Much attention has focused on the aetiology of oxidative damage in cellular and organismal ageing. Especially toxic are the reactive oxygen byproducts of respiration and other biological processes. A mev-1(kn1) mutant of Caenorhabditis elegans has been found to be hypersensitive to raised oxygen concentrations. Unlike the wild type, its lifespan decreases dramatically as oxygen concentrations are increased from 1 to 60%. Strains bearing this mutation accumulate markers of ageing (such as fluorescent materials and protein carbonyls) faster than the wild type. We show here that mev-1 encodes a subunit of the enzyme succinate dehydrogenase cytochrome b, which is a component of complex II of the mitochondrial electron transport chain. We found that the ability of complex II to catalyse electron transport from succinate to ubiquinone is compromised in mev-1 animals. This may cause an indirect increase in superoxide levels, which in turn leads to oxygen hypersensitivity and premature ageing. Our results indicate that mev-1 governs the rate of ageing by modulating the cellular response to oxidative stress.

New polyhydroxylated alkaloids, (2R,3R,4R)-2-hydroxymethyl-3,4-dihydroxypyrrolidine-N-propionamide from the root bark of Morus alba L., and 4-O-alpha-D-galactopyranosyl-calystegine B(2) and 3 beta,6 beta-dihydroxynortropane from the fruits, were isolated by column chromatography using a variety of ion-exchange resins. Fifteen other polyhydroxylated alkaloids were also isolated. 1-Deoxynojirimycin, a potent alpha-glucosidase inhibitor, was concentrated 2.7-fold by silkworms feeding on mulberry leaves. Some alkaloids contained in mulberry leaves were potent inhibitors of mammalian digestive glycosidases but not inhibitors of silkworm midgut glycosidases, suggesting that the silkworm has enzymes specially adapted to enable it to feed on mulberry leaves. The possibility of preventing the onset of diabetes and obesity using natural dietary supplements containing 1-deoxynojirimycin and other alpha-glucosidase inhibitors in high concentration is of great potential interest.

A mev-1(kn1) mutant of the nematode Caenorhabditis elegans is defective in the cytochrome b large subunit (Cyt-1/ceSDHC) in complex II of the mitochondrial electron transport chain. We have previously shown that a mutation in mev-1 causes shortened life span and rapid accumulation of aging markers such as fluorescent materials and protein carbonyls in an oxygen-dependent fashion. However, it remains unclear as to whether this hypersensitivity is caused by direct toxicity of the exogenous oxygen or by the damage of endogenous reactive oxygen species derived from mitochondria. Here we report important biochemical changes in mev-1 animals that serve to explain their abnormalities under normoxic conditions: (i) an overproduction of superoxide anion from mitochondria; and (ii) a reciprocal reduction in glutathione content even under atmospheric oxygen. In addition, unlike wild type, the levels of superoxide anion production from mev-1 mitochondria were significantly elevated under hyperoxia. Under normal circumstances, it is well known that superoxide anion is produced at complexes I and III in the electron transport system. Our data suggest that the mev-1(kn1) mutation increases superoxide anion production at complex II itself rather than at complexes I and III. The mev-1 mutant also had a lactate level 2-fold higher than wild type, indicative of lactic acidosis, a hallmark of human mitochondrial diseases. These data indicate that Cyt-1/ceSDHC plays an important role not only in energy metabolism but also in superoxide anion production that is critically involved in sensitivity to atmospheric oxygen.

Fabry disease is a lysosomal storage disorder caused by the deficiency of alpha-Gal A (alpha-galactosidase A) activity. In order to understand the molecular mechanism underlying alpha-Gal A deficiency in Fabry disease patients with residual enzyme activity, enzymes with different missense mutations were purified from transfected COS-7 cells and the biochemical properties were characterized. The mutant enzymes detected in variant patients (A20P, E66Q, M72V, I91T, R112H, F113L, N215S, Q279E, M296I, M296V and R301Q), and those found mostly in mild classic patients (A97V, A156V, L166V and R356W) appeared to have normal K(m) and V(max) values. The degradation of all mutants (except E59K) was partially inhibited by treatment with kifunensine, a selective inhibitor of ER (endoplasmic reticulum) alpha-mannosidase I. Metabolic labelling and subcellular fractionation studies in COS-7 cells expressing the L166V and R301Q alpha-Gal A mutants indicated that the mutant protein was retained in the ER and degraded without processing. Addition of DGJ (1-deoxygalactonojirimycin) to the culture medium of COS-7 cells transfected with a large set of missense mutant alpha-Gal A cDNAs effectively increased both enzyme activity and protein yield. DGJ was capable of normalizing intracellular processing of mutant alpha-Gal A found in both classic (L166V) and variant (R301Q) Fabry disease patients. In addition, the residual enzyme activity in fibroblasts or lymphoblasts from both classic and variant hemizygous Fabry disease patients carrying a variety of missense mutations could be substantially increased by cultivation of the cells with DGJ. These results indicate that a large proportion of mutant enzymes in patients with residual enzyme activity are kinetically active. Excessive degradation in the ER could be responsible for the deficiency of enzyme activity in vivo, and the DGJ approach may be broadly applicable to Fabry disease patients with missense mutations.

Fabry disease is a lysosomal storage disorder caused by deficient lysosomal alpha-galactosidase A (alpha-Gal A) activity. Deficiency of the enzyme activity results in progressive deposition of neutral glycosphingolipids with terminal alpha-galactosyl residue in vascular endothelial cells. We recently proposed a chemical chaperone therapy for this disease by administration of 1-deoxygalactonojirimycin, a potent inhibitor of the enzyme, at subinhibitory intracellular concentrations [Fan, J.-Q., Ishii, S., Asano, N. and Suzuki, Y. (1999) Nat. Med. 5, 112-115]. 1-Deoxygalactonojirimycin served as a specific chaperone for those mutant enzymes that failed to maintain their proper conformation to avoid excessive degradation. In order to establish a correlation between in vitro inhibitory activity and intracellular enhancement activity of the specific chemical chaperone, a series of 1-deoxygalactonojirimycin derivatives were tested for activity with both alpha-Gal A and Fabry lymphoblasts. 1-Deoxygalactonojirimycin was the most potent inhibitor of alpha-Gal A with an IC50 value of 0.04 microM. alpha-Galacto-homonojirimycin, alpha-allo-homonojirimycin and beta-1-C-butyl-deoxygalactonojirimycin were effective inhibitors with IC50 values of 0.21, 4.3 and 16 microM, respectively. N-Alkylation, deoxygenation at C-2 and epimerization at C-3 of 1-deoxygalactonojirimycin markedly lowered or abolished its inhibition toward alpha-Gal A. Inclusion of 1-deoxygalactonojirimycin, alpha-galacto-homonojirimycin, alpha-allo-homonojirimycin and beta-1-C-butyl-deoxygalactonojirimycin at 100 microM in culture medium of Fabry lymphoblasts increased the intracellular alpha-Gal A activity by 14-fold, 5.2-fold, 2.4-fold and 2.3-fold, respectively. Weaker inhibitors showed only a minimum enhancement effect. These results suggest that more potent inhibitors act as more effective specific chemical chaperones for the mutant enzyme, and the potent competitive inhibitors of alpha-Gal A are effective specific chemical chaperones for Fabry disease.

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Chromatographic separation of an extract of the bulbs of Scilla sibirica resulted in the isolation of five pyrrolidines, two pyrrolidine glycosides, six piperidines, one piperidine glycoside, and eight pyrrolizidines. 2,5-Dideoxy-2,5-imino-glycero-d-manno-heptitol (homoDMDP, 1) is a common alkaloid in all plants of the Hyacinthaceae examined to date and was also found in S. sibirica. The structures of the new alkaloids were elucidated by spectroscopic methods as 7-deoxy-homoDMDP (4), 2,5-dideoxy-2,5-imino-glycero-d-galacto-heptitol (5), the 4-O-beta-d-mannoside (6) and the 4-O-beta-d-mannobioside (7) of 6-deoxy-homoDMDP (2), 7-deoxyhomonojirimycin (12), 7-deoxyhomomannojirimycin (13), and polyhydroxypyrrolizidines, hyacinthacines A(4) (15), A(5) (16), A(6) (17), A(7) (18), B(4) (20), B(5) (21), and B(6) (22). HomoDMDP (1) is a potent inhibitor of beta-glucosidase and beta-galactosidase, while 6-deoxy-homoDMDP (2) showed significantly less inhibition. However, 7-deoxygenation of 1, leading to 4, showed no effect on the inhibitory activity toward both enzymes. Although 2 is not an inhibitor of alpha-l-fucosidase, the monomannoside of 2 shows inhibitory activity toward alpha-l-fucosidase. Elongation of the beta-mannopyranosyl chain of 6 to give 7 enhanced the inhibitory activity.