Transitional states in a chemostat culture of Candida utilis

Transient states of the chemostat Candida utilis 1668-3-37 culture were studied when its growth was limited by ethanol and an abrupt acidification of the medium from pH 5.0 to 2.2 was done or when the dilution rate was rapidly changed from D = 0.1 to 0.3 h-1 and back to 0.07 h-1. The pH shock was found to cause stronger oscillations in a number of parameters (the weight of dry biomass, the content of residual ethanol, the content of RNA in the cells) than a change in the dilution rate. In the latter case the population density changed more smoothly than the content of RNA did. DNA content remained at one and the same level in all of the experiments. All of the oscillations were observed only in the first generation after a shock; there upon, the culture remained for a long time (7 to 10 generations) in a very stable state typical of chemostat cultures. The oscillations induced by the unfavourable pH of the medium were compared with those caused by an abrupt change in the dilution rate. The pH shock brought about multiple damping oscillations of the parameters whereas a change in the dilution rate resulted, most often, in a merely one oscillation.     

The effect of pH on the growth and metabolism of Streptococcus bovis in continuous culture

Streptococcus bovis H13/1 was grown anaerobically at pHs between 5.0 and 6.5 in a glucose-limited chemostat at a dilution rate of 0.05/h. The growth yield and the production of acetate, ethanol and formate decreased at pHs less than 6.5 whereas the production of lactate increased at the lower pH values. When a culture was subjected to sequential pH changes, growth yield and fermentation products were influenced not only by the pH existing in the culture medium but also by the metabolic activity of the cells at the preceding pHs in the sequence. The results are discussed in relation to the mechanisms available for the maintenance of pH homeostasis and for the metabolic control of fermentation pathways in Strep. bovis.     

The effect of pH on the growth and metabolism of Streptococcus bovis in continuous culture

Streptococcus bovis H13/1 was grown anaerobically at pHs between 5.0 and 6.5 in a glucose-limited chemostat at a dilution rate of 0.05/h. The growth yield and the production of acetate, ethanol and formate decreased at pHs less than 6.5 whereas the production of lactate increased at the lower pH values. When a culture was subjected to sequential pH changes, growth yield and fermentation products were influenced not only by the pH existing in the culture medium but also by the metabolic activity of the cells at the preceding pHs in the sequence. The results are discussed in relation to the mechanisms available for the maintenance of pH homeo-stasis and for the metabolic control of fermentation pathways in Strep. bovis.     

Oscillatory metabolism of Saccharomyces cerevisiae in continuous culture

Short-period (40-50 min) synchronized metabolic oscillation was found in a continuous culture of yeast Saccharomyces cerevisiae under aerobic conditions at low-dilution rates. During oscillation, many parameters changed cyclically, such as dissolved oxygen concentration, respiration rate, ethanol and acetate concentrations in the culture, glycogen, ATP, NADH, pyruvate and acetate concentrations in the cells. These changes were considered to be associated with glycogen metabolism. When glycogen was degraded, the respiro-fermentative phase was observed, in which ethanol was produced and the respiration rate decreased. In this phase, the levels of intracellular pyruvate and acetate became minimum, ATP became high and intracellular pH at its lowest level. When glycogen metabolism changed from degradation to accumulation, the respiratory phase started, during which ethanol was re-assimilated from the culture and the respiration rate increased. Intracellular pyruvate and acetate became maximum, ATP decreased and the intracellular pH appeared high. These findings may indicate new aspects of the control mechanism of glycogen metabolism and how respiration and ethanol fermentation are regulated together under aerobic conditions.     

Importance of bicarbonate transport for protection of cardiomyocytes against reoxygenation injury

Isolated cardiomyocytes from adult rats were incubated in anoxic bicarbonate-buffered media at extracellular pH (pH(o)) 6.4 until a cytosolic Ca(2+) overload and intracellular pH (pH(i)) of 6.4 were reached. On reoxygenation, the pH of the medium was changed to 7.4 to activate the Na(+)/H(+)exchanger (NHE) and the Na(+)-HCO(-)(3) symporter (NBS). The reoxygenation was performed in the absence or presence of the NHE inhibitor HOE-642 (3 micromol/l) and/or the NBS inhibitor DIDS (0.5 mmol/l), as in bicarbonate-free media. In reoxygenated control cells pH(i) rapidly recovered to the preanoxic level, and a burst of spontaneous oscillations of cytosolic Ca(2+) occurred, accompanied by the development of hypercontracture. When NBS and NHE were simultaneously inhibited during reoxygenation, pH(i) recovery was prevented, Ca(2+) oscillations were attenuated, and hypercontracture was abolished. Sole inhibition of NBS or NHE showed no protection against hypercontracture. In the absence of cytosolic acidosis, HOE-642 or DIDS did not prevent hypercontracture induced by Ca(2+) overload. The results demonstrate that simultaneous inhibition of NHE and NBS is needed to protect myocardial cells against reoxygenation-induced hypercontracture.     

Relationship between outer membrane protein and lipopolysaccharide profiles and serotypes of enterotoxigenic Escherichia coli isolated in Brazil

Abstract When the yeast Saccharomyces cerevisiae was grown under aerobic continuous culture conditions with a medium containing ethanol as carbon source, an autonomous sustained metabolic oscillation appeared. This oscillation was observed in rates and concentrations of various parameters such as, ethanol, oxygen uptake rate, carbon dioxide evolution rate, NaOH addition rate for pH control, acetate, and intracellular pH. No changes were observed in concentrations of stock carbohydrates. Intracellular pH changes were out of phase with oxygen uptake rate, which was reverse of the results with glucose-based oscillation. These results suggested that changes in glycolytic flux and intracellular pH were not regulating the oscillation. Analysis suggested that one of the oscillatory regulation points was located in the ethanol assimilation pathway.     

Cybernetic model of the growth dynamics of Saccharomyces cerevisiae in batch and continuous cultures.

Growth of Saccharomyces cerevisiae on glucose in aerobic batch culture follows the well-documented diauxic pattern of completely fermenting glucose to ethanol during the first exponential growth phase, followed by an intermediate lag phase and a second exponential growth phase consuming ethanol. In continuous cultures over a range of intermediate dilution rates, the yeast bioreactor exhibits sustained oscillations in all the measured concentrations, such as cell mass, glucose, ethanol, and dissolved oxygen, the amounts of intracellular storage carbohydrates, such as glycogen and trehalose, the fraction of budded cells as well as the culture pH. We present here a structured, unsegregated model for the yeast growth dynamics developed from the 'cybernetic' modeling framework, to simulate the dynamic competition between all the available metabolic pathways. This cybernetic model accurately predicts all the key experimentally observed aspects: (i) in batch cultures, duration of the intermediate lag phase, sequential production and consumption of ethanol, and the dynamics of the gaseous exchange rates of oxygen and carbon dioxide; and (ii) in continuous cultures, the spontaneous generation of oscillations as well as the variations in period and amplitude of oscillations when the dilution rate or agitatin rate are changed.     

Ammonium uptake in carrot cell structures is influenced by pH-dependent cell aggregation

Semicontinuously grown wild carrot ( Daucus carota L.) cells were used in an investigation of the effect of culture medium pH on ammonium uptake in suspension cultures as a first step in exploring the relationship between pH and anthocyanin biosynthesis. In contrast to published data showing decreasing uptake rates with decreasing culture medium pH, ammonium-limited, semicontinuous carrot cell cultures showed a 25% greater ammonium uptake rate at pH 4.5 than at pH 5.5. When cells that had been grown semicontinuously in medium with a pH of 4.5 or 5.5 were grown in batch cultures at pH 4.5, 5.5 or 6.5 the ammonium uptake rates were those of the semicontinuous cultures, indicating that the pH of the batch culture medium had no effect on ammonium uptake rates over 7 days. The cell culture was composed of very small aggregates when it was grown semicontinuously in medium at pH 4.5, but was composed of large aggregates when it was grown semicontinuously in medium at pH 5.5. The aggregation/disaggregation of the cells was pH dependent, as changing the pH of the semicontinuous culture medium altered the extent of the aggregation. We conclude that the change in culture medium pH caused the cells to aggregate or disaggregate which in turn decreased or increased the rate of ammonium uptake from the medium.     

S-Adenosylmethionine,cyclopropane fatty acid synthase,and the production of lactobacillic acid in Lactobacillus plantarum

S-Adenosylmethionine (AdoMet) levels in Lactobacillus plantarum were found to increase concomitantly with the production of membrane cyclopropane fatty acids under normal growth conditions. This increase in AdoMet did not occur when the pH of the culture medium (initially pH 6.5) was not allowed to fall (pH 4 or lower) during growth. When the culture medium was maintained at pH 6.5, cyclopropane fatty acid synthesis also remained low. While the activity of cyclopropane fatty acid synthase is increased as the pH decreases, the activity of AdoMet synthetase is largely unaffected by the variation of pH of the culture medium. The production of cyclopropane fatty acids is also dependent upon continued protein synthesis; in the presence of chloramphenicol cyclopropane fatty acid synthase activity is decreased, resulting in a lowered production of cyclopropane fatty acids. A dramatic increase in AdoMet levels occurs in the presence of chloramphenicol. It is proposed that AdoMet levels, in conjunction with cyclopropane fatty acid synthase activities, regulate cyclopropane fatty acid synthesis in L. plantarum.     

Environmental pH modulation of collagenase in normal human fibroblast cultures

Normal human skin fibroblast cultures have been used to assess the effects of relatively minor changes in environmental pH on collagenase, a major extracellular gene product. Collagenase accumulation in the culture medium, measured both as enzyme activity and immunoreactive material, was 2- to 10-fold greater at pH 7.6–8.2 than at pH 6.8–7.2. The pH-associated increase in collagenase was parallel by an increase in general protein synthesis. Nevertheless, prototypic lysosomal and cytoplasmic enzymes changed very little under identical culturing conditions. Although substantial intracellular protein degradation occurred at all pH values, the small differences either in general protein degradation or in specific collagenase degradation in the medium were of insufficient magnitude to account for the increased accumulation of collagenase.     

Involvement of a cell size control mechanism in the induction and maintenance of oscillations in continuous cultures of budding yeast

Spontaneous oscillations occur in glucose-limited continuous cultures of Saccharomyces cerevisiae under aerobic conditions. The oscillatory behavior is detectable as a periodic change of many bioparameters such as dissolved oxygen, ethanol production, biomass concentration, as well as cellular content of storage carbohydrates and is associated to a marked synchronization of the yeast population. These oscillations may be related to a periodic accumulation of ethanol produced by yeast in the culture medium.The addition of ethanol to oscillating yeast cultures supports this hypothesis: indeed, no effect was observed if ethanol was added when already present in the medium, while a marked phase oscillation shift was obtained when ethanol was added at any other time. Moreover, the addition of ethanol to a nonoscillating culture triggers new oscillations. An accurate analysis performed at the level of nonoscillating yeast populations perturbed by addition of ethanol showed that both the growth rate and the protein content required for cell division increased in the presence of mixed substrate (i.e., ethanol plus limiting glucose). A marked synchronization of the yeast population occurred when the added ethanol was exhausted and the culture resumed growth only on limiting glucose. A decrease of protein content required for cell division was also apparent. These experimental findings support a new model for spontaneous oscillations in yeast cultures in which the alternative growth on limiting glucose and limiting glucose plus ethanol modifies the critical protein content required for cell division.     

A method of direct-viewing comparative analysis of tumour cell motility in vitro; effect of pH and adhesion on cells of different malignancy

A novel method for the comparative analysis of cell motility by direct viewing was developed and used in a preliminary study of sarcoma cells. Three cell lines from the RPS family of sarcomas in inbred LEW/CUB rats which differ in the incidence of spontaneous metastasis were studied. A system of four different culture conditions, designed to mimic the stress within the tumour, was created by changing and combining two variables: the pH of the medium (with 2% calf serum only), which was either physiological at 7.4 or 6.6; and the adhesiveness of the culture substratum, which was either at a standard level or decreased. It was found that changing the pH from 7.4 to 6.6 in a single step slowed the cells down, and also changed the way in which they moved, from «walking« in random directions to moving in a more directional way. Making the culture surface less adhesive sped up cell locomotion. Decreasing the adhesiveness of the culture substratum and the pH simultaneously stimulated the migration of highly metastasizing cells preferentially. Thus we have found a way to distinguish between highly metastasizing A297Nb sarcoma cells and poorly metastasizing T15 and non-metastasizing K2 sarcoma cells, a distinction that could not be made by a simple examination of the cells. It is concluded that a comparison of cell motility by directly viewing the cells under different conditions may be useful when investigating the in vitro motility properties of malignant cells.     

Allosteric Properties of Chorismate Mutase from Quercus pedunculata

Chorismate mutase from Quercus pedunculata Ehrh. leaves has been purified by ammonium sulfate precipitation, molecular sieving and hydroxyapatite chromatography. Some results obtained during the purification suggest the presence, in oak, of two isofunctional forms of the enzyme, the one sensitive, the other insensitive to the action of aromatic amino acids. The regulable form exhibits a molecular weight of about 45,000. It is inhibited by tyrosine and by phenylalanine and is activated by tryptophan. In addition to its activating properties, this latter compound, endowed with a great affinity for the enzyme, reverses the inhibition due to the two other amino acids. The H⁺ concentration of the medium plays an important role in the sensitivity of the enzyme with regard to its effectors. Inhibition by tyrosine and by phenylalanine is maximal at pH 6.5, at which value the two ligands present an identical effect. At alkaline pH values, the rate of inhibition decreases regularly, tyrosine becoming the most effective inhibitor. Activation by tryptophan is particularly acute in a mildly acid medium; at pH 6.5, this effector increases the enzymatic activity threefold. Its action is weak (20% activation) in the optimum pH zone (pH 7.8) and increases towards the more basic pH values. As a result, tryptophan maintains a constant level of enzymatic activity throughout a large pH zone (between 6.5 and pH 9.0). The physiological significance of these results is discussed.     

Cybernetic modeling of spontaneous oscillations in continuous cultures of Saccharomyces cerevisiae.

We have developed a 'cybernetic' model to simulate the dynamic competition between all the available metabolic pathways of yeast Saccharomyces cerevisiae. This computer model predicts all the key experimentally observed aspects of the sustained oscillations in all the measured concentrations in continuous cultures, such as the spontaneous generation of oscillations as well as the variations in period and amplitude of oscillations when the dilution rate or agitation rate are changed.     

Analysis of factors responsible for the regeneration to intact cells from sphaeroplasts of Saccharomyces cerevisiae

The regeneration of Saccharomyces cerevisiae, NCIM 3288, cells from its sphaeroplasts were found to be influenced by a number of factors. The most suitable conditions of regeneration were also dependent on growth medium, that is, using malt-extractglucose-yeast extract-peptone (MGYP) medium: mannitol 0.7 M, pH 6.5, 30 °C and using yeast extract-peptone-glucose (YPG) medium: sucrose 0.7 M, pH 5.0 and 30 °C. The maximum regeneration frequency was observed in YPG medium.     

Characterization and overproduction of the Escherichia coli appA encoded bifunctional enzyme that exhibits both phytase and acid phosphatase activities

The appA gene that was previously shown to code for an acid phosphatase instead codes for a bifunctional enzyme exhibiting both acid phosphatase and phytase activities. The purified enzyme with a molecular mass of 44,708 Da was further separated by chromatofocusing into two isoforms of identical size with isoelectric points of 6.5 and 6.3. The isoforms had identical pH optima of 4.5 and were stable at pH values from 2 to 10. The temperature optimum for both phytase isoforms was 60 degrees C. When heated at different pH values the enzyme showed the greatest thermal resistance at pH 3. The pH 6.5 isoform exhibited K(m) and Vmax values of 0.79 mM and 3165 U.mg-1 of protein for phytase activity and 5.5 mM and 712 U.mg-1 of protein for acid phosphatase, respectively. The pH 6.3 isoform exhibited slightly lower K(m) and Vmax values. The enzyme exhibited similar properties to the phytase purified by Greiner et al. (1993), except the specific activity of the enzyme was at least 3.5-fold less than that previously reported, and the N-terminal amino acid sequence was different. The Bradford assay, which was used by Greiner et al. (1993) for determination of enzyme concentration was, in our hands, underestimating protein concentration by a factor of 14. Phytase production using the T7 polymerase expression system was enhanced by selection of a mutant able to grow in a chemically defined medium with lactose as the carbon source and inducer. Using this strain in fed-batch fermentation, phytase production was increased to over 600 U.mL-1. The properties of the phytase including the low pH optimum, protease resistance, and high activity, demonstrates that the enzyme is a good candidate for industrial production as a feed enzyme.     

Effect of a pH change in the medium and cytoplasm of cultured cells on the rhythm of protein synthesis

A circahoral rhythm of protein syntheses similar to that in monolayer hepatocytes was discovered in cell culture of Chinese hamster fibroblasts. Studies on the effects of pH changes in the culture medium and cultured cells on different parameters of protein synthesis showed some pH-dependent changes of predecessor pool and of its incorporation intensity into proteins. At the same time changes in a relative incorporation of the predecessor into proteins (with a correction for the pool) were insignificant. This value characterizing the productivity of protein synthesis does not seem to be directly associated with pH changes in the cells. The mean period of the rhythm of protein synthesis and intracellular pH was not changed with medium pH alterations.     

pH Change in Culture Medium Induces Reorganization of Intranuclear Actin in Two-Cell Mouse Embryos

Actin is a permanent component of the cell nucleus involved in many nuclear processes. However, some nuclear functions of actin remain insufficiently explored. The role played by various extracellular stimuli in regulation of nuclear actin still remains enigmatic. Deviation of basic parameters of culture medium from optimal values is a member of the group of extracellular stimuli that are very important for mammalian embryos cultured in vitro. Change in culture medium pH from the level optimal for embryo homeostasis is one such signals. The purpose of this study was to investigate the intranuclear actin distribution in nuclei of two-cell mouse embryos under stress conditions induced by changes in extracellular pH. The pattern of actin localization has been tracked after short-term culturing of the embryos at optimal (pH 7.2), increased (pH 7.8), or decreased (pH 6.5) pH conditions. Analysis was carried out with confocal microscopy using methods of direct fluorescent and indirect immunofluorescent identification of actin. It has been shown that the change of culture medium pH from the optimum value is the signal that alters intranuclear actin distribution in nuclei of the embryonic cells. Culture of two-cell mouse embryos in suboptimal pH conditions (pH 6.5 and pH 7.8) induced alterations in the intranuclear actin localization, which, in particular, were expressed in accumulation of monomeric actin and the appearance of phalloidin-stainable actin in the nuclei. These changes, in our opinion, show some signs of similarity with stress-induced changes in nuclear-actin distribution, which, as has been reported earlier by a number of researchers, have been observed in the nuclei of somatic cells.     

Nutritional and physiological factors affecting germination of heterothallic Saccharomyces cerevisiae ascospores.

Saccharomyces cerevisiae strain AP-3 was examined with respect to those nutritional requirements and physiological conditions which influence its germination rate. It was found that glucose as a carbon source supported the most rapid rate of germination for this heterothallic strain. In contrast, strain AP-3 spore germination was supported the least by the carbon sources potassium acetate and lactose. Of the nitrogen sources tested in culture medium containing glucose, the complex nitrogen sources peptone and casein hydrolysate appeared to be capable of stimulating germination better than a control culture containing ammonium sulphate. None of the amino acids screened were found to stimulate strain AP-3 germination compared with ammonium sulphate. The optimal culture medium pH for ascospore germination was 4.5 although spore germination could still be initiated by glucose between pH 3.0 and pH 7.5. Germination initiation by glucose was observed over a temperature range from 25 degrees C to 50 degrees C, but the optimal temperature appeared to be 40 degrees C.     

Modes of secretagogue-induced [Ca]i responses in individual chromaffin cells of the perfusedrat adrenal medulla

Chromaffin cells in the perfused rat adrenal medulla were loaded with indo-1 for confocal image analyses. Resting levels of [Ca(2+)](i) in chromaffin cells were similar and were stable with time. This is in contrast to the situation in isolated rat chromaffin cells, in which spontaneous oscillations of [Ca(2+)](i) are known to occur. When chromaffin cells were stimulated for 3-4 min by high K(+) or nicotine, [Ca(2+)](i) increased to a peak in 20-30 s and then declined rather smoothly. In contrast, chromaffin cells stimulated by muscarine or low pH (6.5) commonly exhibited irregular oscillations in [Ca(2+)](i). This provides additional evidence supporting the previous claim that muscarine and low pH evoke catecholamine secretion using partly shared mechanisms. Although muscarine and low pH were speculated to produce weaker responses in noradrenaline-secreting cells due to their selective stimulation of adrenaline secretion, no clear indications for segregation of cell types from [Ca(2+)](i) responses to these stimulants were found. The perfused adrenal medulla loaded with Indo-1 was also employed for simultaneously monitoring integrated changes in [Ca(2+)](i)(Ca responses) by conventional microfluorometry and in catecholamine secretion from a whole medulla (secretory responses). When the profiles of secretory responses were approximated by the kth power of the profiles of Ca responses, the k-values were estimated to be 2.2 and 2.3 for high-K(+)- and nicotine-elicited responses, respectively, whereas a k-value of 1.4 was obtained for both muscarine- and low-pH-elicited responses. An analysis showed that the significant difference in the k-value with these two classes of stimulants is accounted for by the stimulant-dependent patterns of [Ca(2+)](i) responses found in confocal image analysis.