Roles of glycerol and glycerol-3-phosphate dehydrogenase (NAD+) in acquired osmotolerance of Saccharomyces cerevisiae.

In a cell culture of Saccharomyces cerevisiae exponentially growing in basal medium, only 0.02% of the cells were osmotolerant, i.e., survived transfer to medium containing 1.4 M NaCl. Short-time conditioning in 0.7 M NaCl medium transformed the whole population into an osmotolerance phenotype. During this conditioning, the rate of formation of glycerol, the main compatible solute in S. cerevisiae, increased threefold and the specific activity of glycerol-3-phosphate dehydrogenase (NAD+) (GPDH) (EC 1.1.1.8) was enhanced sixfold. The apparent flux control coefficient for GPDH in the formation of glycerol was estimated to be 0.6. Glycerol production was also favored by regulated activities of alcohol dehydrogenase (EC 1.1.1.1) and aldehyde dehydrogenase [NAD(P)]+ (EC 1.2.1.5). About 50% of the total glycerol produced during conditioning in 0.7 M NaCl was retained intracellularly, and the increased glycerol accumulation was shown to be not merely a result of enhanced production rate but also of increased retention of glycerol. Washing the cells with solutions of lower salinities resulted in loss of glycerol, with retained levels proportional to the concentration of NaCl in the washing solution. Cycloheximide addition inhibited the development of acquired osmotolerance and conditioned cells washed free of glycerol retained a high degree of osmotolerance, which indicate that protein synthesis was required to establish the osmotolerance state.     

Use of microcalorimetric monitoring in establishing continuous energy balances and in continuous determinations of substrate and product concentrations of batch-grown Saccharomyces cerevisiae

Energy balance calculations were performed for different physiological states during batch growth of Saccharomyces cerevisiae with glucose as carbon and energy source. For the different physiological states, energy recoveries close to one were obtained, which permitted a continuous control that the constantly changing growth process was quantified accurately. During the respiro-fermantative phase of growth, during which glucose served as the carbon and energy source, a low-heat-yield value (DeltaQ(x)) of -8.6 kJ/g dry biomass formed was obtained. This low-heat-yield value was due to the mainly fermentative metabolism during the middle of this phase of growth. After a transition phase, the ethanol produced during the respiro-fermentative growth was respired. During this respiratory phase, the heat yield values increased markedly, resulting in a lowest value of -42.7 kJ/g. The low-heat-yield values of the respiro-fermentative growth is not a reflection of the most efficient metabolism of S. cerevisiae. On the contrary, during the middle of this phase, 74% of the energy input was dissipated as ethanol, 6% was dissipated as heat, and the energy conserved as biomass was just 13%, while during the early respiratory phase, 69% of the energy input was dissipated as heat, and 22% of the energy input was conserved as biomass. By mathematical modeling and direct monitoring on-line of the rate of heat production, continuous calculations of (1) glucose consumption, and (3) biomass production were performed, and were shown to correlate closely with measured values for the continuously changing growth process.     

Comparative study of some parameters of energy metabolism in two strains of Saccharomyces cerevisiae

A comparative study of energy metabolism in two strains Saccharomyces cerevisiae (the initial strain N 73 and laser-irradiated mutant strain Y-503) was performed. In all growth phases, the rates of oxygen consumption by cells of Y-503 were higher than in the initial strain. The maximum (threefold) increase in the rate of oxygen consumption was observed in the linear phase. The effects of respiratory chain inhibitors rotenone, antimycin A, and cyanide on cellular and mitochondrial respiration were identical. There are two sites of energy coupling in the respiratory chain of mitochondria in S. cerevisiae N 73 and Y-503, and electron flow mainly is mainly mediated by cytochrome oxidase. The data suggest that a higher respiratory activity of S. cerevisiae Y-503 cells in comparison with N 73 is associated with greater amounts of mitochondria and total surface area of coupling mitochondrial membranes, which appears to be a factor contributing to a high physiological and biochemical activity of this strain.     

Influence of the physiological state of the inoculum on fermentation of musts from Pedro Ximénez grapes by Saccharomyces cerevisiae

Two inocula in different physiological states, namely in the exponential growth phase and in the declining phase were prepared from a strain of Saccharomyces cerevisiae. With these inocula were fermented musts from grapes of the Pedro Ximénez variety, sterilized by filtration. Cell growth and the activity of the enzymes alcohol dehydrogenase and aldehyde dehydrogenase [NADP+ and NAD(P)+] were found to vary with the state of the inoculum. This was reflected in the specific rate of production and, in some instances, in the final concentration of acetaldehyde, acetic acid, ethanol, isoamyl alcohols, phenethyl alcohol and their esters in the wine.     

Ornithine decarboxylase activity and cell cycle regulation in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the specific activity of the enzyme ornithine decarboxylase (ODC) was correlated with overall growth status. The activity of ODC was highest in actively growing cells, whereas the specific activity was lower in slow-growing cultures limited for nitrogen or inhibited by low concentrations of cycloheximide. Specific activities of ODC were also low in cultures arrested in the stationary phase (in the G1 portion of the cell cycle) by starvation for required nutrients. Although correlated with overall growth, ODC activity was not required for growth or cell cycle regulation. Cells continued to grow in the presence of the polyamine spermidine or spermine, which markedly reduced ODC specific activities. Thus, high levels of ODC activity were not necessary for growth, nor were decreased ODC specific activities sufficient to cause cells to arrest in G1. Conversely, one agent (o-phenanthroline) which causes growing cells to arrest in G1 did so with no effect on ODC specific activity. Therefore, ODC specific activity changes are not necessary for cell cycle regulation but simply reflect the normal growth status of cells.     

Reversible inhibition of the hydrocortisone induction of glycerol phosphate dehydrogenase by cytochalasin B in rat glial C6 cells

The hydrocortisone (HC) induction of glycerol phosphate dehydrogenase (GPDH; EC 1.1.1.8) in rat glial C6 cells was inhibited reversibly and in a dose-dependent manner by cytochalasin B (CB). CB had no effect on basal level GPDH, total cellular RNA, DNA or protein content nor did it act as a general inhibitor of the rate of protein synthesis. CB did not appear to be acting via dissociation of microtubules since colcemid had no effect on the induction process. The addition of an alternate energy source (sodium pyruvate) did not relieve the CB inhibition of GPDH induction suggesting that CB is not exerting its effect by blocking glucose utilization. The inhibition by CB is not dependent on the temporal sequence of the induction process since it specifically inhibited GPDH induction at any time it was added. CB did not alter the rate of degradation of GPDH in these cells and direct measurements of the specific rate of synthesis of GPDH demonstrated that CB decreased the induced rate of GPDH synthesis by about 60%. The site of inhibition was more precisely defined by experiments which demonstrated a 60% decrease in specific nuclear binding of ³H-HC even though total cellular uptake of ³H-HC was unaffected. This effect on nuclear binding of HC is sufficient to account for the decreased accumulation of GPDH activity in CB-treated cells.     

Interferon-gamma and interleukin-1 beta inhibit adipoconversion in cultured rodent preadipocytes.

Cytokines like tumor necrosis factor (TNF), interferon-gamma (IFN-gamma), and interleukin-1 (IL-1) are known to interfere with the differentiation of cultured cell lines of adipocyte precursors. In the present study, the effect of mouse and rat IFN-gamma, as well as human IL-1 beta, was investigated on rodent preadipocytes in primary cultures, either in the presence of fetal bovine serum (FBS, 10%) or in serum-free defined medium. IFN-gamma exerted an antiproliferative action that was more pronounced when cells reached confluency than during the growth phase of the culture. Morphological observation and quantifications of undifferentiated and differentiating cells revealed that IFN-gamma caused a decrease in the proportion of cells devoid of lipid droplets which would correspond to fibroblast-like cells, whereas preadipocytes remained unaffected. IFN-gamma induced a marked retardation of adipoconversion, resulting in a partial inhibition of lipoprotein lipase (LPL) activity and a severe decrease in glycerol-3-phosphate dehydrogenase (GPDH) activity. The antiproliferative and anti-LPL effects of IFN-gamma were neutralized by adding anti-IFN-gamma antibodies, while these antibodies prevented only partially the depressing effect of IFN-gamma on GPDH activity. Contrary to IFN-gamma, IL-1 beta slightly enhanced the proliferation in preadipocyte cultures. IL-1 beta also depressed adipoconversion, inhibited markedly LPL activity, and partially reduced GPDH activity. These results show that the influence of cytokines on adipoconversion observed in preadipocyte cell lines can be found in normal preadipocytes in culture.     

Hormonal regulation of the differentiation of rat adipocyte precursor cells in primary culture

A reproducible cell culture system is described that allows the study of adipose conversion in fibroblast-like cells isolated by collagenase digestion of epididymal and perirenal adipose tissue from male rats weighing 70-200 g. Adipose conversion as measured by lipid accumulation and increase in glycerophosphate dehydrogenase (GPDH) activity during differentiation strongly depends on the density at which cells are inoculated and starts only when cells are confluent and when physiological amounts of corticosterone and insulin are added. beta-Estradiol, testosterone, thyroxine, triiodothyronine, and growth hormone do not affect the differentiation process. Methylisobutylxanthine added during the first 2 days after confluence, added with insulin and corticosterone, potentiates the effect of insulin on GPDH activity and accelerates triglyceride accumulation. The effect of methyl-isobutylxanthine seems to be mediated by increased cyclic AMP concentrations, inasmuch as it may be replaced by forskolin.     

Regulation of phosphoenolpyruvate carboxykinase and pyruvate kinase in Saccharomyces cerevisiae grown in the presence of glycolytic and gluconeogenic carbon sources and the role of mitochondrial function on gluconeogenesis

Phosphoenolpyruvate carboxykinase (PEPCKase) and pyruvate kinase (PKase) were measured in Saccharomyces cerevisiae grown in the presence of glycolytic and gluconeogenic carbon sources. The PEPCKase activity was highest in ethanol-grown cells. However, high PEPCKase activity was also observed in cells grown in 1% glucose, especially as compared with the activity of sucrose-, maltose-, or galactose-grown cells. Activity was first detected after 12 h when glucose was exhausted from the growth medium. The PKase activity was very high in glucose-grown cells; considerable activity was also present in ethanol- and pyruvate-grown cells. The absolute requirement of respiration for gluconeogenesis was demonstrated by the absence or significantly low levels of PEPCKase and fructose-1,6-bisphosphatase activities observed in respiratory deficient mutants, as well as in wild-type S. cerevisiae cells grown in the presence of glucose and antimycin A or chloramphenicol. Obligate glycolytic and gluconeogenic enzymes were present simultaneously only in stationary phase cells, but not in exponential phase cells; hence futile cycling could not occur in log phase cells regardless of the presence of carbon source in the growth medium.     

Regulation of the quiescence-induced genes: quiescin Q6, decorin, and ribosomal protein S29

The transition from growth to quiescence is deeply deranged in cancer cells. Expression of the quiescence-induced genes, quiescin Q6, decorin, and S29, was examined in important physiological states and in several cell types. Senescent fibroblasts expressed neither Q6 nor decorin mRNAs. The quiescins were induced in serum-deprived cultures. Trypsinized cells, which rapidly reattached to the culture dish, expressed Q6, S29, and decorin mRNAs at reduced levels, compared to those that remained in suspension. Expression of Q6 and S29 mRNAs in endothelial cells was low in growth phase and high in quiescent cells. Q6 and S29 mRNAs were found in a large variety of human tissues. The quiescin Q6 protein was detected in WI38 cell extracts and in conditioned medium from quiescent cells. A complex regulation of the quiescins by growth and attachment status in specific cell types may be of importance in pathological growth regulation and the development of cancer.     

The freeze-thaw stress response of the yeast Saccharomyces cerevisiae is growth phase specific and is controlled by nutritional state via the RAS-cyclic AMP signal transduction pathway.

The ability of cells to survive freezing and thawing is expected to depend on the physiological conditions experienced prior to freezing. We examined factors affecting yeast cell survival during freeze-thaw stress, including those associated with growth phase, requirement for mitochondrial functions, and prior stress treatment(s), and the role played by relevant signal transduction pathways. The yeast Saccharomyces cerevisiae was frozen at -20 degrees C for 2 h (cooling rate, less than 4 degrees C min-1) and thawed on ice for 40 min. Supercooling occurred without reducing cell survival and was followed by freezing. Loss of viability was proportional to the freezing duration, indicating that freezing is the main determinant of freeze-thaw damage. Regardless of the carbon source used, the wild-type strain and an isogenic petite mutant ([rho 0]) showed the same pattern of freeze-thaw tolerance throughout growth, i.e., high resistance during lag phase and low resistance during log phase, indicating that the response to freeze-thaw stress is growth phase specific and not controlled by glucose repression. In addition, respiratory ability and functional mitochondria are necessary to confer full resistance to freeze-thaw stress. Both nitrogen and carbon source starvation led to freeze-thaw tolerance. The use of strains affected in the RAS-cyclic AMP (RAS-cAMP) pathway or supplementation of an rca1 mutant (defective in the cAMP phosphodiesterase gene) with cAMP showed that the freeze-thaw response of yeast is under the control of the RAS-cAMP pathway. Yeast did not adapt to freeze-thaw stress following repeated freeze-thaw treatment with or without a recovery period between freeze-thaw cycles, nor could it adapt following pretreatment by cold shock. However, freeze-thaw tolerance of yeast cells was induced during fermentative and respiratory growth by pretreatment with H2O2, cycloheximide, mild heat shock, or NaCl, indicating that cross protection between freeze-thaw stress and a limited number of other types of stress exists.     

Growth and maturation of primary-cultured adipocytes from lean and ob/ob mice

Stromal vascular cells from epididymal fat pads of lean and obese mice were cultured in a medium (α-MEM) containing fetal bovine serum (FBS) and cell replication followed for 11 days. In both types of cells, confluence occurred at 4–5 days, after which virtual growth arrest occurred in lean-mouse cells while replication continued, albeit at a slower rate in obese-mouse cells. Little or no lipid accumulation or glycerol-3-phosphate dehydrogenase (GPDH) activity was observed under these conditions. When a differentiation mixture consisting of insulin, corticosterone and isobutylmethylxanthine was added to the serum-containing α-MEM, a proportion of the lean-mouse cells accumulated triglycerides and GPDH activity increased significantly, indicating differentiation. By contrast, little or no differentiation occurred in obese-mouse cells. When cells grown in serum-containing α-MEM were transferred to a serum-free defined medium at confluence, extensive differentiation and maturation occurred in lean-mouse cells but not in obese-mouse cells. Similar experiments were conducted in cells isolated from the retroperitoneal fat pad. Although the growth pattern was similar to that of epididymal preadipocytes, the retroperitoneal lean- and obese-mouse cells differentiated more readily than epididymal cells, as shown by the GPDH specific activity. These data suggest that cells from obese mice are resistant to differentiation under conditions that support extensive differentiation in lean-mouse cells.     

Susceptibility of individual cells of Saccharomyces cerevisiae to the killer toxin K1

The susceptibility of sensitive yeast to killer toxins is known to depend on various factors, such as the selected killer toxin, the exposed yeast strain, its growth phase and the state of culture under given experimental conditions. The aim of this paper was to find whether individual cells from one culture are equally susceptible to the impact of the killer toxin. For this purpose the rhodamine B assay in a modified form was used. In order to observe the fate of individual cell the method of fluorescence video microscopy with a digital picture analysis was applied. Four selected groups of specific cells (with no, small, medium, and large bud, respectively) were investigated. Different sensitivity of Saccharomyces cerevisiae cells to the killer toxin K1 was observed in these cell groups. The most susceptible appeared to be the cells which were in S-phase (cells with the small buds); the least susceptible were the M-phase cells with large buds. The enhanced susceptibility in S-phase results probably from coincidence in higher porosity of the cell wall, accumulation of surface receptors, and enlarged growth activity at the surface cell structures.     

Comparison of three different promoter systems for secretory alpha-amylase production in fed-batch cultures of recombinant Saccharomyces cerevisiae

Cloned gene expression in recombinant Saccharomyces cerevisiae 20B-12 containing three different plasmids was compared in batch and fed-batch cultures. The plasmids pNA3, pNA7, and pNA9 contain the alpha-amylase gene under the control of SUC2, PGK, and GAL7 Promoters, respectively. The synthesis of alpha-amylase was therefore induced by low glucose concentration for the SUC2 and PGK promoters, and by galactose for GAL7 promoter. The specific cell growth rates were similar among cells harboring the three different plasmids; they decreased from 0.35 to 0.38 h(-1) during the cell growth phase to 0.03 to 0.06h(-1) during the production phase. The secretory alpha-amylase activity of cells harboring plasmid pNA7 was 129 U/mL in fed-batch culture, which was 1.4 and 2 times as high as the activities of cells harboring plasmids pNA3 and pNA9, respectively. The secretion ratios (amount of extracellular alpha-amylase activity/amounts of total alpha-amylase activity) of cells harboring plasmids pNA3, pNA7, and pNA9 were 91.4%, 94.5%, and 95.3%, respectively. (c) 1993 Wiley & Sons, Inc.     

渗透剂对酿酒酵母生长的影响及其模型分析

考察了Saccharomyces cerevisiae FL1酵母菌株在固体平板上的生长动力学过程及2种渗透剂对不同生理阶段的酵母细胞增殖行为的影响．建立了一个数学模型,该模型可以预测固体培养过程中生物量随时间的变化情况,结果表明,模型预测值与实际值能够很好符合,将该模型应用于考察氯化钠和甘油对细胞增殖行为的影响,结果揭示,氯化钠显著抑制指数生长期和平衡期细胞分裂,降低酵母比生长速率和生物量;甘油对平衡期细胞增殖有促进作用,提高比生长速率和生物量;甘油与氯化钠之间存在协同作用,0．15M甘油不能减弱高盐的应激作用。     

Glycerol Phosphate Dehydrogenase Activity of Oligodendrocytes Isolated from Adult Pig Brain: Its Inducibility by Hydrocortisone

Developing oligodendrocytes cultured in vitro express glycerol phosphate dehydrogenase (GPDH; EC 1.1.1.8) and are known to respond to glucocorticoid treatment by increased activity of GPDH. We present evidence that GPDH is enriched in white matter and oligodendrocytes of adult pig brain. Bulk-isolated oligodendrocytes maintained in culture for several weeks exhibit an almost constant level of GPDH activity. Furthermore, a 4-day stimulation with hydrocortisone induces GPDH specific activity of long-term cultured oligodendrocytes from adult pig brain.     

Development of a chemically defined serum-free medium for differentiation of rat adipose precursor cells

Stromal-vascular cells from the epididymal fat pad of 4-week-old rats, when cultured in a medium containing insulin or insulin-like growth factor, IGF-I, triiodothyronine and transferrin, were able to undergo adipose conversion. Over ninety percent of the cells accumulated lipid droplets and this proportion was reduced in serum-supplemented medium. The adipose conversion was assessed by the development of lipoprotein lipase (LPL) and glycerol-3-phosphate dehydrogenase (GPDH) activities, [14C]glucose incorporation into polar and neutral lipids, triacylglycerol accumulation and lipolysis in response to isoproterenol. Similar results were obtained with stromal-vascular cells from rat subcutaneous and retroperitoneal adipose tissues. Stromal-vascular cells required no adipogenic factors in addition to the components of the serum-free medium. Insulin was required within a physiological range of concentrations for the emergence of LPL and at higher concentrations for that of GPDH. When present at concentrations ranging from 2 to 50 nM, IGF-I was able to replace insulin for the expression of both LPL and GPDH. The development of a serum-free, chemically defined medium for the differentiation of diploid adipose precursor cells opens up the possibility of characterizing inhibitors or activators of the adipose conversion process.     

Thermal inactivation of Enterococcus faecium: effect of growth temperature and physiological state of microbial cells

AIMS: To provide data on the effects on culture temperature and physiological state of cells on heat resistance of Enterococcus faecium, which may be useful in establishing pasteurization procedures. METHODS AND RESULTS: The heat resistance of this Ent. faecium (ATCC 49624 strain) grown at different temperatures was monitored at various stages of growth. In all cases, the bacterial cells in the logarithmic phase of growth were more heat sensitive. For cells which had entered in the stationary phase, D70 values of 0.53 min at 5 degrees C, 0.74 min at 10 degrees C, 0.83 min at 20 degrees C, 0.79 min at 30 degrees C, 0.63 min at 37 degrees C, 0.48 min at 40 degrees C and 0.41 min at 45 degrees C were found. By extending the incubation times cells were more heat resistant as stationary phase progressed, although a different pattern was observed for cells grown at different temperatures. At the lower temperatures heat resistance increased progressively, reaching D70 values of 1.73 min for cells incubated at 5 degrees C for 50 days and 1.04 min for those grown at 10 degrees C for 16 days. At other temperatures assayed heat resistance became stable for late stationary phase cells, reaching D70 values of 1.05, 1.08 and 1.01 min for cultures incubated at 20, 30 and 37 degrees C. Heat resistance of cells obtained at higher temperatures, 40 and 45 degrees C, was significantly lower, with D70 values of 0.76 and 0.67 min, respectively. Neither the growth temperature nor the growth phase modified the z-values significantly. CONCLUSIONS: D70 values obtained for Ent. faecium (ATCC 49624) varies from 0.33 to 1.73 min as a function of culture temperature and physiological state of cells. However, z values calculated were not significantly influenced by these factors. A mean value of 4.50 +/- 0.39 degrees C was found. SIGNIFICANCE AND IMPACT OF THE STUDY: Overall results strongly suggest that, to establish heat processing conditions of pasteurized foods ensuring elimination of Ent. faecium, it is advisable to take into account the complex interaction of growth temperature and growth phase of cells acting on bacterial thermal resistance.