A Yeast Mitogenic Factor Active on Human Peripheral Leucocytes in Culture

Abstract

WE have observed that the addition of zymosan-treated sera to short-term human peripheral leucocyte cultures leads to a slight but significant increase in mitotic frequency. Inactivation of serum complement by zymosan¹ as an important factor in the increase in mitotic frequency was ruled out, and it could be shown that an active mitogenic factor was extracted from zymosan during treatment of the sera. It was found that a more active mitogenic factor could be extracted directly from whole frozen yeast (Saccharomyces cerevisiae) and this extract was then used exclusively. The method of extraction consisted of overnight incubation at 37° of equal volumes of saline washed cells and 0.2 N potassium hydroxide, followed by centrifugation at 2,000 r.p.m. for 30 min. The resulting supernatant was neutralized with perchloric acid, and then passed through a ‘Sephadex’ DEAE column which had been washed with 0.2 M tris buffer at pH 7.1. Before use, the yeast extract (YAF) was sterilized in boiling water for 30 min. Leucocyte suspensions were obtained by dextran sedimentation (2.5 ml. 6 per cent dextran per 10 ml. heparinized blood at 37°). Colchicine (0.02 µg/ml. culture) was added 8–10 h before collecting and chromosome preparations were made according to the method of Hungerford et al.².