An aqueous polysaccharide extract from the edible mushroom Pleurotus ostreatus induces anti-proliferative and pro-apoptotic effects on HT-29 colon cancer cells.

Anti-proliferative and pro-apoptotic activities of fractions of Pleurotus ostreatus were examined using HT-29 colon cancer cells in vitro. A hot-water-soluble fraction of the mycelium of the liquid cultured mushroom was partially isolated and chemically characterized as a low-molecular-weight alpha-glucan. HT-29 cells were exposed to the different isolates and significant inhibition of proliferation was obtained in a dose-dependent manner. Proliferation inhibition was shown to be the result of apoptotic induction because the pro-apoptotic molecules Bax and cytosolic cytochrome-c were upregulated. Fluorescence-activated cell sorter analyses of polysaccharide-treated HT-29 cells showed a high percentage of Annexin-positive cells. Here, we describe a newly identified low-molecular-weight alpha-glucan with promising anti-tumorigenic properties, and demonstrate its direct effect on colon cancer cell proliferation via induction of programmed cell death.

Effect of copper and manganese ions on activities of laccase and peroxidases in three Pleurotus species grown on agricultural wastes.

Copper (Cu2+) and manganese (Mn2+) ions influenced laccase (Lac) and peroxidase production in Pleurotus eryngii, Pleurotus ostreatus, and Pleurotus pulmonarius. In P. eryngii, the optimum Cu2+ concentration for Lac production was 1 mM and for peroxidases 10 mM, and Mn2+ concentration of 5 mM led to peaks of Lac and peroxidase activity. In P. ostreatus HAI 493, the highest level of Lac activity was at Cu2+ concentrations of 1 and 10 mM and Mn2+ concentration of 1 mM, respectively. The absence of Cu2+ and Mn2+ caused the highest levels of peroxidase production. In P. ostreatus HAI 494, the highest level of Lac activity was at a Cu2+ concentration of 5 mM and at Mn2+ concentration of 1 mM, respectively. High levels of peroxidase activity were found in the medium without and with 1 mM Cu2+, and at 1 and 5 mM Mn2+, respectively. In P. pulmonarius, the highest Lac activity was found in the presence of 5 mM Cu2+ and 5 mM Mn2+, respectively. The absence of Cu2+ and Mn2+ as well as their presence at a concentration of 1 mM led to the peaks of peroxidase activities.

Regulation of two homodimer hexosaminidases in the mycoparasitic fungus Trichoderma asperellum by glucosamine.

Trichoderma asperellum is a mycoparasitic fungus which is used as a biocontrol agent against plant pathogens. Its hydrolytic enzymes take part in its parasitic interaction, degrading the pathogen cell wall and thereby helping to control disease. One of those enzymes, beta- N-acetyl- d-glucosaminidase (GlcNAcase), degrades chitin, which is a major component of the cell wall of many plant-pathogenic fungi. Two GlcNAcases of T. asperellum T203, designated EXC1Y and EXC2Y, were purified, their genes and their promoters were sequenced, and their regulation was studied. The enzymes share homology (59% identity) but are easily distinguished by PAGE assay. Biochemical characterization, Edman degradation, and mass spectrometry demonstrated that EXC1Y and EXC2Y are both active as homodimers. Both genes are up-regulated by glucosamine (GlcN), in contrast to two endochitinases of this fungus. GlcN induces the secretion of several proteins (including a beta-glucosidase), among which EXC1Y is the most abundant. An exc2y knockout was constructed, to study the regulation of EXC1Y expression and secretion. The fungus has the ability to store a high amount of this enzyme in an active form and secrete it into the medium later.

Expression regulation of the endochitinase chit36 from Trichoderma asperellum (T. harzianum T-203).

The presence of the endochitinase CHIT36 from Trichoderma harzianum TM was assessed in several antagonistic Trichoderma strains belonging to different molecular taxonomic groups. CHIT37 from T. harzianum CECT 2413 was sequenced and found to display 89% homology with CHIT36 at the amino acid level. Northern analysis showed that chit36Y from T. asperellum is regulated both by glucose and nitrogen levels. Stress conditions, colloidal chitin and N-acetyl-glucosamine are effective inducers of this gene. The promoter of chit36Y was cloned and was used to direct expression of a gfp reporter gene in Trichoderma transformants. Confrontation experiments with the plant pathogen Rhizoctonia solani revealed that direct contact between the fungi is not necessary for gfp expression. The R. solani-inducing factor appears to be a soluble molecule capable of diffusing through a dialysis membrane (<12 kDa). CHIT36 recombinant protein from the yeast Pichia pastoris was active against different phytopathogens, confirming the importance of this endochitinase in the mycoparasitic activity of Trichoderma antagonistic strains.