Metabolic characterization of Lactococcus lactis deficient in lactate dehydrogenase using in vivo 13C-NMR.

The metabolism of glucose by nongrowing cells of Lactococcus lactis strain FI7851, constructed from the wild-type L. lactis strain MG1363 by disruption of the lactate dehydrogenase (ldh) gene [Gasson, M.J., Benson, K., Swindel, S. & Griffin, H. (1996) Lait 76, 33-40] was studied in a noninvasive manner by 13C-NMR. The kinetics of the build-up and consumption of the pools of intracellular intermediates mannitol 1-phosphate, fructose 1,6-bisphosphate, 3-phosphoglycerate, and phosphoenolpyruvate as well as the utilization of [1-13C]glucose and formation of products (lactate, acetate, mannitol, ethanol, acetoin, 2,3-butanediol) were monitored in vivo with a time resolution of 30 s. The metabolism of glucose by the parental wild-type strain was also examined for comparison. A clear shift from typical homolactic fermentation (parental strain) to a mixed acid fermentation (lactate dehdydrogenase deficient; LDHd strain) was observed. Furthermore, high levels of mannitol were transiently produced and metabolized once glucose was depleted. Mannitol 1-phosphate accumulated intracellularly up to 76 mM concentration. Mannitol was formed from fructose 6-phosphate by the combined action of mannitol-1-phosphate dehydrogenase and phosphatase. The results show that the formation of mannitol 1-phosphate by the LDHd strain during glucose catabolism is a consequence of impairment in NADH oxidation caused by a highly reduced LDH activity, the transient production of mannitol 1-phosphate serving as a regeneration pathway for NAD+ regeneration. Oxygen availability caused a drastic change in the pattern of intermediates and end-products, reinforcing the key-role of the fulfilment of the redox balance. The flux control coefficients for the step catalysed by mannitol-1-phosphate dehydrogenase were calculated and the implications in the design of metabolic engineering strategies are discussed.     

Studies on anaerobic glucose and glutamate metabolism in larvae of Callitroga macellaria

Anaerobic metabolism was investigated in Callitroga macellaria larvae, using isotopic, chromatographic and enzymatic methods after in vivo and in vitro incubations.The study of anaerobic glucose metabolism shows that the classical pathway from glucose to lactate may not be the only pathway of anaerobic energy gain. End products of anaerobic [¹⁴C]-glucose catabolism are lactate, acetate, alanine, pyruvate and polyols.During four hours of anaerobiosis the concentrations of glutamate, glutamine, aspartate, citrate and isocitrate, malate decreased, whereas asparagine, oxaloacetate and α-ketoglutarate concentrations remained constant and the succinate concentration increased. In vitro incubations with [¹⁴C]-glutamate confirmed that glutamate utilization occurred anaerobically. The pathway is suggested to proceed from glutamate via α-ketoglutarate to succinate, to proline and to acetate possibly via citrate.The results indicate that anaerobic metabolism in C. macellaria larvae may differ significantly from other known pathways.     

Isolation and characterization of adult rat hearts cells.

Adult rat heart muscle cells were isolated after simultaneous perfusion of multiple (two to eight) hearts with buffered salt solutions containing collagenase and hyaluronidase. Yields (35 to 50% of ventricular weight with approximately 70% viability) are quantitatively suitable for metabolic studies. Viability has been determined by the ability of intact cells to exclude trypan blue and the inability of intact cells to oxidize exogenous succinate. Micrographs show that the fine structure of the isolated cells is well ordered. Cell concentrations of glycogen, glucose 6-phosphate, citrate, and various enzymes were similar to those of intact heart. ATP and creatine phosphate concentrations were lower than in whole hearts. Adenosine 3′,5′-monophosphate concentrations were somewhat elevated. Deoxyribonucleic acid was lower than in whole tissue. The isolated cells retain certain metabolic control mechanisms. The uncoupler of oxidative phosphorylation, 2,4-dinitrophenol, increased oxygen consumption severalfold, whereas exogenous ADP had no effect on respiration. Under anaerobic conditions the rates of glucose utilization and lactate production were faster than in the presence of oxygen, indicating retention of the Pasteur effect. The addition of glucose and insulin caused a decrease in oxygen uptake or the Crabtree effect. Exogenously added pyruvate decreased glycolytic flux and produced a pronounced increase in intracellular citrate and glucose 6-phosphate. Isoproterenol stimulated adenylate cyclase activity of the isolated cells at the same concentrations effective with intact heart preparations. Isoproterenol and glucagon caused the activation of phosphorylase. The cells deteriorated as a function of incubation time, as indicated by a decrease in ATP content and a loss of lactate dehydrogenase into the medium. Cell deterioration was greatly accelerated by Ca²⁺ at concentrations greater than 10^?5m.     

Inactivation of the Pta-AckA Pathway Causes Cell Death in Staphylococcus aureus

During growth under conditions of glucose and oxygen excess, Staphylococcus aureus predominantly accumulates acetate in the culture medium, suggesting that the phosphotransacetylase-acetate kinase (Pta-AckA) pathway plays a crucial role in bacterial fitness. Previous studies demonstrated that these conditions also induce the S. aureus CidR regulon involved in the control of cell death. Interestingly, the CidR regulon is comprised of only two operons, both encoding pyruvate catabolic enzymes, suggesting an intimate relationship between pyruvate metabolism and cell death. To examine this relationship, we introduced ackA and pta mutations in S. aureus and tested their effects on bacterial growth, carbon and energy metabolism, cid expression, and cell death. Inactivation of the Pta-AckA pathway showed a drastic inhibitory effect on growth and caused accumulation of dead cells in both pta and ackA mutants. Surprisingly, inactivation of the Pta-AckA pathway did not lead to a decrease in the energy status of bacteria, as the intracellular concentrations of ATP, NAD⁺, and NADH were higher in the mutants. However, inactivation of this pathway increased the rate of glucose consumption, led to a metabolic block at the pyruvate node, and enhanced carbon flux through both glycolysis and the tricarboxylic acid (TCA) cycle. Intriguingly, disruption of the Pta-AckA pathway also induced the CidR regulon, suggesting that activation of alternative pyruvate catabolic pathways could be an important survival strategy for the mutants. Collectively, the results of this study demonstrate the indispensable role of the Pta-AckA pathway in S. aureus for maintaining energy and metabolic homeostasis during overflow metabolism.     

Effects of carbonylcyanide-m-chlorophenylhydrazone (CCCP) and acetate on Escherichia coli O157:H7 and K-12: uncoupling versus anion accumulation

Non-growing cells of Escherichia coli O157:H7 and K-12 that were incubated anaerobically in sodium phosphate buffer at pH 6.5 consumed glucose at a rate of approximately 8 μmol·(mg protein)⁻¹·h⁻¹ and had intracellular pH values of 7.3 and 7.5, respectively. The uncoupler, carbonylcyanide-m-chlorophenylhydrazone (CCCP), caused a marked decrease in intracellular pH, ATP and potassium of both strains. Low concentrations of CCCP stimulated glucose consumption rate, but higher concentrations were inhibitory. Acetate also caused a decrease in intracellular pH, but it never caused a large decrease in glucose consumption rate. Acetate decreased the intracellular ATP of E. coli K-12, but it had no effect on the ATP of O157:H7. Acetate had no effect on the intracellular potassium of E. coli O157:H7, and acetate-treated K-12 cells had even more potassium than untreated controls. Based on these results, acetate and CCCP appear to have different effects on E. coli. The comparison of E. coli O157:H7 and K-12 indicated that intracellular pH, acetate accumulation and intracellular potassium were related. E. coli K-12 maintained a higher intracellular pH than O157:H7, accumulated more acetate and had a greater intracellular potassium.     

Cross-Feeding of Lactate Between Streptococcus lactis and Bacteroides sp. Isolated from Termite Hindguts

Streptococcus lactis and Bacteroides sp., isolated from hindguts of Reticulitermes flavipes termites, were grown anaerobically in monoculture and coculture. When grown in a glucose medium, S. lactis monoculture produced lactate as the major fermentation product, with small amounts of formate, acetate, ethanol, and CO₂. In coculture, glucose was completely consumed during growth of S. lactis. Lactate, produced by S. lactis, then supported much of the growth of Bacteroides and was fermented to propionate, acetate, and CO₂. Small amounts of succinate were formed during growth of Bacteroides in the coculture, but little change in the formate or ethanol concentration was observed. Monoculture growth of Bacteroides in a tryptone-yeast extract medium revealed that incorporation of 20 to 40 mM lactate increased cell yields and production of organic acids. However, initial lactate concentrations greater than 40 mM suppressed not only growth of Bacteroides but also acidic product formation. Results suggest that cross-feeding of lactate between streptococci and bacteroides constitutes one aspect of the overall hindgut fermentation in termites.     

Effects of oxygen on Pseudomonas nautica growth on n-alkane with or without nitrate

The denitrifying marine bacterium, Pseudomonas nautica 617, can grow on lactate aerobically or anaerobically in presence of nitrate with generation times of 1.5 and 3 h respectively. The growth on heptadecane occurs only in presence of oxygen whatever its concentration with a genrration time of 8.5 h. The influence of oxygen, carbon sources (lactate or heptadecane) and nitrate was examined on O₂, NO₃ ^-, NO₂ ^- consumption, on nitrate and nitrite reductases activities, on cell yields, and on the ratio of CO₂ produced per unit of biomass. Pseudomonas nautica metabolizes hydrocarbons under denitrifying conditions in the presence of oxygen. Nitrate and nitrite are used during growth on lactate and heptadecane up to oxygen concentrations corresponding to 50 and 30% of air-saturation, respectively. When growth on n-alkane was not oxygen-limited (above 50% of air-saturation) the catabolism decreases in favour of carbon incorporation into the cell. Nitrate and nitrite reductases were strongly inhibited after 20% of airsaturation in the presence of lactate as growth substrate. With n-alkane, only the nitrate reductase activity was greatly reduced.     

Engineering of sugar metabolism of Corynebacterium glutamicum for production of amino acid l-alanine under oxygen deprivation

Corynebacterium glutamicum was genetically engineered to produce l-alanine from sugar under oxygen deprivation. The genes associated with production of organic acids in C. glutamicum were inactivated and the alanine dehydrogenase gene (alaD) from Lysinibacillus sphaericus was overexpressed to direct carbon flux from organic acids to alanine. Although the alaD-expressing strain produced alanine from glucose under oxygen deprivation, its productivity was relatively low due to retarded glucose consumption. Homologous overexpression of the gapA gene encoding glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in the alaD-expressing strain stimulated glucose consumption and consequently improved alanine productivity. In contrast gapA overexpression did not affect glucose consumption under aerobic conditions, indicating that oxygen deprivation engendered inefficient regeneration of NAD⁺ resulting in impaired GAPDH activity and reduced glucose consumption in the alanine-producing strains. Inactivation of the alanine racemase gene allowed production of l-alanine with optical purity greater than 99.5%. The resulting strain produced 98 g l⁻¹ of l-alanine after 32 h in mineral salts medium. Our results show promise for amino acid production under oxygen deprivation.     

Engineering of Corynebacterium glutamicum for High-Yield l-Valine Production under Oxygen Deprivation Conditions

We previously demonstrated efficient l-valine production by metabolically engineered Corynebacterium glutamicum under oxygen deprivation. To achieve the high productivity, a NADH/NADPH cofactor imbalance during the synthesis of l-valine was overcome by engineering NAD-preferring mutant acetohydroxy acid isomeroreductase (AHAIR) and using NAD-specific leucine dehydrogenase from Lysinibacillus sphaericus. Lactate as a by-product was largely eliminated by disrupting the lactate dehydrogenase gene ldhA. Nonetheless, a few other by-products, particularly succinate, were still produced and acted to suppress the l-valine yield. Eliminating these by-products therefore was deemed key to improving the l-valine yield. By additionally disrupting the phosphoenolpyruvate carboxylase gene ppc, succinate production was effectively suppressed, but both glucose consumption and l-valine production dropped considerably due to the severely elevated intracellular NADH/NAD⁺ ratio. In contrast, this perturbed intracellular redox state was more than compensated for by deletion of three genes associated with NADH-producing acetate synthesis and overexpression of five glycolytic genes, including gapA, encoding NADH-inhibited glyceraldehyde-3-phosphate dehydrogenase. Inserting feedback-resistant mutant acetohydroxy acid synthase and NAD-preferring mutant AHAIR in the chromosome resulted in higher l-valine yield and productivity. Deleting the alanine transaminase gene avtA suppressed alanine production. The resultant strain produced 1,280 mM l-valine at a yield of 88% mol mol of glucose⁻¹ after 24 h under oxygen deprivation, a vastly improved yield over our previous best.     

Staphylococcus aureus Alpha-Toxin Mediates General and Cell Type-Specific Changes in Metabolite Concentrations of Immortalized Human Airway Epithelial Cells

Staphylococcus aureus alpha-toxin (Hla) is a potent pore-forming cytotoxin that plays an important role in the pathogenesis of S. aureus infections, including pneumonia. The impact of Hla on the dynamics of the metabolome in eukaryotic host cells has not been investigated comprehensively. Using ¹H-NMR, GC-MS and HPLC-MS, we quantified the concentrations of 51 intracellular metabolites and assessed alterations in the amount of 25 extracellular metabolites in the two human bronchial epithelial cell lines S9 and 16HBE14o⁻ under standard culture conditions and after treatment with sub-lethal amounts (2 µg/ml) of recombinant Hla (rHla) in a time-dependent manner. Treatment of cells with rHla caused substantial decreases in the concentrations of intracellular metabolites from different metabolic pathways in both cell lines, including ATP and amino acids. Concomitant increases in the extracellular concentrations were detected for various intracellular compounds, including nucleotides, glutathione disulfide and NAD⁺. Our results indicate that rHla has a major impact on the metabolome of eukaryotic cells as a consequence of direct rHla-mediated alterations in plasma membrane permeability or indirect effects mediated by cellular signalling. However, cell-specific changes also were observed. Glucose consumption and lactate production rates suggest that the glycolytic activity of S9 cells, but not of 16HBE14o⁻ cells, is increased in response to rHla. This could contribute to the observed higher level of resistance of S9 cells against rHla-induced membrane damage.     

Very Low Ethanol Concentrations Affect the Viability and Growth Recovery in Post-Stationary-Phase Staphylococcus aureus Populations

Pharmaceuticals, culture media used for in vitro diagnostics and research, human body fluids, and environments can retain very low ethanol concentrations (VLEC) (≤0.1%, vol/vol). In contrast to the well-established effects of elevated ethanol concentrations on bacteria, little is known about the consequences of exposure to VLEC. We supplemented growth media for Staphylococcus aureus strain DSM20231 with VLEC (VLEC⁺ conditions) and determined ultramorphology, growth, and viability compared to those with unsupplemented media (VLEC⁻ conditions) for prolonged culture times (up to 8 days). VLEC⁺-grown late-stationary-phase S. aureus displayed extensive alterations of cell integrity as shown by scanning electron microscopy. Surprisingly, while ethanol in the medium was completely metabolized during exponential phase, a profound delay of S. aureus post-stationary-phase recovery (>48 h) was observed. Concomitantly, under VLEC⁺ conditions, the concentration of acetate in the culture medium remained elevated while that of ammonia was reduced, contributing to an acidic culture medium and suggesting decreased amino acid catabolism. Interestingly, amino acid depletion was not uniformly affected: under VLEC⁺ conditions, glutamic acid, ornithine, and proline remained in the culture medium while the uptake of other amino acids was not affected. Supplementation with arginine, but not with other amino acids, was able to restore post-stationary-phase growth and viability. Taken together, these data demonstrate that VLEC have profound effects on the recovery of S. aureus even after ethanol depletion and delay the transition from primary to secondary metabolite catabolism. These data also suggest that the concentration of ethanol needed for bacteriostatic control of S. aureus is lower than that previously reported.     

Carbon Flux Analysis by 13C Nuclear Magnetic Resonance To Determine the Effect of CO2 on Anaerobic Succinate Production by Corynebacterium glutamicum

Wild-type Corynebacterium glutamicum produces a mixture of lactic, succinic, and acetic acids from glucose under oxygen deprivation. We investigated the effect of CO₂ on the production of organic acids in a two-stage process: cells were grown aerobically in glucose, and subsequently, organic acid production by nongrowing cells was studied under anaerobic conditions. The presence of CO₂ caused up to a 3-fold increase in the succinate yield (1 mol per mol of glucose) and about 2-fold increase in acetate, both at the expense of l-lactate production; moreover, dihydroxyacetone formation was abolished. The redistribution of carbon fluxes in response to CO₂ was estimated by using ¹³C-labeled glucose and ¹³C nuclear magnetic resonance (NMR) analysis of the labeling patterns in end products. The flux analysis showed that 97% of succinate was produced via the reductive part of the tricarboxylic acid cycle, with the low activity of the oxidative branch being sufficient to provide the reducing equivalents needed for the redox balance. The flux via the pentose phosphate pathway was low (∼5%) regardless of the presence or absence of CO₂. Moreover, there was significant channeling of carbon to storage compounds (glycogen and trehalose) and concomitant catabolism of these reserves. The intracellular and extracellular pools of lactate and succinate were measured by in vivo NMR, and the stoichiometry (H⁺:organic acid) of the respective exporters was calculated. This study shows that it is feasible to take advantage of natural cellular regulation mechanisms to obtain high yields of succinate with C. glutamicum without genetic manipulation.     

Physiological Role of β-Phosphoglucomutase in Lactococcus lactis

A β-phosphoglucomutase (β-PGM) mutant of Lactococcus lactis subsp. lactis ATCC 19435 was constructed using a minimal integration vector and double-crossover recombination. The mutant and the wild-type strain were grown under controlled conditions with different sugars to elucidate the role of β-PGM in carbohydrate catabolism and anabolism. The mutation did not significantly affect growth, product formation, or cell composition when glucose or lactose was used as the carbon source. With maltose or trehalose as the carbon source the wild-type strain had a maximum specific growth rate of 0.5 h⁻¹, while the deletion of β-PGM resulted in a maximum specific growth rate of 0.05 h⁻¹ on maltose and no growth at all on trehalose. Growth of the mutant strain on maltose resulted in smaller amounts of lactate but more formate, acetate, and ethanol, and approximately 1/10 of the maltose was found as β-glucose 1-phosphate in the medium. Furthermore, the β-PGM mutant cells grown on maltose were considerably larger and accumulated polysaccharides which consisted of α-1,4-bound glucose units. When the cells were grown at a low dilution rate in a glucose and maltose mixture, the wild-type strain exhibited a higher carbohydrate content than when grown at higher growth rates, but still this content was lower than that in the β-PGM mutant. In addition, significant differences in the initial metabolism of maltose and trehalose were found, and cell extracts did not digest free trehalose but only trehalose 6-phosphate, which yielded β-glucose 1-phosphate and glucose 6-phosphate. This demonstrates the presence of a novel enzymatic pathway for trehalose different from that of maltose metabolism in L. lactis.     

Glucose Metabolism in Sediments of a Eutrophic Lake: Tracer Analysis of Uptake and Product Formation

The uptake of glucose and the formation of end products from glucose catabolism have been measured for sediments of eutrophic Wintergreen Lake with a combination of tritiated and ¹⁴C-labeled tracers. Time course analyses of the loss of [³H]glucose from sediments were used to establish rate constants for glucose uptake at natural substrate concentrations. Turnover times from these analyses were about 1 min for littoral and profundal sediments. No seasonal or site differences were noted in turnover times. Time course analyses of [U-¹⁴C]glucose uptake and ¹⁴C-labeled end product formation indicated that glucose mass flow could not be calculated from end product formation since the specific activity of added [¹⁴C]glucose was significantly diluted by pools of intracellular glucose and glucose metabolites. Mass flow could only be accurately estimated by use of rates of uptake from tracer studies. Intermediate fermentation end products included acetate (71%), propionate (15%), lactate (9%), and only minor amounts of butyrates or valerates. Addition of H₂ to sediments resulted in greater production of lactate (28%) and decreased formation of acetate (50%), but did not affect glucose turnover. Depth profiles of glucose uptake indicated that rates of uptake decreased with depth over the 0- to 18-cm interval and that glucose uptake accounted for 30 to 40% of methanogenesis in profundal sediments.     

Engineering Lactococcus lactis for Production of Mannitol: High Yields from Food-Grade Strains Deficient in Lactate Dehydrogenase and the Mannitol Transport System

Mannitol is a sugar polyol claimed to have health-promoting properties. A mannitol-producing strain of Lactococcus lactis was obtained by disruption of two genes of the phosphoenolpyruvate (PEP)-mannitol phosphotransferase system (PTS^Mtl). Genes mtlA and mtlF were independently deleted by double-crossover recombination in strain L. lactis FI9630 (a food-grade lactate dehydrogenase-deficient strain derived from MG1363), yielding two mutant (ΔldhΔmtlA and ΔldhΔmtlF) strains. The new strains, FI10091 and FI10089, respectively, do not possess any selection marker and are suitable for use in the food industry. The metabolism of glucose in nongrowing cell suspensions of the mutant strains was characterized by in vivo ¹³C-nuclear magnetic resonance. The intermediate metabolite, mannitol-1-phosphate, accumulated intracellularly to high levels (up to 76 mM). Mannitol was a major end product, one-third of glucose being converted to this hexitol. The double mutants, in contrast to the parent strain, were unable to utilize mannitol even after glucose depletion, showing that mannitol was taken up exclusively by PEP-PTS^Mtl. Disruption of this system completely blocked mannitol transport in L. lactis, as intended. In addition to mannitol, approximately equimolar amounts of ethanol, 2,3-butanediol, and lactate were produced. A mixed-acid fermentation (formate, ethanol, and acetate) was also observed during growth under controlled conditions of pH and temperature, but mannitol production was low. The reasons for the alteration in the pattern of end products under nongrowing and growing conditions are discussed, and strategies to improve mannitol production during growth are proposed.     

Effect of aerobic and anaerobic growth on the cell wall ofStaphylococcus aureus

Electron micrographs ofStaphylococcus aureus 7167 which had been grown anaerobically showed that the cell wall was approximately 5 times thicker than the wall of bacteria after aerobic growth. Cell walls prepared from anaerobically grownS. aureus were more sensitive to the bacteriolytic enzymes: lysostaphin, lysozyme, and the wall-associated autolytic enzyme ofB. subtilis 168 I^?. Our findings are interpreted as evidence that the cell wall or surface of anaerobically grownS. aureus 7167 is different from that of aerobically grownS. aureus 7167. The findings suggest that the cell wall peptidoglycan of the anaerobe is a more loosely formed network, resulting in a more rapid solubilization by the bacteriolytic enzymes.     

The dynamics of carbohydrate metabolism in Candida albicans

The total cellular concentrations of the intermediary metabolites and the carbohydrate end products were determined for starved Candida albicans yeast cells and cells forming germ tubes during a 60-min incubation in imidazole-HCl buffer in the absence and presence of 2.5 mM glucose, 2.5 mM glutamine, and 0.2% serum at 37°C. These cells were also incubated in the presence of tracer [U-¹⁴C]glucose and the specific radioactivities of the metabolites and end products determined. The labeling data indicated (1) a minimum of two metabolically independent pools of glucose 6-phosphate, fructose 6-phosphate, glucose 1-phosphate, and uridine diphosphoglucose; (2) compartmentation of the pathways of catabolism and anabolism; (3) channeling of the exogenous tracer glucose into the anabolic pathway compartments of the starved cells; and (4) a significant rate of turnover of cell wall carbohydrates in cells incubated under nongrowth conditions and rapid turnover of these pools in germ tube forming cells. The labeling data will be used to construct kinetic models of carbohydrate metabolism in C. albicans.     

Arctic ground squirrel neuronal progenitor cells resist oxygen and glucose deprivation-induced death

AIM: To investigate the influence of ischemia/reperfusion on arctic ground squirrel(AGS) neuronal progenitor cells(NPCs), we subjected these cultured cells to oxygen and glucose deprivation.METHODS: AGS NPCs were expanded and differentiated into NPCs and as an ischemia vulnerable control, commercially available human NPCs(hNPCs) were seeded from thawed NPCs. NPCs, identified by expression of TUJ1 were seen at 14-21 d in vitro(DIV). Cultures were exposed to control conditions, hypoxia, oxygen and glucose deprivation or glucose deprivation alone or following return to normal conditions to model reperfusion. Cell viability and death were assessed from loss of ATP as well as from measures of alamarB lue~ and lactate dehydrogenase in the media and from counts of TUJ1 positive cells using immunocytochemistry. Dividing cells were identified by expression of Ki67 and phenotyped by double labeling with GFAP, MAP2 ab or TUJ1. RESULTS: We report that when cultured in NeuraLife~(TM), AGS cells remain viable out to 21 DIV, continue to express TUJ1 and begin to express MAP2 ab. Viability of hN PCs assessed by fluorescence alamarB lue(arbitrary units) depends on both glucose and oxygen availability [viability of hNPCs after 24 h oxygen glucose deprivation(OGD) with return of oxygen and glucose decreased from 48151 ± 4551 in control cultures to 43481 ± 2413 after OGD, P 0.05]. By contrast, when AGS NPCs are exposed to the same OGD with reperfusion at 14 DIV, cell viability assessed by alamar Blue increased from 165305 ± 11719 in control cultures to 196054 ± 13977 after OGD. Likewise AGS NPCs recovered ATP(92766 ± 6089 in control and 92907 ± 4290 after modeled reperfusion; arbitrary luminescence units), and doubled in the ratio of TUJ1 expressing neurons to total dividing cells(0.11 ± 0.04 in control cultures vs 0.22 ± 0.2 after modeled reperfusion, P 0.05). Maintaining AGS NPCs for a longer time in culture lowered resistance to injury, however, did not impair proliferation of NPCs relative to other cell lineages after oxygen deprivation followed by re-oxygenation.CONCLUSION: Ischemic-like insults decrease viability and increase cell death in cultures of human NPCs. Similar conditions have less affect on cell death and promote proliferation in AGS NPCs.     

Evolutionary engineering of a glycerol‐3‐phosphate dehydrogenase‐negative,acetate‐reducing Saccharomyces cerevisiae strain enables anaerobic growth at high glucose concentrations

Glycerol production by Saccharomyces cerevisiae, which is required for redox-cofactor balancing in anaerobic cultures, causes yield reduction in industrial bioethanol production. Recently, glycerol formation in anaerobic S. cerevisiae cultures was eliminated by expressing Escherichia coli (acetylating) acetaldehyde dehydrogenase (encoded by mhpF) and simultaneously deleting the GPD1 and GPD2 genes encoding glycerol-3-phosphate dehydrogenase, thus coupling NADH reoxidation to reduction of acetate to ethanol. Gpd^– strains are, however, sensitive to high sugar concentrations, which complicates industrial implementation of this metabolic engineering concept. In this study, laboratory evolution was used to improve osmotolerance of a Gpd^– mhpF-expressing S. cerevisiae strain. Serial batch cultivation at increasing osmotic pressure enabled isolation of an evolved strain that grew anaerobically at 1 M glucose, at a specific growth rate of 0.12 h⁻¹. The evolved strain produced glycerol at low concentrations (0.64 ± 0.33 g l⁻¹). However, these glycerol concentrations were below 10% of those observed with a Gpd⁺ reference strain. Consequently, the ethanol yield on sugar increased from 79% of the theoretical maximum in the reference strain to 92% for the evolved strains. Genetic analysis indicated that osmotolerance under aerobic conditions required a single dominant chromosomal mutation, and one further mutation in the plasmid-borne mhpF gene for anaerobic growth.     

The effects of glucose and oxygen on the cytochromes and metabolic activity of yeast batch cultures. I. Saccharomyces spp.

Saccharomyces cerevisiae and Saccharomyces carlsbergensis were grown in batch culture with and without oxygen control. The concentrations of A-, B- and C-type cytochromes of both yeasts were dependent on the oxygen concentration during growth as well as on the initial glucose concentration of the growth medium. S. cerevisiae cytochromes were maximal after growth in low glucose and low oxygen; S. carlsbergensis cytochromes were maximal after growth in low glucose and high oxygen. Except when glucose was in very low concentration, its catabolism by S. carlsbergensis was directed predominantly towards ethanolic fermentation regardless of the oxygen concentration. Growth rate, total cell mass and yield were maximal, and anabolism was closely balanced with catabolism, when glucose and oxygen of S. carlsbergensis cultures were both high. Under these conditions neither catabolism, respiratory or ethanolic, nor glucose uptake were maximal.