A promising class of compounds for DNA transfection have been designed by conjugating various potyamines to bile-acid-based amphiphiles. Formulations containing these compounds were tested for their ability to facilitate the uptake of a 3-galactosidase reporter plasmid into COS-7 cells. Dioleoyl phosphatidyl ethanolamine (DOPE) formulations of some of the compounds were several times better than Lipofectin at promoting DNA uptake. The most active compounds contained the most hydrophilic bile acid components.The activity is clearly not related to affinity for DNA: the hydrophobic bile acid conjugates were found to form stable complexes with DNA at lower charge ratios than the hydrophilic conjugates. We suggest that the high activity of the best compounds is related to their facial amphiphilicity, which may confer an ability to destabilize membranes. The success of these unusual cationic transfection agents may inspire the design of even more effective gene delivery agents.Gene therapy is an exciting approach to the treatment of genetic defects, as well as diseases such as cancer and chronic viral infections (1-3). Unfortunately, the enthusiasm initially displayed for gene therapy has been tempered by the realization that there are no easy solutions to the problem of how to get genes into cells. The most efficient methods for transferring DNA across cell membranes involve the use of viral vectors (1, 4, 5); however, there are growing concerns about both the short-and long-term risks ofviral vectors. These concerns have prompted a search for other strategies for DNA delivery, and in the past few years, a variety of nonviral gene delivery systems have been investigated (6-9). Although some success in getting DNA into cells has been achieved, gene delivery with nonviral vectors remains an inefficient process. To make gene therapy a reality, more efficient DNA delivery systems are needed. In this paper, we report the design and preliminary evaluation of a promising class of DNA delivery agents. In designing these delivery agents, we started by considering the properties of existing nonviral delivery systems. Of all the nonviral DNA delivery systems that have been explored, cationic lipids have shown the most promise based on a combination of efficacy, stability, and toxicity. Lipofectin (Fig.
An in situ transglycosylase assay has been developed using endogenously synthesized lipid II. The assay involves the preferential synthesis and accumulation of lipid II in a reaction mixture containing the cell wall membrane material isolated from Escherichia coli, exogenously supplied UDP-MurNAc-pentapeptide, and radiolabeled UDP-GlcNAc. In the presence of Triton X-100, the radiolabeled product formed is almost exclusively lipid II, while the subsequent formation of peptidoglycan is inhibited. Removal of the detergent resulted in the synthesis of peptidoglycan (25% incorporation of radiolabeled material) from the accumulated lipid II. This reaction was inhibited by moenomycin, a known transglycosylase inhibitor. In addition, tunicamycin, which affects an earlier step of the pathway by inhibiting MraY, had no effect on the formation of peptidoglycan in this assay, as expected. Similarly, ampicillin and bacitracin did not inhibit the formation of peptidoglycan under the conditions established.
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