Genetic organization and regulation of the xylose degradation genes in Streptomyces rubiginosus.

The xylose isomerase (xylA) and the xylulose kinase (xylB) genes from Streptomyces rubiginosus were isolated, and their nucleotide sequences were determined. The xylA and xylB genes encode proteins of 388 and 481 amino acids, respectively. These two genes are transcribed divergently from within a 114-nucleotide sequence separating the coding regions. Regulation of the xyl genes in S. rubiginosus was examined by fusing their promoters to the Pseudomonas putida catechol dioxygenase gene and integrating the fusions into the minicircle integration site on the S. rubiginosus chromosome. The expression of catechol dioxygenase was then measured under a variety of conditions. The results indicated that transcription of the xyl genes was induced by D-xylose and repressed by glucose. Data from quantitative S1 mapping were consistent with this conclusion and suggested that xylA had one and xylB had two transcription initiation sites. The transcription initiation site of xylA was 40 bp upstream of the coding region. The two transcription initiation sites of xylB were 20 and 41 bp 5' of its translation initiation codon. Under control of appropriate regulatory elements, the cloned xyl genes are capable of complementing either Escherichia coli xylose isomerase- or xylulose kinase-deficient strains. The deduced amino acid sequence of the S. rubiginosus xylA protein is highly homologous to sequences of other microbial xylose isomerases.

Age-related changes in the redox status of the housefly, Musca domestica.

The objective of this study was to further test the hypothesis that aging in the housefly is associated with increased oxidative stress. Age-related changes in the concentration of glutathione, NAD and NADP, which undergo oxidation-reduction reactions, and of H2O2, a potent cellular oxidant, were examined in the homogenates of adult male houseflies at 4, 8, 12 and 16 days of age. Sixteen days of age represents the beginning of the dying phase of the population when about 20% mortality usually occurs. Results indicate that the ratios of reduced/oxidized forms decline with age; H2O2 concentration steadily increases with age. Results suggest that the intracellular redox potential of the housefly becomes progressively more pro-oxidizing or less reducing during the aging process.

Correlation between life expectancy, flightlessness and actomyosin adenosine triphosphatase activity in the housefly, Musca domestica.

The objective of this investigation was to determine if actomyosin ATPase activity in flight muscles is correlated with life expectancy of houseflies. All houseflies lose flying ability before death which permits the identification of shorter-lived flightless 'crawlers' from their longer-lived cohorts, the 'fliers'. Life expectancy of crawlers is about one-third shorter than that of the fliers. Flying performance of houseflies, as measured by the total duration of flying activity during 1 h periods, average duration of flights and the number of rest stops, was highest at 4 days of age and declined thereafter. Actomyosin ATPase activity was higher in the fliers than in the crawlers of the same age. Abolition of flight, by surgical removal of wings at 1 day of age, had no effect on the enzyme activity. Results are interpreted to suggest that actomyosin ATPase activity is correlated with physiological rather than chronological age of flies.

Relationship between life expectancy, endogenous antioxidants and products of oxygen free radical reactions in the housefly, Musca domestica.

The general objective of this study was to identify biochemical correlates of life expectancy in the adult male housefly. All houseflies lose flying ability prior to death, whereby, in an aging population, shorter-lived flies can be identified as flightless 'crawlers' from their longer-lived cohorts, the 'fliers'; the average lifespan of crawlers is about one-third shorter than the fliers. Neither crawlers nor fliers exhibited any physical damage to their chemoreceptive tarsi, thereby ruling out starvation as a probable cause of death. Levels of antioxidant defenses (superoxide dismutase, catalase and glutathione) and products of oxygen free radical reactions (inorganic peroxides and thiobarbituric acid [TBA]-reactants) were compared between crawlers and fliers. The fliers showed higher superoxide dismutase and catalase activities and glutathione concentration than crawlers, whereas, the amount of inorganic peroxides (H2O2) and TBA-reactants was higher in the crawlers than in fliers. Results of this study demonstrate, for the first time, that longer life expectancy of organisms belonging to the same cohort group is associated with relatively higher levels of antioxidants and lower concentrations of products of oxygen free radical reactions.

Effects of exogenous antioxidants on the levels of endogenous antioxidants, lipid-soluble fluorescent material and life span in the housefly, Musca domestica.

Effects of exogenous antioxidant administration (0.5% and 2% ascorbate, beta-carotene and alpha-tocopherol in sucrose) on life-span, metabolic rate, activities of superoxide dismutase and catalase, levels of glutathione, inorganic peroxides and chloroform-soluble fluorescent material (lipofuscin) were examined in adult male houseflies. Administration of antioxidants at a level of 0.5% did not affect life-span, whereas, 2% ascorbate and alpha-tocopherol decreased average life-span. Metabolic rate of flies was unaffected, except by 2% ascorbate, which caused a decrease. Superoxide dismutase activity was depressed by 2% ascorbate at all ages, and by beta-carotene and alpha-tocopherol in older flies. Catalase activity was unaffected except by alpha-tocopherol at younger ages. Glutathione concentration was decreased by ascorbate and beta-carotene at both concentrations administered. Inorganic peroxides (H2O2) were increased by 2% beta-carotene and alpha-tocopherol. Only high concentrations of ascorbate and beta-carotene decreased the level of soluble fluorescent material. Results suggest that administration of exogenous antioxidants causes a compensatory depression of endogenous defenses.

Effects of experimentally altered glutathione levels on life span, metabolic rate, superoxide dismutase, catalase and inorganic peroxides in the adult housefly, Musca domestica.

Effects of varied levels of glutathione, an intracellular redox buffer, were examined in the adult male housefly in order to study the inter-relationship between enzymic and non-enzymic antioxidant defenses. An increase of over 100% in the concentrations of glutathione was induced by the administration of 3 mM L-2-oxothiazolidine-4-carboxylate (LOC), which increases the intracellular level of cysteine. A decrease in glutathione concentration of up to 85% was achieved by the administration of L-buthionine-SR-sulfoximine (BUS), which irreversibly inhibits glutamylcysteine synthetase. Life spans of houseflies were shortened by a decrease in the glutathione concentration, but were not prolonged by augmentation of glutathione. Metabolic rate and superoxide dismutase activity were independent of glutathione concentration. H2O2 was increased by both experimental regimes, whereas catalase activity was decreased by BUS. Results suggest that catalase activity is influenced by glutathione concentration.

Involvement of Glutathione in the Differentiation of the Slime Mold Physarum polycephalum: (cellular differentiation/Physarum/oxy-free radicals/superoxide dismutase/glutathione).

The effects of experimentally-altered glutathione concentration on differentiation of the slime mold, Physarum polycephalum were examined. Spherulation was induced by transfer of Physarum from growth medium to a salts-only starvation medium. As differentiation proceeded, superoxide dismutase (SOD) activity in control cultures increased by as much as 21-fold. This increase in SOD activity paralleled the rate of differentiation. Glutathione (GSH) concentration decreased during differentiation by more than 80% in all cultures, regardless of the initial concentration. The rate of differentiation was inversely related to the initial GSH concentration and directly proportional to the SOD activity. These observations suggest that a free radical mechanism may be involved in the differentiation of Physarum microplasmodia into spherules.