RESUMO
Pleurotus ostreatus, one of the most widely cultivated edible mushrooms, produces high numbers of spores causing severe respiratory health problems for people, clogging of filters and spoilage of produce. A non-sporulating commercial variety (SPOPPO) has been successfully introduced into the market in 2006. This variety was generated by introgression breeding of a natural mutation into a commercial variety. Our cytological studies revealed that meiosis in the natural and derived sporeless strains was blocked in metaphase I, apparently resulting in a loss of spore formation. The gene(s) underlying this phenotype were mapped to an 80 kb region strongly linked to sporelessness and identified by transformation of wild type genes of this region into a sporeless strain. Sporulation was restored by re-introduction of the DNA sequence encoding the P. ostreatus meiotic recombination gene MSH4 homolog (poMSH4). Subsequent molecular analysis showed that poMSH4 in the sporeless P. ostreatus was interrupted by a DNA fragment containing a region encoding a CxC5/CxC6 cysteine cluster associated with Copia-type retrotransposons. The block of meiosis in metaphase I by a poMSH4 null mutant suggests that this protein plays an essential role in both Class I and II crossovers in mushrooms, similar to animals (mice), but unlike in plants. MSH4 was previously shown to be a target for breeding of sporeless varieties in P. pulmonarius, and the null mutant of the MSH4 homolog of S. commune (scMSH4) confers an extremely low level of spore formation. We propose that MSH4 homologs are likely to be a breeding target for sporeless strains both within Pleurotus sp. and in other Agaricales.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Meiose , Pleurotus/fisiologia , Esporos Fúngicos/genética , Troca Genética , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Ligação Genética , Metáfase , Fenótipo , Pleurotus/genética , Retroelementos/genéticaRESUMO
BACKGROUND: Food is a potential source of immunomodulating compounds that may be used to steer immune responses towards a desired status such as reducing inflammatory disorders. However, to identify and characterize such bioactive compounds, biologically relevant and standardized assays are required. Macrophages play an important role in immunomodulation and are suited for developing cell-based assays. An assay was developed based on macrophages, in a homogeneous differentiation state, using the human monocytic cell line THP-1 previously used to assess immunomodulatory properties of low-molecular-weight allergens, hormones, dietary supplements and therapeutic drugs. RESULTS: Zymosan and mushroom polysaccharide extracts lead to a heterogeneous differentiation state of THP-1 monocytes, and these cells secrete low levels of cytokines upon stimulation. Differentiation into macrophages using a low concentration of phorbol 12-myristate 13-acetate improved responsiveness. Elevated levels of cytokines were secreted by cells in a homogenous differentiation state. In addition, it was determined that the assay performs best when using cells at a concentration of (2.5-5) × 10(5) cells mL(-1). CONCLUSION: An assay was developed suitable to distinguish the immunomodulatory properties of food compounds in a reproducible manner. It was evaluated using eight mushroom species by measuring the secretion of relevant cytokines TNF-α, IL-1ß, IL-6 and IL-10.
Assuntos
Agaricus/química , Coprinus/química , Citocinas/metabolismo , Fatores Imunológicos/farmacologia , Macrófagos/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Polissacarídeos/farmacologia , Agaricales/química , Produtos Biológicos/farmacologia , Produtos Biológicos/uso terapêutico , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Humanos , Fatores Imunológicos/uso terapêutico , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Interleucina-10/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Macrófagos/metabolismo , Monócitos/metabolismo , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Zimosan/farmacologiaRESUMO
Mushrooms are known for their immune-modulating and anti-tumour properties. The polysaccharide fraction, mainly beta-glucans, is responsible for the immune-modulating effects. Fungal beta-glucans have been shown to activate leukocytes, which depend on structural characteristics of beta-glucans. As edible mushrooms come in contact with the intestinal immune system, effects on enterocytes are also interesting. Our aim was to evaluate the effect of mushroom polysaccharide extracts varying in beta-glucan structure on nitric oxide production by bone marrow-derived macrophages (BMMs) from mice and on nuclear factor-kappaB transactivation in human intestinal Caco-2 cells. We demonstrated that extracts from Agaricus bisporus stimulated nitric oxide production by BMM, whereas extracts from Coprinus comatus and spores of Ganoderma lucidum had only minor effects. Furthermore, extracts of A. blazei Murill and Phellinus linteus had no effect at all. Almost all mushroom extracts lowered nuclear factor-kappaB transactivation in Caco-2 cells. Structural analysis of A. bisporus compared with A. blazei Murill suggests that branching of the beta-glucan chain is essential for immune-stimulating activity. In conclusion, extracts from A. bisporus activate BMM, without activating enterocytes. These characteristics make A. bisporus an attractive candidate as a nutritional compound to stimulate the immune response in depressed states of immunity.