The noncoding control region of the mitochondrial DNA of various gallinaceous birds was studied with regard to its restriction fragment length polymorphism (RFLP) and sequences of the first 400 bases. Tandem duplication of the 60-base unit was established as a trait unique to the genus

S-Alk(en)yl-L-cysteine sulfoxides are pharmaceutically important secondary metabolites produced by plants that belong to the genus Allium. Biosynthesis of S-alk(en)yl-L-cysteine sulfoxides is initiated by S-alk(en)ylation of glutathione, which is followed by the removal of glycyl and γ-glutamyl groups and S-oxygenation. However, most of the enzymes involved in the biosynthesis of S-alk(en)yl-L-cysteine sulfoxides in Allium plants have not been identified. In this study, we identified three genes, AsGGT1, AsGGT2, and AsGGT3, from garlic (Allium sativum) that encode γ-glutamyl transpeptidases (GGTs) catalyzing the removal of the γ-glutamyl moiety from a putative biosynthetic intermediate of S-allyl-L-cysteine sulfoxide (alliin). The recombinant proteins of AsGGT1, AsGGT2, and AsGGT3 exhibited considerable deglutamylation activity toward a putative alliin biosynthetic intermediate, γ-glutamyl-S-allyl-L-cysteine, whereas these proteins showed very low deglutamylation activity toward another possible alliin biosynthetic intermediate, γ-glutamyl-S-allyl-L-cysteine sulfoxide. The deglutamylation activities of AsGGT1, AsGGT2, and AsGGT3 toward γ-glutamyl-S-allyl-L-cysteine were elevated in the presence of the dipeptide glycylglycine as a γ-glutamyl acceptor substrate, although these proteins can act as hydrolases in the absence of a proper acceptor substrate, except water. The apparent Km values of AsGGT1, AsGGT2, and AsGGT3 for γ-glutamyl-S-allyl-L-cysteine were 86 μM, 1.1 mM, and 9.4 mM, respectively. Subcellular distribution of GFP-fusion proteins transiently expressed in onion cells suggested that AsGGT2 localizes in the vacuole, whereas AsGGT1 and AsGGT3 possess no apparent transit peptide for localization to intracellular organelles. The different kinetic properties and subcellular localizations of AsGGT1, AsGGT2, and AsGGT3 suggest that these three GGTs may contribute differently to the biosynthesis of alliin in garlic.

SUMMARYS-Alk(en)yl-L-cysteine sulfoxides are cysteine-derived secondary metabolites highly accumulated in the genus Allium. Despite pharmaceutical importance, the enzymes that contribute to the biosynthesis of S-alk-(en)yl-Lcysteine sulfoxides in Allium plants remain largely unknown. Here, we report the identification of a flavin-containing monooxygenase, AsFMO1, in garlic (Allium sativum), which is responsible for the S-oxygenation reaction in the biosynthesis of S-allyl-L-cysteine sulfoxide (alliin). Recombinant AsFMO1 protein catalyzed the stereoselective S-oxygenation of S-allyl-L-cysteine to nearly exclusively yield (R C S S )-S-allylcysteine sulfoxide, which has identical stereochemistry to the major natural form of alliin in garlic. The S-oxygenation reaction catalyzed by AsFMO1 was dependent on the presence of nicotinamide adenine dinucleotide phosphate (NADPH) and flavin adenine dinucleotide (FAD), consistent with other known flavin-containing monooxygenases. AsFMO1 preferred S-allyl-L-cysteine to c-glutamyl-S-allyl-L-cysteine as the S-oxygenation substrate, suggesting that in garlic, the S-oxygenation of alliin biosynthetic intermediates primarily occurs after deglutamylation. The transient expression of green fluorescent protein (GFP) fusion proteins indicated that AsFMO1 is localized in the cytosol. AsFMO1 mRNA was accumulated in storage leaves of pre-emergent nearly sprouting bulbs, and in various tissues of sprouted bulbs with green foliage leaves. Taken together, our results suggest that AsFMO1 functions as an S-allyl-L-cysteine S-oxygenase, and contributes to the production of alliin both through the conversion of stored c-glutamyl-S-allyl-L-cysteine to alliin in storage leaves during sprouting and through the de novo biosynthesis of alliin in green foliage leaves.

Because colorectal cancer is likely to develop in many people at some point during their lives, prevention has become a high priority. Diet and nutrition play an important role during the multistep colon carcinogenic process. Garlic has been traditionally used as a spice and is well known for its medicinal properties; several studies have indicated its pharmacologic functions, including its anticarcinogenic properties. However, the mechanisms by which garlic can prevent colorectal cancer remain to be elucidated. This study investigated the effect of aged garlic extract (AGE) on the growth of colorectal cancer cells and their angiogenesis, which are important microenvironmental factors in carcinogenesis. AGE suppressed the proliferation of 3 different colorectal cancer cell lines-HT29, SW480, and SW620-in the same way, but its effects on the invasive activities of these 3 cell lines were different. the invasive activities of SW480 and SW620 cells were inhibited by AGE, whereas AGE had no effect on the invasive activity of Ht29 cells. The action of AGE appears to be dependent on the type of cancer cell. On the other hand, AGE enhanced the adhesion of endothelial cells to collagen and fibronectin and suppressed cell motility and invasion. AGE also inhibited the proliferation and tube formation of endothelial cells potently. These results suggest that AGE could prevent tumor formation by inhibiting angiogenesis through the suppression of endothelial cell motility, proliferation, and tube formation. AGE would be a good chemopreventive agent for colorectal cancer because of its antiproliferative action on colorectal carcinoma cells and inhibitory activity on angiogenesis.

1,2,3,4-Tetrahydro-beta-carboline derivatives (THbetaCs) are formed through Pictet-Spengler chemical condensation between tryptophan and aldehydes during food production, storage and processing. In the present study, in order to identify the antioxidants in aged garlic extract (AGE), we fractionated it and identified four THbetaCs; 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acids (MTCC) and 1-methyl-1,2,3,4-tetrahydro-beta-carboline-1,3-dicarboxylic acid (MTCdiC) in both diastereoisomers using liquid chromatography mass spectrometry (LC-MS). Interestingly, these compounds were not detected in raw garlic, but the contents increased during the natural aging process of garlic. In in vitro assay systems, all of these compounds have shown strong hydrogen peroxide scavenging activities. (1S, 3S)-MTCdiC was found to be stronger than the common antioxidant, ascorbic acid. MTCC and MTCdiC inhibited AAPH-induced lipid peroxidation. Both MTCdiCs also inhibited LPS-induced nitrite production from murine macrophages at 10-100 microM. Our data suggest that these compounds are potent antioxidants in AGE, and thus may be useful for prevention of disorders associated with oxidative stress.