Complexes of Adenovirus with Polycationic Polymers and Cationic Lipids Increase the Efficiency of Gene Transfer in Vitro and in Vivo

Abstract

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...

The extra- and intracellular barriers to lipid and adenovirus-mediated pulmonary gene transfer in native sheep airway epithelium

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Transgene expression is increased by photochemically mediated transduction of polycation-complexed adenoviruses

“…[6][7][8] One strategy for obtaining CAR-independent entry in Ad5-resistant cells is to make noncovalent complexes of adenovirus (Ad) with cationic agents. [9][10][11][12][13][14][15][16] The cationic components can associate with the Ad particles due to the net negative viral surface charge, and hence, the attachment to the negatively charged cell membrane is facilitated. This method enables potent gene delivery via CAR-independent cell infection, demonstrating that the interaction with the primary receptor is not crucial for cellular entry.…”

In vitro and in vivo asessment of adenovirus 41 as a vector for gene delivery to the intestine

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