they were stimulated by fungal components. HALD also suppressed the phagocytosis of fungal components by macrophages. These results suggest that HALD can suppress the function of macrophages without causing cytotoxicity.
Assuntos
Bioquímica/métodos , Ácido Homogentísico/química , Levodopa/química , Macrófagos/imunologia , Melaninas/imunologia , Animais , Lacase/química , Masculino , Melaninas/química , Camundongos , Camundongos Endogâmicos C57BL , Polimerização , SolubilidadeRESUMO
Laccases are enzymes that oxidize various aromatic compounds, and therefore they have attracted much attention from the standpoints of medical and industrial applications. We previously isolated the cDNA that codes for a laccase isozyme (Lac2a) from the medicinal mushroom Agaricus brasiliensis (Matsumoto-Akanuma et al., Int. J. Med. Mushrooms, 16, 375-393, 2014). In this study, we first attempted heterologous expression of the wild-type laccase using a Pichia pastoris secretory expression system. However, the trial was unsuccessful most likely because the enzyme was too unstable and degraded immediately after production. Therefore, we improved the stability of the laccase by using a phylogeny-based design method. We created a mutant laccase in which sixteen original residues were replaced with those found in the phylogenetically inferred ancestral sequence. The resulting mutant protein was successfully produced using the P. pastoris secretory expression system and then purified. The designed laccase showed catalytic properties similar to those of other fungal laccases. Moreover, the laccase is highly thermally stable at acidic and neutral pH and is also stable at alkaline pH at moderate temperatures. We expect that the laccase will serve as a useful tool for enzymatic polymerization of di-phenolic compounds.
Assuntos
Agaricus/enzimologia , Lacase/química , Lacase/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação , Filogenia , Agaricus/genética , Biocatálise , DNA Complementar/genética , Estabilidade Enzimática/genética , Concentração de Íons de Hidrogênio , Lacase/genética , Lacase/isolamento & purificação , Proteínas Mutantes/genética , Proteínas Mutantes/isolamento & purificação , Oxirredução , Pichia/genética , Pichia/metabolismo , Engenharia de Proteínas , TemperaturaRESUMO
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.