Isolation of protoplast from aerial mycelia and fruit-body formation from regenerated mycelia in Pleurotus cornucopiae.

We have developed a simple and efficient method for protoplast isolation from Pleurotus cornucopiae. Protoplasts were isolated from aerial mycelia cultured on potato dextrose agar medium without time-consuming propagation in liquid culture. Protoplast yield was significantly increased by means of a decompressing pretreatment of mycelia in enzyme solution and a subsequent enzyme reaction with vibrational mixing. The isolated protoplasts regenerated mycelia and these mycelia formed fruit-bodies without any morphological abnormalities.

Stimulative effects of (22E,24R)-ergosta-7,22-diene-3beta,5alpha,6beta-triol from fruiting bodies of Tricholoma auratum, on a mouse osteoblastic cell line, MC3T3-E1.

We screened the differentiation-inducing activities of 39 mushroom extracts from Akita prefecture, Japan, on the mouse osteoblastic cell line, MC3T3-E1. Sixteen phosphate buffered saline (PBS), 8 boiled PBS, 14 ethanol and 12 methanol extracts induced alkaline phosphatase (ALP) activities, an indicator of MC3T3-E1 cell differentiation. The enzyme activities were markedly induced by extracts of Tricholoma auratum, and we isolated the active compound from methanol extracts of this mushroom. Physical data for the isolated active compound were identical to those for (22E,24R)-ergosta-7,22-diene-3beta,5alpha,6beta-triol (1). 1 induced ALP activities of MC3T3-E1 cells and promoted cell proliferation. To investigate the relationships between the chemical structure and differentiation-inducing activity of the compound, ALP-inducing activities of MC3T3-E1 cells by 1, ergosterol (2), ergocalciferol (3), cholesta-3beta3,5alpha6beta-triol (4), 7-dehydrocholesterol (5) and cholecalciferol (6) were tested. The enzyme activities of MC3T3-E1 cells were increased 3.0-fold by 10 microM 1 and 2.4-fold by 10 microM 4. However, 2, 3, 5 and 6 did not induce MC3T3-E1 cell ALP activity at 0.1-10 microM. These results suggested that the OH groups at C-5 and/or C-6 of 1 and 4 played an important role in their differentiation-inducing activities on MC3T3-E1 cells. Furthermore, 1 suppressed induction of MC3T3-E1 cell apoptosis by serum starvation.