INTRODUCTIONhemispheres from 16 to 18-day old rat embryo were treated with a solution of 0 .125% (wt/vol) trypsin for 5 min, and the resulting cell suspension was aspirated through a Pasteur pipette to complete the dissociation . The cells were centrifuged at 400 g for 5 min and the supernate was discarded . The pellet was then resuspended in the growth medium (13), and the suspension was centrifuged for I min at 200 g . The supernate containing the dissociated single cells was transferred to a sterile bottle, final cell concentration was adjusted to 0 .1 0 .3 x 106 cells per I ml medium, and 3 ml of the above suspension was placed into polylysine-coated Petri dishes . After 30 min incubation at 37üC in an atmosphere of 95% air, 5% CO 2 , the medium containing the nonadhered cells was removed and replaced by fresh medium which was then changed every 3-4 days, depending on its acidity . Phase-contrast microscopy was employed for observation of cultures during their growth period .
The reaction between allicin (diallylthiosulfinate), the active component of garlic and reduced glutathione was investigated. The product of this reaction, mixed disulfide S-allylmercaptoglutathione (GSSA) was separated by high performance liquid chromatography and identified by 1H and (13)C nuclear magnetic resonance and mass spectroscopy. The reaction is fast (with an apparent bimolecular reaction rate constant of 3.0 M(-1) s(-1)). It is pH-dependent, which reveals a direct correlation to the actual concentration of mercaptide ion (GS(-)). Both GSSA and S-allylmercaptocysteine (prepared from allicin and cysteine) reacted with SH-containing enzymes, papain and alcohol dehydrogenase from Thermoanaerobium brockii yielding the corresponding S-allylmercapto proteins, and caused inactivation of the enzymes. The activity was restored with dithiothreitol or 2-mercaptoethanol. In addition, GSSA also exhibited high antioxidant properties. It showed significant inhibition of the reaction between OH radicals and the spin trap 5,5'-dimethyl-1-pyroline N-oxide in the Fenton system as well as in the UV photolysis of H2O2. In ex vivo experiments done with fetal brain slices under iron-induced oxidative stress, GSSA significantly lowered the production levels of lipid peroxides. The similar activity of GSSA and allicin as SH-modifiers and antioxidants suggests that the thioallyl moiety has a key role in the biological activity of allicin and its derivatives.
Docosahexaenoic acid (DHA) (22:6) is a polyunsaturated fatty acid of the n - 3 series which is believed to be a molecular target for lipid peroxides (LPO) formation. Its ubiquitous nature in the nervous tissue renders it particularly vulnerable to oxidative stress, which is high in brain during normal activity because of high oxygen consumption and generation of reactive oxygen species (ROS). Under steady state conditions potentially harmful ROS and LPO are maintained at low levels due to a strong antioxidant defense mechanism, which involves several enzymes and low molecular weight reducing compounds. The present review emphasizes a paradox: a discrepancy between the expected high oxidability of the DHA molecule due to its high degree of unsaturation and certain experimental results which would indicate no change or even decreased lipid peroxidation when brain tissue is supplied or enriched with DHA. The following is a critical review of the experimental data relating DHA levels in the brain to lipid peroxidation and oxidative damage there. A neuroprotective role for DHA, possibly in association with the vinyl ether (VE) linkage of plasmalogens (pPLs) in combating free radicals is proposed.
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