Cationic lipids are widely used for gene transfer in vitro and show promise as a vector for in vivo gene therapy applications. However, there is limited understanding of the cellular and molecular mechanisms involved. We investigated the individual steps in cationic lipid-mediated gene transfer to cultured cell lines. We used DMRIE/DOPE (a 1:1 mixture of N-[1-(2,3-dimyristyloxy) propyl]-N,N-dimethyl-N-(2-hydroxyethyl)ammonium bromide (DMRIE) and dioleoyl phosphatidylethanolamine (DOPE) as a model lipid because of its efficacy and because it is being used for clinical trials in humans. The data show that cationic lipid-mediated gene transfer is an inefficient process. Part of the inefficiency may result from the fact that the population of lipid-DNA complexes was very heterogeneous, even under conditions that have been optimized to produce the best transfection. Inefficiency was not due to inability of the complex to enter the cells because most cells took up the DNA. However, in contrast to previous speculation, the results indicate that endocytosis was the major mechanism of entry. After endocytosis, the lipid-DNA aggregated into large perinuclear complexes, which often showed a highly ordered tubular structure. Although much of the DNA remained aggregated in a vesicular compartment, there was at least a small amount of DNA in the cytoplasm of most cells. That observation plus results from direct injection of DNA and lipid-DNA into the nucleus and cytoplasm indicate that movement of DNA from the cytoplasm to the nucleus may be one of the most important limitations to successful gene transfer. Finally, before transcription can occur, the data show that lipid and DNA must dissociate. These results provide new insights into the physical limitations to cationic lipid-mediated gene transfer and suggest that attention to specific steps in the cellular process may further improve the efficiency of transfection and increase its use in a number of applications.

We have investigated the lignin peroxidase-catalyzed oxidation of guaiacol and the role of veratryl alcohol in this reaction by steady-state and pre-steady-state methods. Pre-steady-state kinetic analyses demonstrated that guaiacol is a good substrate for both compounds I and II, the two-and one-electron oxidized enzyme intermediates, respectively, of lignin peroxidase. The rate constant for the reaction with compound I is 1.2 ؋ 10 6 M ؊1 s ؊1. The reaction of guaiacol with compound II exhibits a K d of 64 M and a first-order rate constant of 17 s ؊1 . Oxidation of guaiacol leads to tetraguaiacol formation. This reaction exhibits classical Michaelis-Menten kinetics with a K m of 160 M and a k cat of 7.7 s ؊1. Veratryl alcohol, a secondary metabolite of ligninolytic fungi, is capable of mediating the oxidation of guaiacol. This was shown by steady-state inhibition studies. Guaiacol completely inhibited the oxidation of veratryl alcohol, whereas veratryl alcohol had no corresponding inhibitory effect on guaiacol oxidation. In fact, at low guaiacol concentrations, veratryl alcohol stimulated the rate of guaiacol oxidation. These results collectively demonstrate that veratryl alcohol can serve as a mediator for phenolic substrates in the lignin peroxidase reaction.This study investigates the ability of 3,4-dimethoxybenzyl (veratryl) alcohol to mediate the lignin peroxidase-catalyzed oxidation of guaiacol. Lignin peroxidases are hemeproteins secreted by the white rot fungus Phanerochaete chrysosporium during secondary metabolism (1, 2). These isozymes catalyze the oxidation of lignin and a large number of phenolic and non-phenolic substrates (3, 4). The catalytic cycle of lignin peroxidase is similar to that of other peroxidases (5, 6) where ferric enzyme is first oxidized by H 2 O 2 to generate the twoelectron oxidized intermediate, compound I (7). Compound I is then reduced by one electron donated by a substrate molecule, yielding the 1-electron oxidized enzyme intermediate, compound II, and a free radical product. The catalytic cycle is completed by the one-electron reduction of compound II by a second substrate molecule.In the absence of a reducing substrate, the enzyme can undergo a series of reactions with H 2 O 2 to form compound III, oxyperoxidase (6,8). It is also well documented that prolonged incubation of enzyme with H 2 O 2 in the absence of a reducing substrate such as veratryl alcohol can cause irreversible inactivation of the enzyme (9). In the presence of veratryl alcohol, however, lignin peroxidase undergoes multiple turnovers without any detectable inactivation. Because veratryl alcohol is normally produced by ligninolytic cultures of P. chrysosporium (10), workers have proposed that its physiological function is to protect the enzyme from H 2 O 2 -dependent inactivation (11).An alternate role for veratryl alcohol in lignin biodegradation has been proposed by Harvey et al. (12). These workers observed that substrates that are not oxidized by lignin peroxidase such as anisyl alcohol and 4-methoxyman...

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