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Oxidative stress induced by cumene hydroperoxide was studied in cultured neonatal rat myocytes. A progressive increase of irreversible cell injury as determined by leakage of the cytoplastic enzyme alpha-hydroxybutyrate dehydrogenase (alpha-HBDH) from the cells was noted at concentrations ranging from 25-100 microM cumene hydroperoxide (incubation time 90 min). Cumene hydroperoxide-induced damage was reduced or prevented by several compounds: the application of Trolox C, a water-soluble vitamin E analogue, and of phospholipase A2 inhibitors chlorpromazine and (to a lesser extent) quinacrine prevented alpha-HBDH release. ICRF-159, a chelator of divalent cations, ascorbic acid, a potent antioxidant, and the cysteine protease inhibitor leupeptin did not reduce the cumene hydroperoxide-induced cytotoxicity. Detoxification of hydroperoxides by the glutathione peroxidase system results in an increased flux through the pentose phosphate shunt and loss of NADPH. Glucose inhibited the cumene hydroperoxide-induced alpha-HBDH release, probably by replenishing NADPH. These results indicate that cumene hydroperoxide, after exhaustion of the glutathione system, induces irreversible injury in cultured myocytes by a mechanism that depends to a large extent on deterioration of cellular membranes caused by lipid peroxidation and phospholipase activation.
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PMID:Prevention of cumene hydroperoxide induced oxidative stress in cultured neonatal rat myocytes by scavengers and enzyme inhibitors. 209 37

When grown anaerobically on L-rhamnose, Salmonella typhimurium excreted 1,2-propanediol as a fermentation product. Upon exhaustion of the methyl pentose, 1,2-propanediol was recaptured and further metabolized, provided the culture was kept under anaerobic conditions. n-Propanol and propionate were found in the medium as end products of this process at concentrations one-half that of 1,2-propanediol. As in Klebsiella pneumoniae (T. Toraya, S. Honda, and S. Fukui, J. Bacteriol. 139:39-47, 1979), a diol dehydratase which transforms 1,2-propanediol to propionaldehyde and the enzymes involved in a dismutation that converts propionaldehyde to n-propanol and propionate were induced in S. typhimurium cultures able to transform 1,2-propanediol anaerobically.
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PMID:Anaerobic metabolism of the L-rhamnose fermentation product 1,2-propanediol in Salmonella typhimurium. 328 5

Gluconate which was produced in cultures of Aspergillus niger with glucose as the sole source of carbon, or which was added after exhaustion of glucose, was utilized by this mold. In cell-free extracts from gluconate degrading mycelia gluconokinase and five enzymes of the pentose phosphate pathway were identified. Enzymes of the Entner-Doudoroff pathway and of a modified (non-phosphorylating) Entner-Doudoroff system, as well as a gluconate dehydrogenase could not be detected. It is concluded that gluconate after its phosphorylation to 6-phosphogluconate is metabolized via the pentose phosphate pathway by the strain used.
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PMID:Utilization of gluconate by Aspergillus niger. I. Enzymes of phosphorylating and nonphosphorylating pathways. 407 56

Succinate and several other compounds which induce sphere to rod morphogenesis of A. crystallopoietes were found to suppress both catabolism and assimilation of glucose. Diauxic growth patterns resulted from growth on glucose plus any one of these compounds. Glutamate stimulated growth but was not an inducer of morphogenesis. With this compound, diauxic growth and suppression of glucose catabolism or assimilation did not occur. Glucose permease was studied with alpha-methylglucoside as substrate. The entry system for glucose was found to involve active transport and to have a K(m) of 8 x 10(-4)m. It was inducible, was repressed in succinate-grown cells, and was also inhibited by succinate. The exit system was constitutive and appeared to be less sensitive than the entry system to inhibition by azide. The properties of the glucose permease system may account for the slow growth of the organism on glucose and the preferred use of other substrates for growth. Studies of metabolic pathways for glucose metabolism indicated the operation of the Embden-Meyerhof-Parnas (EMP) and pentose phosphate pathways and of the tricarboxylic acid cycle. Cells grown on glucose plus limiting amounts of succinate or other inducers of morphogenesis metabolized the glucose only after exhaustion of the inducers. Under these circumstances, the organisms employed the EMP pathway to a greater extent than when growing on glucose as sole carbon source.
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PMID:Alteration of glucose metabolism of Arthrobacter crystallopoietes by compounds which induce sphere to rod morphogenesis. 577 9

IOGEN Corporation of Ottawa, Canada, has recently built a 40t/d biomass-to-ethanol demonstration plant adjacent to its enzyme production facility. It has partnered with the University of Toronto to test the C6/C5 cofermenta-tion performance characteristics of the National Renewable Energy Labora-tory's metabolically engineered Zymomonas mobilis using various biomass hydrolysates. IOGEN's feedstocks are primarily agricultural wastes such as corn stover and wheat straw. Integrated recombinant Z. mobilis strain AX101 grows on D-xylose and/or L-arabinose as the sole carbon/energy sources and ferments these pentose sugars to ethanol in high yield. Strain AX101 lacks the tetracycline resistance gene that was a common feature of other recombinant Zm constructs. Genomic integration provides reliable cofermentation performance in the absence of antibiotics, another characteristic making strain AX101 attractive for industrial cellulosic ethanol production. In this work, IOGEN's biomass hydrolysate was simulated by a pure sugar medium containing 6% (w/v) glucose, 3% xylose, and 0.35% arabinose. At a level of 3 g/L (dry solids), corn steep liquor with inorganic nitrogen (0.8 g/L of ammonium chloride or 1.2 g/L of diammonium phosphate) was a cost-effective nutritional supplement. In the absence of acetic acid, the maximum volumetric ethanol productivity of a continuous fermentation at pH 5.0 was 3.54 g/L x h. During prolonged continuous fermentation, the efficiency of sugar-to-ethanol conversion (based on total sugar load) was maintained at >85%. At a level of 0.25% (w/v) acetic acid, the productivity decreased to 1.17 g/L x h at pH 5.5. Unlike integrated, xylose-utilizing rec Zm strain C25, strain AX101 produces less lactic acid as byproduct, owing to the fact that the Escherichia coli arabinose genes are inserted into a region of the host chromosome tentatively assigned to the gene for D-lactic acid dehydrogenase. In pH-controlled batch fermentations with sugar mixtures, the order of sugar exhaustion from the medium was glucose followed by xylose and arabinose. Both the total sugar load and the sugar ratio were shown to be important determinants for efficient cofermentation. Ethanol at a level of 3% (w/v) was implicated as both inhibitory to pentose fermentation and as a potentiator of acetic acid inhibition of pentose fermentation at pH 5.5. The effect of ethanol may have been underestimated in other assessments of acetic acid sensitivity. This work underscores the importance of employing similar assay conditions in making comparative assessments of biocatalyst fermentation performance.
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PMID:Performance testing of Zymomonas mobilis metabolically engineered for cofermentation of glucose, xylose, and arabinose. 1201 70

Vertebrate female reproduction is limited by the oocyte stockpiles acquired during embryonic development. These are gradually depleted over the organism's lifetime through the process of apoptosis. The timer that triggers this cell death is yet to be identified. We used the Xenopus egg/oocyte system to examine the hypothesis that nutrient stores can regulate oocyte viability. We show that pentose-phosphate-pathway generation of NADPH is critical for oocyte survival and that the target of this regulation is caspase-2, previously shown to be required for oocyte death in mice. Pentose-phosphate-pathway-mediated inhibition of cell death was due to the inhibitory phosphorylation of caspase-2 by calcium/calmodulin-dependent protein kinase II (CaMKII). These data suggest that exhaustion of oocyte nutrients, resulting in an inability to generate NADPH, may contribute to ooctye apoptosis. These data also provide unexpected links between oocyte metabolism, CaMKII, and caspase-2.
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PMID:Metabolic regulation of oocyte cell death through the CaMKII-mediated phosphorylation of caspase-2. 1621 6

Natural habitats of yeasts were examined for the presence of strains able to produce ethanol from d-xylose. Black knots, insect frass, and tree exudates were screened by enrichment in liquid d-xylose-yeast extract medium. These and each d-xylose-assimilating yeast in a collection from cactus fruits and Drosophila spp. were tested for alcohol production from this sugar. Among the 412 isolates examined, 36 produced more than 1 g of ethanol liter from 20 g of d-xylose liter, all under aerated conditions. Closer examination of the strains indicated that their time courses of d-xylose fermentation followed different patterns. Some strains produced more biomass than ethanol, and among these, ethanol may or may not be assimilated rapidly after depletion of d-xylose. Others produced more ethanol than biomass, but all catabolized ethanol after carbohydrate exhaustion. Ethanol production appeared best at low pH values and under mild aeration. Possible correlations between the nutritional profiles of the yeasts and their ability to produce ethanol from d-xylose were explored by multivariate analysis. d-Xylose appeared slightly better utilized by yeasts which rate poorly in terms of fermentation. The fermentation of d-glucose had no bearing on d-xylose fermentation. No specific nutritional trait could discriminate well between better d-xylose fermentors and other yeasts.
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PMID:Isolation and Screening of Yeasts That Ferment d-Xylose Directly to Ethanol. 1634 47

Saccharomyces cerevisiae and Candida shehatae were co-immobilized in an agar sheet which was introduced in an original two-chambered bioreactor asymmetrically fed in a batch mode with a mixture of glucose and xylose in a ratio of 35:15. The two sugars were consumed simultaneously. All glucose was fermented but only 20% of xylose. After incubation, yeast cells recovered from different areas of the agar sheet (close to, called Hi, and distant from, called Ho, the substrate chamber) were cultured as suspended cells in fresh culture medium provided with xylose or the sugar mixture. Xylose utilization by gel released Hi yeasts was significantly delayed compared to the Ho culture. Ethanol consumption by Hi yeasts in the two-substrate medium occurred after glucose exhaustion despite the presence of xylose. The waste medium resulting from incubation of the immobilized-cell structure inhibited xylose utilization by C. shehatae. Our results suggested that batch fermentation most probably favoured this incomplete xylose fermentation.
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PMID:Long-term incomplete xylose fermentation, after glucose exhaustion, with Candida shehatae co-immobilized with Saccharomyces cerevisiae. 1695 80

Zygosaccharomyces bailii is the most tolerant yeast species to acetic acid-induced toxicity, being able to grow in the presence of concentrations of this food preservative close to the legal limits. For this reason, Z. bailii is the most important microbial contaminant of acidic food products but the mechanisms behind this intrinsic resistance to acetic acid are very poorly characterized. To gain insights into the adaptive response and tolerance to acetic acid in Z. bailii, we explored an expression proteomics approach, based on quantitative 2DE, to identify alterations occurring in the protein content in response to sudden exposure or balanced growth in the presence of an inhibitory but nonlethal concentration of this weak acid. A coordinate increase in the content of proteins involved in cellular metabolism, in particular, in carbohydrate metabolism (Mdh1p, Aco1p, Cit1p, Idh2p, and Lpd1p) and energy generation (Atp1p and Atp2p), as well as in general and oxidative stress response (Sod2p, Dak2p, Omp2p) was registered. Results reinforce the concept that glucose and acetic acid are coconsumed in Z. bailii, with acetate being channeled into the tricarboxylic acid cycle. When acetic acid is the sole carbon source, results suggest the activation of gluconeogenic and pentose phosphate pathways, based on the increased content of several proteins of these pathways after glucose exhaustion.
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PMID:Adaptive response to acetic acid in the highly resistant yeast species Zygosaccharomyces bailii revealed by quantitative proteomics. 2268 79

The diauxic shift in Saccharomyces cerevisiae is an ideal model to study how eukaryotic cells readjust their metabolism from glycolytic to gluconeogenic operation. In this work, we generated time-resolved physiological data, quantitative metabolome (69 intracellular metabolites) and proteome (72 enzymes) profiles. We found that the diauxic shift is accomplished by three key events that are temporally organized: (i) a reduction in the glycolytic flux and the production of storage compounds before glucose depletion, mediated by downregulation of phosphofructokinase and pyruvate kinase reactions; (ii) upon glucose exhaustion, the reversion of carbon flow through glycolysis and onset of the glyoxylate cycle operation triggered by an increased expression of the enzymes that catalyze the malate synthase and cytosolic citrate synthase reactions; and (iii) in the later stages of the adaptation, the shutting down of the pentose phosphate pathway with a change in NADPH regeneration. Moreover, we identified the transcription factors associated with the observed changes in protein abundances. Taken together, our results represent an important contribution toward a systems-level understanding of how this adaptation is realized.
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PMID:Temporal system-level organization of the switch from glycolytic to gluconeogenic operation in yeast. 2354 79


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