The Effect of Sodium Azide on the Thermotolerance of the Yeasts Saccharomyces cerevisiae and Candida albicans

The addition of sodium azide (a mitochondrial inhibitor) at a concentration of 0.15 mM to glucose-grown Saccharomyces cerevisiae or Candida albicans cells before exposing them to heat shock increased cell survival. At higher concentrations of azide, its protective effect on glucose-grown cells decreased. Furthermore, azide, even at low concentrations, diminished the thermotolerance of galactose-grown yeast cells. It is suggested that azide exerts a protective effect on the thermotolerance of yeast cells when their energy requirements are met by the fermentation of glucose. However, when cells obtain energy through respiratory metabolism, the azide inhibition of mitochondria enhances the damage inflicted on the cells by heat shock.     

The effect of sodium azide on the thermotolerance of the yeast Saccharomyces cerevisiae and Candida albicans

The addition of sodium azide (a mitochondrial inhibitor) at a concentration of 0.15 mM to glucosegrown Saccharomyces cerevisiae or Candida albicans cells before exposing them to heat shock increased cell survival. At higher concentrations of azide, its protective effect on glucose-grown cells decreased. Furthermore, azide, even at low concentrations, diminished the thermotolerance of galactose-grown yeast cells. It is suggested that azide exerts a protective effect on the thermotolerance of yeast cells when their energy requirements are met by the fermentation of glucose. However, when cells obtain energy through respiratory metabolism, the azide inhibition of mitochondria enhances damage inflicted on the cells by heat shock.     

Adenosine phosphates and the control of glycolysis and gluconeogenesis in yeast

1. Changes in dry weight, protein, RNA and DNA were measured in yeast during adaptation to glycolytic metabolism. 2. Only RNA increased significantly during the lag phase, but during the exponential phase all these cellular components increased in parallel. 3. The concentrations of ATP, ADP, AMP and glucose 6-phosphate were measured in respiring yeast and during the transition to glycolytic metabolism. 4. In respiring cells the concentration of AMP was at its highest and that of ATP was at its lowest; this relationship was reversed in glycolysing cells. 5. ADP concentration was similar in respiring and glycolysing cells, but glucose 6-phosphate concentration was much higher in the glycolysing cells. 6. A possible reason for mitochondrial repression is suggested. 7. It is concluded that adenosine phosphates do not control the direction of glycolytic flux in yeast and an alternative control of glycolysis and gluconeogenesis by enzyme activation and inactivation is suggested.     

Development of mitochondrial membranes in anaerobically grown yeast cells.

Biochemical analyses of mitochondrial marker substances, especially cardiolipin and oligomycin-sensitive ATPase [EC 3.6.1.3], as well as electron microscopic observations were carried out to eludicate the process of mitochondrial development in annaerobic yeast cells. Cardiolipin was found to be localized in the mitochondria in anaerobic cells. Its cellular content was a little higher in the stationary phase than in the exponential phase in glucose-grown cells and increased further in galactose-grown cells. The lipid content of the mitochondrial preparation obtained from glucose-grown stationary cells was nearly as high as that from galactose-grown cells. It was also comparable to that of aerobic cells in the stationary phase, where mitochondria are fully developed. Both cellular and mitochondrial levels of oligomycin-sensitive ATPase activity were also found to rise markedly in galactose-grown anaerobic cells, although not in stationary phase cells grown anaerobically on glucose. These high levels of the mitochondrial markers indicate a developmental change in mitochondrial structure even in anaerobically grown cells, which lack mitochondrial cytochromes. In the process of aerobic adaptation, respiratory system formation was observed to occur much faster in galactose-grown cells than in glucose-grown cells, and not to be inhibited by chloramphenicol and high concentrations of glucose structure in anaerobic cells. The developmental change was also corroborated by electron microscopic observations, which revealed the occurrence of two types of mitochondria in anaerobic cells. One was found in glucose-repressed cells and was characterized by the presence of numerous electron-dense granules in the matrix. In contrast, the other type, found in glucose-derepressed cells, had an electron-lucent matrix. No crista membrane was seen in either type of mitochondria in anaerobic cells, although the infoldings of the inner membrane, which partition the matrix into two parts and therefore are called "septum membranes," appeared frequently in the stationary phase cells. On the basis of these results, the process of mitochondrial development in yeast cells is discussed.     

Optimized analysis of intracellular adenosine and guanosine phosphates in Escherichia coli.

To investigate the intracellular concentrations of adenosine phosphates in Escherichia coli, especially during bioreactor cultivations, a method that enables reproducible determination of adenosine phosphates in culture solutions containing at least 0.25 g dry cell weight/L has been developed. The detection limits of AMP, ADP, and ATP were found to be as low as 1 pmol. The method involves fast sampling, instantaneous inactivation of cell metabolism, extraction of nucleotides, and quantitative analysis by ion-pair reversed-phase HPLC.     

Effects of culture conditions on the in vitro infection of fibroblasts by Candida albicans.

The effects of yeast culture age, carbon source, growth temperature, and germ-tube inducers on adherence to primary fibroblast cultures was studied in conjunction with the determination of adherence-mediated mammalian cell damage by measuring chromium-51 release from fibroblast monolayers. The results indicated that yeast culture age affected adherence only when the yeasts were grown at 37 degrees C, not after growth at 28 degrees C. At 37 degrees C, quantitatively fewer exponential-phase, glucose- or galactose-grown yeasts adhered to fibroblasts than did yeasts that were in lag or stationary phases. The reduced adherence correlated with less chromium-51 release and reduced germ-tube formation. The addition of germ-tube inducers, such as N-acetyl-D-glucosamine or serum, to exponential-phase yeasts caused an increase in germ-tube formation with a concomitant increase in yeast adherence and release of chromium-51 from the monolayers. Exponential-phase galactose-grown yeasts were more responsive to serum-induced germ-tube formation, germ-tube elongation, and fibroblast adherence than were exponential-phase glucose-grown yeasts. In addition, exponential-phase galactose-grown yeasts caused more chromium-51 release from monolayers in the presence of serum than did glucose-grown yeasts. Overall, conditions that enhanced germ-tube formation and elongation resulted in greatest adherence-mediated damage to the monolayers.     

Galactose fermentation by Streptococcus lactis and Streptococcus cremoris: pathways, products, and regulation

All of the lactic streptococci examined except Streptococcus lactis ML8 fermented galactose to lactate, formate, acetate, and ethanol. The levels of pyruvate-formate lyase and lactate dehydrogenase were elevated and reduced, respectively, in galactose-grown cells compared with glucose- or lactose-grown cells. Reduced intracellular levels of both the lactate dehydrogenase activator (fructose, 1,6-diphosphate) and pyruvate-formate lyase inhibitors (triose phosphates) appeared to be the main factors involved in the diversion of lactate to the other products. S. lactis ML8 produced only lactate from galactose, apparently due to the maintenance of high intracellular levels of fructose 1,6-diphosphate and triose phosphates. The growth rates of all 10 Streptococcus cremoris strains examined decreased markedly with galactose concentrations below about 30 mM. This effect appeared to be correlated with uptake predominantly by the low-affinity galactose phosphotransferase system and initial metabolism via the D-tagatose 6-phosphate pathway. In contrast, with four of the five S. lactis strains examined, galactose uptake and initial metabolism involved more extensive use of the high-affinity galactose permease and Leloir pathway. With these strains the relative flux of galactose through the alternate pathways would depend on the exogenous galactose concentration.     

Interaction of Candida albicans with neutrophils: effect of phenotypic changes in yeast cell-surface composition

The susceptibility of four strains of Candida albicans to phagocytosis and intracellular killing by rabbit peritoneal neutrophils was investigated. Two of the strains, isolated from active infections, were known to synthesize a surface layer of mannoprotein fibrils in response to growth on 500 mm-galactose; the other strains, from asymptomatic carriers, lacked this capability. The presence of serum opsonins greatly enhanced phagocytosis of all four strains and, following opsonization, phagocytosis of an infective strain was equally rapid after growth on either 500 mm-galactose or 50 mm-glucose. In the absence of opsonins, galactose-grown infective strains were phagocytosed faster than either glucose-grown infective strains or galactose-grown carrier strains. These differences in phagocytic uptake were paralleled by differences in neutrophil chemiluminescence response. Intracellular killing of galactose-grown infective strains was only half that of glucose-grown infective strains or galactose-grown carrier strains after incubation for 60 min. Pretreatment of neutrophils with extracellular polymeric material, which contains the surface fibrils, completely inhibited intracellular killing. These results indicate that production of the fibrillar layer promotes yeast virulence by increasing resistance to intracellular killing, although it may enhance phagocytosis in locations where opsonic activity is poor.     

Correlation between cell-surface hydrophobicity of Candida albicans and adhesion to buccal epithelial cells

Adhesion of four isolates of Candida albicans to buccal epithelial cells was determined after growth of the yeasts in defined medium containing 50 mM glucose or 500 mM galactose as the carbon source. With each isolate, adhesion of galactose-grown yeasts was significantly higher than that of glucose-grown organisms. Yeast cell-surface hydrophobicity was assessed by two methods, a modified hydrocarbon adhesion assay and a more sensitive polystyrene microsphere assay. All four isolates were significantly more hydrophobic after growth on 500 mM galactose than after growth on 50 mM glucose. Overall, a strong positive correlation between adhesion and surface hydrophobicity was observed (r = 0.965). These results are discussed in relation to the role of yeast-surface hydrophobicity in pathogenesis.     

Inhibition of glucose metabolism by n-hexadecane in Cladosporium (Amorphotheca) resinae.

When Cladosporium resinae is provided with n-hexadecane and glucose, n-hexadecane is used preferentially. Studies using [14C]glucose indicated that n-hexadecane did not inhibit glucose uptake but did retard oxidation of glucose to CO2 and assimilation of glucose carbon into trichloroacetic acid-insoluble material. Glucose could be recovered quantitatively from hydrocarbon-grown cells that had been transferred to glucose. Four enzymes that may be involved in glucose metabolism, hexokinase, glucose-6-phosphate dehydrogenase, glucose-phosphate isomerase, and succinate dehydrogenase, were not detected in cells grown on hexadecane but were present in cells grown on glucose. Addition of hexadecane to extracts of glucose-grown cells resulted in immediate loss of activity for each of the four enzymes, but two other enzymes did not directly involved in glucose metabolism, adenosine triphosphatase and alanine-ketoacid aminotransferase, were not inhibited by hexadecane in vitro. Cells grown on hexadecane and transferred to glucose metabolize intracellular hexadecane; after 1 day, activity of hexokinase, glucose-6-phosphate dehydrogenase, glucosephosphate isomerase, and succinate dehydrogenase could be detected and 22% of the intracellular hydrocarbon had been metabolized. Hexadecane-grown cells transferred to glucose plus cycloheximide showed the same level of activity of all the four enzymes as cells transferred to glucose alone. Thus, intracellular n-hexadecane or a metabolite of hexadecane can inthesis of those enzymes is not inhibited.     

Role of guanine nucleotides in the regulation of the Ras/cAMP pathway in Saccharomyces cerevisiae

The CDC25 gene product is a guanine nucleotide exchange factor for Ras proteins in yeast. Recently it has been suggested that the intracellular levels of guanine nucleotides may influence the exchange reaction. To test this hypothesis we measured the levels of nucleotides in yeast cells under different growth conditions and the relative amount of Ras2-GTP. The intracellular GTP/GDP ratio was found to be very sensitive to growth conditions: the ratio is high, close to that of ATP/ADP during exponential growth, but it decreases rapidly before the beginning of stationary phase, and it drops further under starvation conditions. The addition of glucose to glucose-starved cells causes a fast increase of the GTP/GDP ratio. The relative amount of Ras2-GTP changes in a parallel way suggesting that there is a correlation with the cytosolic GTP/GDP ratio. In addition 'in vitro' mixed-nucleotide exchange experiments done on purified Ras2 protein demonstrated that the GTP and GDP concentrations influence the extent of Ras2-GTP loading giving further support to their possible regulatory role.     

Adenosine triphosphate-linked control of Pseudomonas aeruginosa glucose-6-phosphate dehydrogenase

Extracts of Pseudomonas aeruginosa (ATCC 7700) cells grown on glucose, gluconate, or glycerol had enzyme activities related to the Entner-Doudoroff pathway. These activities were present in no more than trace amounts when the bacteria were grown on succinate. Fructose-1,6-diphosphate aldolase could not be detected in extracts of the bacteria grown on any of the above carbon sources. Therefore, it appears that P. aeruginosa degrades glucose via an inducible Entner-Doudoroff pathway. The apparent absence of fructose-1,6-diphosphate aldolase in cells growing on succinate suggests that the bacteria can form hexose and pentose phosphates from succinate by an alternate route. d-Glucose-6-phosphate dehydrogenase, a branch-point enzyme of the Entner-Doudoroff pathway, was purified 50-fold from glucose-grown cells. Its molecular weight, estimated by sucrose density gradient centrifugation, was found to be approximately 190,000. The enzyme was strongly inhibited by adenosine triphosphate, guanosine triphosphate, and deoxyguanosine triphosphate, which decreased the apparent binding of glucose-6-phosphate to the enzyme. It is suggested that adenine nucleotide-linked control of glucose-6-phosphate dehydrogenase may regulate the overall catabolism of hexose phosphates and prevent their wasteful degradation under certain conditions requiring gluconeogenesis.     

The Effect of Nonanal on Dipodascus aggregatus IV. Studies with Different Carbon Sources

Nonanal (80 μ) in ethanolic solution stimulated the growth of Dipodascus aggregatus with fructose (55.5 mM) as carbon source (inoculum grown with fructose or glucose). If the inoculum had been grown with galactose, neither growth with galactose nor growth with glucose was affected by nonanal. If the inoculum had been grown with glucose, growth with galactose was weakly. stimulated. —Growth with galactose (galactose-grown inoculum) was strongly stimulated by nonanal if xylose at a low concentration (0.53 mM) was added. — The oxygen uptake of glucose grown cells with glucose as substrate was stimulated by 200 μM nonanal in the absence of ethanol. The respiratory activity of galactose-grown cells was also stimulated with galactose as well as with glucose as substrate. In the absence of exogenous substrate the oxygen uptake of glucose-grown cells was weakly stimulated by nonanal whereas that of galactose-grown cells was strongly stimulated.     

Growth inhibition of transformed mouse fibroblasts by adenine nucleotides occurs via generation of extracellular adenosine

The growth of transformed mouse fibroblasts (3T6 cells) in medium containing 5% fetal bovine serum was inhibited after treatment with concentrations greater than 50 microM ATP, ADP, or AMP. Adenosine, the common catabolite of the nucleotides, had no effect on cell growth at concentrations below 1 mM. However, the following results indicate that the toxicity of ATP, ADP, and AMP is mediated by serum- and cell-associated hydrolysis of the nucleotides to adenosine. 1) ADP and AMP, but not ATP, were toxic to 3T6 cells grown in serum-free medium or medium in which phosphohydrolase activity of serum was inactivated. Under these conditions, the cells exhibited cell-associated ADPase and 5'-nucleotidase activity, but little ecto-ATPase activity. 2) Inhibition of adenosine transport in 3T6 cells by dipyridamole or S-(p-nitrobenzyl)-6-thioinosine prevented the toxicity of ATP in serum-containing medium and of ADP and AMP in serum-free medium. 3) A 16-24-h exposure to 125 microM AMP or ATP was needed to inhibit cell growth under conditions where serum- and cell-associated hydrolysis of the nucleotides generated adenosine in the medium continuously over the same time period. In contrast, 125 microM adenosine was completely degraded to inosine and hypoxanthine within 8-10 h. Furthermore, multiple doses of adenosine added to the cells at regular intervals over a 16-h period were significantly more toxic than an equivalent amount of adenosine added in one dose. Treatment of 3T6 cells with AMP elevated intracellular ATP and ADP levels and reduced intracellular UTP levels, effects which were inhibited by extracellular uridine. Uridine also prevented growth inhibition by ATP, ADP, and AMP. These and other results indicate that serum- and cell-associated hydrolysis of adenine nucleotides to adenosine suppresses growth by adenosine-dependent pyrimidine starvation.     

Steady-state kinetics of formaldehyde dehydrogenase and formate dehydrogenase from a methanol-utilizing yeast, Candida boidinii.

Initial velocity studies and product inhibition studies were conducted for the forward and reverse reactions of formaldehyde dehydrogenase (formaldehyde: NAD oxidoreductase, EC 1.2.1.1) isolated from a methanol-utilizing yeast Candida boidinii. The data were consistent with an ordered Bi-Bi mechanism for this reaction in which NAD+ is bound first to the enzyme and NADH released last. Kinetic studies indicated that the nucleoside phosphates ATP, ADP and AMP are competitive inhibitors with respect to NAD and noncompetitive inhibitors with respect to S-hydroxymethylglutathione. The inhibitions of the enzyme activity by ATP and ADP are greater at pH 6.0 and 6.5 than at neutral or alkaline pH values. The kinetic studies of formate dehydrogenase (formate:NAD oxidoreductase, EC 1.2.1.2) from the methanol grown C. boidinii suggested also an ordered Bi-Bi mechanism with NAD being the first substrate and NADH the last product. Formate dehydrogenase the last enzyme of the dissimilatory pathway of the methanol metabolism is also inhibited by adenosine phosphates. Since the intracellular concentrations of NADH and ATP are in the range of the Ki values for formaldehyde dehydrogenase and formate dehydrogenase the activities of these main enzymes of the dissimilatory pathway of methanol metabolism in this yeast may be regulated by these compounds.     

The influence of adenosine on intermediary metabolism of isolated hepatocytes.

The effect of adenosine was tested on the energetic metabolism of fed rat liver cells after isolation. The cells were incubated in a buffered saline medium with glucose (5 mM) and adenosine (1 mM) for 30 minutes at 37 degrees C. This increased the concentration of the adenylic nucleotides ATP (+57 per cent, ADP (+39 per cent). Cyclic AMP was increased (+50 per cent) and the intracellular inorganic phosphate decreased (-22 per cent). These changes were accompaned by a decrease of glycogenolysis, glucose consumption and lactate production. Measurement of glycolytic intermediates showed decreased concentrations of fructose 1,6-bis-phosphate and 3-phosphoglycerate proportional to the increase in ATP concentration. The near-equilibrium of the glyceraldehyde 3-phosphate dehydrogenase-phosphoglycerate kinase system was not modified by adenosine. The decrease of the NAD+/NADH ratio along with the increase of the ATP/ADP X PO4 ratio explains the decrease of 3-phosphoglycerate. The decrease in glucose consumption can be explained by the cross over at the phosphofructokinase stage with the decrease of fructose 1,6-bisphosphate. The major part of adenosine was deaminated as indicated by an increase in the production of ammonia and urea. The effects of inosine, or adenosine along with an inhibitor of adenosine deaminase (pentostatin) suggest that adenosine acts on the glucose consumption through adenylic nucleotides. However the increase of the adenylic nucleotide level cannot totally explain the other metabolic changes: decrease of the NAD+/NADH cytoplasmic ratio, constancy of this ratio in mitochondria, decrease of gluconeogenesis from lactate. A direct action of adenosine can therefore be expected.     

Poly-β-hydroxybutyrate biosynthesis and the regulation of glucose metabolism in Azotobacter beijerinckii

Azotobacter beijerinckii possesses the enzymes of both the Entner-Doudoroff and the oxidative pentose phosphate cycle pathways of glucose catabolism and both pathways are subject to feedback inhibition by products of glucose oxidation. The allosteric glucose 6-phosphate dehydrogenase utilizes both NADP(+) and NAD(+) as electron acceptors and is inhibited by ATP, ADP, NADH and NADPH. 6-Phosphogluconate dehydrogenase (NADP-specific) is unaffected by adenosine nucleotides but is strongly inhibited by NADH and NADPH. The formation of pyruvate and glyceraldehyde 3-phosphate from 6-phosphogluconate by the action of the Entner-Doudoroff enzymes is inhibited by ATP, citrate, isocitrate and cis-aconitate. Glyceraldehyde 3-phosphate dehydrogenase is unaffected by adenosine and nicotinamide nucleotides but the enzyme is non-specific with respect to NADP and NAD. Citrate synthase is strongly inhibited by NADH and the inhibition is reversed by the addition of AMP. Isocitrate dehydrogenase, a highly active NADP-specific enzyme, is inhibited by NADPH, NADH, ATP and by high concentrations of NADP(+). These findings are discussed in relation to the massive synthesis of poly-beta-hydroxybutyrate that occurs under certain nutritional conditions. We propose that synthesis of this reserve material, to the extent of 70% of the dry weight of the organism, serves as an electron and carbon ;sink' when conditions prevail that would otherwise inhibit nitrogen fixation and growth.     

Differential metabolism of cellobiose and glucose by Clostridium thermocellum and Clostridium thermohydrosulfuricum.

Clostridium thermohydrosulfuricum consumed glucose in preference to cellobiose as an energy source for growth. The rates of substrate uptake in glucose- and cellobiose-grown cell suspensions were 45 and 24 nmol/min per mg (dry weight), respectively, at 65 degrees C. The molar growth yields (i.e., grams of cells per mole of glucose equivalents) were similar on cellobiose and glucose (19 and 16, respectively). Both glucose- and cellobiose-grown cells contained a glucose permease activity and high levels of hexokinase (greater 0.34 mumol/min per mg of protein at 40 degrees C). Growth on cellobiose was associated with induction of a cellobiose permease activity. In contrast, Clostridium thermocellum metabolized cellobiose in preference to glucose as an energy source and displayed lower growth rates on both substrates. The substrate uptake rates in cellobiose- and glucose-grown cell suspensions were 18 and 17 nmol/min per mg (dry weight), respectively. The molar yields were 38 on cellobiose and 20 on glucose. Extracts of glucose- and cellobiose-grown cells both contained cellobiose phosphorylase and phosphoglucomutase activities, whereas only glucose-grown cells contained detectable levels of glucose permease and hexokinase activities. The general catalytic and kinetic properties of the glucose- and cellobiose-catabolizing enzymes in the two species are described, and a model is proposed to distinguish differential saccharide metabolism by these thermophilic ethanologens.     

Glucose-6-Phosphate Dehydrogenase from the Chemolithotroph Thiobacillus ferrooxidans

Glucose-6-phosphate dehydrogenase was partially purified from both glucose-grown and iron-glucose-grown Thiobacillus ferrooxidans. The enzyme possesses a dual nucleotide specificity for either nicotinamide adenine dinucleotide phosphate (NADP) or nicotinamide adenine dinucleotide (NAD) and has a molecular weight of 110,000 as determined by gel electrophoresis. Evidence is presented that T. ferrooxidans glucose-6-phosphate dehydrogenase is identical when isolated from cells grown mixotrophically (iron-glucose grown) or cells grown heterotrophically (glucose-grown cells). The enzyme is activated by Mg(2+), and to a lesser extent by low concentrations of Mn(2+). Reduced NAD inhibits the enzyme from T. ferrooxidans. No deviation from normal Michaelis-Menten kinetics was observed in velocity versus substrate concentration experiments. Adenosine triphosphate exerted a profound inhibition of the enzyme; the effect was 10 times more pronounced in the presence of NAD as compared to NADP. The physiological significance of this inhibition is discussed.