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.

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.

Rapid assays for Escherichia coli were developed by using the compound 4methylumbelliferone glucuronide (MUG), which is hydrolyzed by glucuronidase to yield a fluorogenic product. The production of glucuronidase was limited to strains of E. coli and some Salmonella and Shigella strains in the family Enterobacteriaceae. For immediate confirmation of the presence of E. coli in most-probable-number tubes, MUG was incorporated into lauryl tryptose broth at a final concentration of 100 ,ug/ml. Results of both the presumptive test (gas production) and the confirmed test (fluorescence) for E. coli were obtained from a variety of food, water, and milk samples after incubation for only 24 h at 35°C. Approximately 90% of the tubes showing both gas production and fluorescence contained fecal coliforms (they were positive in EC broth incubated at 45°C). Few false-positive reactions were observed. The lauryl tryptose broth-MUG-mostprobable-number assay was superior to violet red bile agar for the detection of heatand chlorine-injured E. coli cells. Anaerogenic strains produced positive reactions, and small numbers of E. coli could be detected in the presence of large numbers of competing bacteria. The fluorogenic assay was sensitive and rapid; the presence of one viable cell was detected within 20 h. E. coli colonies could be distinguished from other coliforms on membrane filters and plates of violet red bile agar if MUG was incorporated into the culture media. A rapid confirmatory test for E. coli that is amenable to automation was developed by using microtitration plates filled with a nonselective medium containing MUG. Pure or mixed cultures containing E. coli produced fluorescence within 4 h (most strains) to 24 h (a few weakly positive strains).

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A small, anaerobic, gram-positive coccobacillus that reduces cholesterol to coprostanol was isolated from a hog sewage lagoon. This isolate, strain HLT (T = type strain) does not require cholesterol for growth, but it requires lecithin and has phospholipase activity. Much acid is produced by the fermentation of amygdalin, lactose, and salicin. Arabinose, cellobiose, fructose, glucose, mannose, and melibiose are fermented weakly. Acetic, formic, and succinic acids are produced, as is hydrogen, The isolate does not reduce nitrate, produce indole, or hydrolyze starch and gelatin. Esculin is hydrolyzed. The properties of strain HLT are most similar to those of members of the genus Eubacterium. Because strain HL (= ATCC 51222) has unique morphological and physiological properties, we propose that it should be the type strain of a new species in the genus Eubacterium, Eubacterium coprostanoligenes.To date, 13 strains of cholesterol-reducing bacteria have been isolated and characterized (2, 4, 12,14). All of these organisms were isolated from the fecal contents of rats, humans, or baboons and were classified as strains of Eubactenurn species.Because of the methods used and the sources sampled, most of the strains of cholesterol-reducing bacteria that have been characterized have similar properties. These bacteria require strict anaerobic conditions for growth, and all but two strains require a plasmalogen (plasmenylethanolamine) to reduce cholesterol to coprostanol (1). Many also require cholesterol or a related sterol for growth (4, 12,14).Coprostanol is not absorbed by the human gastrointestinal system. Therefore, the use of bacteria that can convert cholesterol to coprostanol holds promise for medical reasons and is of interest to food industries. New methods and a wide range of sample types have been tested to facilitate the isolation of new cholesterol-reducing bacteria (5, 6). In this paper, we describe characterization of a cholesterol-reducing bacterium isolated from a hog sewage lagoon. Because strain H L has unique morphological and physiological properties, including the capacity to reduce cholesterol to coprostanol, we propose that it should be the type strain of a new species, Eubactenum coprostanoligenes. MATERIALS AND METHODSMedia and culture conditions. Base cholesterol medium (BCM) contained (per liter) 2 g of Casitone (Difco Laboratories, Detroit, Mich.), 10 g of yeast extract, 2 g of cholesterol (ash-free; Sigma Chemical Co., St. Louis, Mo.), 1 g of lecithin (type IV-S; Sigma), 0.5 g of sodium thioglycolate, 1 g of CaCl, . 2H,O, and 1 mg of resazurin. Plasmenylethanolamine was not added to this medium. BCM was prepared as described by Brinkley and coworkers (l), with the following modifications: cholesterol and lecithin were homogenized in distilled, purified water in a Waring blender under a stream of 0,-free N, gas (7); other components were added; and the pH was adjusted to 7.2 with 3 N KOH. The medium was boiled under an N, atmosphere and cooled, and 9-ml aliquots were dispensed into culture tube...