Improved maintenance of adult rat alveolar type II cell differentiation in vitro: effect of serum-free, hormonally defined medium and a reconstituted basement membrane.

We have developed a serum-free, hormonally defined medium for maintenance of differentiation of adult type II cells cultured on Engelbreth-Holm-Swarm (EHS) tumor basement membrane gels. This defined medium consists of 1:1 (vol/vol) mixture of Ham's F12 and Dulbecco's modified Eagle's media supplemented with insulin, dibutyryl cyclic AMP, hydrocortisone, epidermal growth factor, selenium, and albumin/linoleic acid complex. Compared to cells cultured on EHS gels in serum-supplemented medium, type II cells cultured on EHS gels in this defined medium showed increased acetate incorporation into total lipids (10-fold) and an increase in the relative percentage of acetate incorporated into phosphatidylcholine (PC) (87.8 +/- 0.4% versus 78.5 +/- 1.0% [mean +/- SE]; P less than 0.01), saturated phosphatidylcholine (SPC) (61.4 +/- 0.5% versus 55.2 +/- 0.9%; P less than 0.01), and phosphatidylglycerol (PG) (5.3 +/- 0.3% versus 0.8 +/- 0.1%; P less than 0.01) and decreased acetate incorporation into neutral lipids (9.7 +/- 0.8% versus 62.6 +/- 1.9%; P less than 0.01). No response to this defined medium was seen when type II cells were cultured on tissue culture plastic. Type II cells cultured on EHS gels in serum-supplemented medium for 4 d had numerous neutral lipid droplets in their cytoplasm. In contrast, neutral lipid droplets were not commonly observed within the cytoplasm of the cells cultured in serum-free, hormonally defined medium on EHS gels. This morphologic finding was consistent with the result that cells cultured in serum-supplemented medium significantly increased the relative percentage of acetate incorporated into neutral lipids. These data indicate that adult type II cells cultured on a reconstituted basement membrane (EHS gels) can be maintained in synthetic culture medium without serum. These culture conditions permit the expression of a pattern of differentiated phospholipid biosynthesis and cell morphology more similar to normal type II cell differentiation.

Bactericidal effect of plaunotol, a cytoprotective antiulcer agent, against Helicobacter pylori.

In order to investigate the bactericidal effect of plaunotol, an oily antiulcer agent, against Helicobacter pylori, comparative studies were conducted using its derivatives, M-4, M-5, and M-6, whose hydrophobicity decreased in the order of plaunotol > M-6 > M-5 > M-4 by log P determination. Plaunotol rapidly reduced the viability of H. pylori in vitro, and cell death was associated with cell lysis. In addition, plaunotol showed eightfold stronger bactericidal activity against H. pylori than M-6 and M-5, while the compound with the lowest hydrophobicity, M-4, showed no bactericidal activity. The bactericidal activities of plaunotol and its derivatives were related to the hydrophobicity of these compounds. To investigate a possible interaction between these compounds and the cell membrane of H. pylori, their effects on liposomal membranes prepared from phosphatidylethanolamine and cardiolipin, which are known to be present in the membrane of H. pylori, were determined by detection of glucose release from the liposomes. Plaunotol showed eight-fold higher activity than M-6 and M-5, while M-4 showed no activity. The effects of plaunotol and its derivatives on liposomal membrane were therefore related to their bactericidal activities. In addition, it was confirmed that the bactericidal effect of plaunotol against H. pylori was neutralized by the liposomal membrane, and that plaunotol led to an increase in permeability of the membrane, as evidenced by measurement of the leakage of 260 nm absorbing-material from H. pylori. These results suggest that the bactericidal effect of plaunotol against H. pylori is due to the interaction between this compound and the bacterial cell membrane.