Replicative aging of the yeast does not require DNA replication

Glucose addition to a stationary culture of wild-type Saccharomyces cerevisiae BY4742 cells with zero activity of MDR pumps resuspended in a fresh medium causes pump resynthesis (measured as pump-effected diS-C3(3) efflux). In a stationary culture in its original growth medium, this glucose-induced pump resynthesis fails to occur due to depletion of essential nutrients or to extracellular metabolites produced by cells during growth. Direct pump inactivation by metabolites is excluded since exponential cells with high MDR pump activity cultured in a medium with high concentration of extracellular metabolites retain this activity for at least 2 h. The metabolites also do not affect pump synthesis on the level of gene expression as addition of concentrated growth medium or an amino acid mixture to stationary cells in spent growth medium restores glucose-induced pump synthesis. The block of MDR pump synthesis is therefore due to the lack of essential nutrients in spent medium.     

Modeling growth and biochemical activities of Azospirillum spp.

An unstructured mathematical model was developed and used in the evaluation of biochemical activities of four Azospirillum spp. strains grown in batch cultures in a high C/N-ratio medium. The strains were evaluated for their ability to grow on fructose and produce exo-polysaccharide, and to sustain nitrogenase activity by using fructose or polysaccharides. Quantitative expression of the regulation of polysaccharide synthesis and nitrogenase (acetylene reduction) activity from the mineral nitrogen and sugar concentration in the culture medium was achieved. It was found that, during growth, Azospirillum spp. produced significant quantities of exocellular and capsular polysaccharide, whereas after depletion of the carbon source from the culture medium polysaccharides were consumed, especially in A. lipoferum strains. Significant nitrogenase activity was detected during polysaccharide degradation. Oxygen uptake was high during assimilation of fructose and low during polysaccharide degradation.     

The instability of physiological properties used as criteria in the taxonomy of yeasts

Summary Stock cultures of yeast strains which have acquired the ability to ferment and assimilate various sugars often represent a mixed population in regard to these features.Usually de-adaptation was not found after cultivation in glucose medium, indicating that the saltations are stable in this medium.The lyophilized cultures revived in glucose medium have the original characteristics of the strains.When single-cell colonies of various strains unable to utilize the appropriate sugar were grown in a liquid medium containing either galactose, sucrose or maltose, cells fermenting the sugar offered were found in the cultures. The acquisition of maltose-fermenting ability and the simultaneous acquisition of ability to utilize either galactose and raffinose, sucrose and raffinose, maltose and sucrose, or maltose, sucrose and -methyl-d-glucoside were observed.By an identification according to the acquired features the yeasts concerned have to be classified in other species than the original strain.     

The effect of growth medium on the antioxidant defense of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

We compared the oxidation of dihydrorhodamine 123, glutathione contents and activities of superoxide dismutase (SOD) and catalase for three wild-type strains of Saccharomyces cerevisiae grown on media with different carbon sources. The rate of oxidation of dihydrorhodamine 123 was much higher in respiring cells grown on ethanol or glycerol media than in fermenting cells grown on glucose medium. The total SOD activity was highest on glycerol medium and lowest on ethanol medium, while the catalase activity was highest on glycerol medium. The sequence of glutathione content values was: glucose > ethanol > glycerol.     

Proton-ATPase activity in cells of lactobacilli grown in the presence of propionate

Lactobacillus helveticus ATCC 15009 and CRL 581, and Lact. casei LC3 were grown in a complex medium with and without 15 mmol 1^-1 of neutralized propionic acid and assayed for proton-translocating ATPase activity. The enzyme activity was higher when the medium contained fatty acid than in its absence for all strains studied. Characteristics of this increased ATPase were identical to those of the enzyme located on the membrane of normal cells. The substrate consumption rate of resting cells was increased by propionate. This effect was reverted by the specific H⁺-ATPase inhibitor N,N '-dicyclohexylcarbodiimide indicating that the increment of fermentative activity was related to the H⁺-ATPase activity. These results suggest that the amplification of H⁺-ATPase activity could be involved in the inhibition of lactobacilli growth in cultures where propionic acid is unavoidably present, such as some mixed cultures with propionibacteria.     

The Influence of Calcium or Manganese on the Resistance to EDTA, Polymyxin B or Cold Shock, and the Composition of Pseudomonas aeruginosa Grown in Glucose- or Magnesium-depleted Batch Cultures

Cultures were batch grown in simple salts media in which growth was limited either by depletion of glucose and magnesium (C/Mg-dep) or by glucose alone (C-dep). Cultures were also grown in these media supplemented by calcium and/or manganese.
All cultures grown in the C-dep media were sensitive to ethylenediaminetetraacetic acid (EDTA), polymyxin and also to cold shock but were relatively resistant to ethyleneglycol-bis(2-aminoethyl ether)-N, N-tetraacetic acid (EGTA). Inclusion of calcium or manganese in the growth medium enhanced lysis by EDTA. Cultures grown in the basic C/Mg-dep media were resistant to EDTA, EGTA, polymyxin and to cold shock. Sensitivity to these agents was retained by cultures grown in C/Mg-dep media supplemented with Ca²⁺ and/or Mn²⁺. Cells grown in C/Mg-dep media with added Mn²⁺ were more sensitive to EDTA and polymyxin than those from the unsupplemented C/Mg-dep media but still resistant compared with C-dep cultures. All cultures from supplemented C/Mg-dep media were more sensitive to EGTA than those from any of the C-dep media.
Whole cells and cell walls from these various media had differing amounts of cell wall, phosphorus, amino sugar, carbohydrates, readily extractable lipid (REL), total phospholipid (PL), and especially differences in cell wall divalent metal cation content.
The differences in PL, REL and amino sugars and carbohydrate did not correlate with the response of C-dep and C/Mg-dep bacteria to EDTA, EGTA or polymyxin. The results are discussed in relation to the hypothesis that the sensitivity of Pseudomonas aeruginosa to polymyxin and EDTA is more dependent on outer membrane cation content rather than on other components, e.g. PL and lipopolysaccharide.     

The influence of certain growth conditions on the phosphatase activity of Streptococcus mutans grown in batch and continuous culture.

Acid phosphatase activity was detected in Streptococcus mutans strain NCTC 10832, and both acid and alkaline phosphatase in strains 2M2 and K1R. In batch culture, activity was maximal by mid exponential phase for 2M2 and at the end of this phase for NCTC 10832. Alkaline, but not acid, phosphatase activity of 2M2 and K1R increased when the inorganic phosphate in the medium was low; this was considered due, at least partly, to inducible or derepressible enzymes. In continuous culture, acid phosphatase activity of NCTC 10832 varied with the sugar substrate. The activity was increased by cell disruption and the degree of this increase for cells grown on different sugars parallelled the amounts of extracellular, insoluble polysaccharide produced on those sugars. Activity was highest for glucose-grown whole cells and for sucrose-grown disrupted cells.     

Effects of acetic acid on the kinetics of xylose fermentation by an engineered, xylose-isomerase-based Saccharomyces cerevisiae strain

Acetic acid, an inhibitor released during hydrolysis of lignocellulosic feedstocks, has previously been shown to negatively affect the kinetics and stoichiometry of sugar fermentation by (engineered) Saccharomyces cerevisiae strains. This study investigates the effects of acetic acid on S. cerevisiae RWB 218, an engineered xylose-fermenting strain based on the Piromyces XylA (xylose isomerase) gene. Anaerobic batch cultures on synthetic medium supplemented with glucose–xylose mixtures were grown at pH 5 and 3.5, with and without addition of 3 g L⁻¹ acetic acid. In these cultures, consumption of the sugar mixtures followed a diauxic pattern. At pH 5, acetic acid addition caused increased glucose consumption rates, whereas specific xylose consumption rates were not significantly affected. In contrast, at pH 3.5 acetic acid had a strong and specific negative impact on xylose consumption rates, which, after glucose depletion, slowed down dramatically, leaving 50% of the xylose unused after 48 h of fermentation. Xylitol production was absent (<0.10 g L⁻¹) in all cultures. Xylose fermentation in acetic –acid-stressed cultures at pH 3.5 could be restored by applying a continuous, limiting glucose feed, consistent with a key role of ATP regeneration in acetic acid tolerance.     

Trehalose promotes the survival of Saccharomyces cerevisiae during lethal ethanol stress, but does not influence growth under sublethal ethanol stress

Trehalose is known to protect cells from various environmental assaults; however, its role in the ethanol tolerance of Saccharomyces cerevisiae remains controversial. Many previous studies report correlations between trehalose levels and ethanol tolerance across a variety of strains, yet variations in genetic background make it difficult to separate the impact of trehalose from other stress response factors. In the current study, investigations were conducted on the ethanol tolerance of S. cerevisiae BY4742 and BY4742 deletion strains, tsl1 Δ and nth1 Δ, across a range of ethanol concentrations. It was found that trehalose does play a role in ethanol tolerance at lethal ethanol concentrations, but not at sublethal ethanol concentrations; differences of 20–40% in the intracellular trehalose concentration did not provide any growth advantage for cells incubated in the presence of sublethal ethanol concentrations. It was speculated that the ethanol concentration-dependent nature of the trehalose effect supports a mechanism for trehalose in protecting cellular proteins from the damaging effects of ethanol.     

Effects of pH and type of sugar in the medium on tyrosinase activity in cultured melanoma cells.

The tyrosinase (EC 1.14.18.1) activity of cultured mouse melanoma cells B16 in the stationary phase of growth, depends greatly on the pH of the medium and the kind of sugar present. The enzyme activity of a homogenate of cells grown at pH 7.2 in Eagles's MEM supplemented with 10% new born calf serum and con taining galactose in place of glucose, was about ten times that of a homogenate of cells cultured at pH 6.3 in the same medium. The tyrosinase activity changed reversibly on changing the pH of the culture medium. When cultured at a constant pH of 7.2, cells grown with 1 mM galactose had about five times higher tyrosinase activity than cells grown with 1 mM glucose. Only a small amount of lactate accumulated in cultures with glucose and it had little effect on the enzyme activity. These two findings explain the very low tyrosinase activity of cells cultured in medium with 5 mM glucose: the low activity is due to the presence of glucose and to the low pH resulting from conversion of glucose to lactic acid.     

Sensitivity of selected Frankia isolates from Casuarina, Allocasuarina and North American host plants to sodium chloride

Three experiments examined the effects of NaCl concentrations 0 to 500 mM on the growth of isolates of Frankia from Casuarinaceae and selected North American host plants. Four Casuarina isolates grew well in defined medium (pyruvate-BAP) but not in a yeast extract medium. Conversely the non-Casuarina isolates preferred the yeast-extract medium, although two of them grew in the defined medium. When grown in their preferred medium, the Casuarina isolates were little affected by NaCl concentrations up to 200 m M but did not grow at 500 m M . The non-Casuarina isolates, with the exception of an isolate from Purshia tridentata . were severely affected above 50 m M NaCl.
Nitrogenase activity (C₂H₂ reduction) by the non-Casuarina isolates could not be detected in low-N medium although protein determinations indicated that a low level of nitrogen fixation had occurred. All four Casuarina isolates showed nitrogenase activity in culture, up to 200 m M NaCl, although at that concentration of NaCl, growth was affected more than that of cultures in N-supplemented medium. All four strains showed a marked increase in nitrogenase activity up to 72 h after the addition of C₂H₂, with the magnitude of the effect and their subsequent behaviour being strain dependent.
The results indicate that the isolates of Frankia from Casuarina and Allocasuarina , and that from Purshia tridentata , are more tolerant of NaCl than isolates from species not normally growing under sodic conditions. They provide optimism that these strains could successfully establish in saline soils if introduced with species of host plants tolerant to these soils.     

Influence of propionate on growth and fermentative activity of lactobacilli

Propionic acid was added to cultures of Lactobacillus helveticus and Lact. casei growing in a complex medium. The effect of different concentrations of this fatty acid on the growth kinetic, substrate consumption rate and coefficient and product formation rate was examined. Low concentrations of propionic acid produced a low increase or no modification of the growth rate of lactobacilli. By contrast, higher concentrations reduced the growth rate and substrate yields in all the strains. The fermentative activity of lactobacilli increased in all the cultures with propionate. When the concentration of glucose in the medium was low, the population density of all the strains at the stationary growth phase was lower in the presence of propionate than in control cultures. An increase in the concentration of glucose in the medium counteracted the inhibitory effect of propionate. The addition of lithium lactate to the medium gave greater inhibition even at low propionate concentrations. The inhibition results were explained by assuming that propionic acid could diffuse through cell membranes in the undissociated form, increasing the inward leak of H⁺ in the cells. The extrusion of H⁺ by the H⁺-ATPase is possible by increasing the fermentative activity of the cells. When metabolism cannot supply the ATP required for proton extrusion, growth rate and biomass production decrease.     

Biosynthesis of cellulolytic enzymes in semi-continuous cultures

The aim of the research was to check the possibility of the semicontinuous cultivation of two Fusarium sp. strains producing cellulolytic enzymes. The cultures were grown in 5 1 fermentor on the mineral medium with addition of 1% cellulose cotton. In these cultures lasting 9 to 16 days the total activity was about 13 mg of reducing sugar/ml culture filtrate/hour. The comparison of the results of semi-continuous and periodic cultures pointed to the productivity of cellulolytic enzymes which was 1.2 to 2 times higher during one hour in semi-continuous cultures.     

Changes in electrophoretic mobility and lytic enzyme activity associated with development of flocculating ability in Saccharomyces cerevisiae.

Cells from stationary-phase cultures of two strains of Saccharomyces cerevisiae (3 and 20) failed to flocculate when grown in a complex or a chemically defined medium, while those of two other strains (11 and 13) flocculated when grown in either medium. Strain 30 flocculated when grown in complex but not defined medium and harvested from stationary-phase cultures. pH-electrophoretic mobility measurements on all five strains showed that mobility attributable to carboxyl groups usually increased as cultures progressed from the exponential to the stationary phase, while that caused by phosphate groups tended to decline. Acquisition of flocculating ability was accompanied in strains 11 and 30 by a slight increase in amidase activity, and greater increases compared with nonflocculent populations in activities of leucine aminopeptidase. alpha-mannosidase, and proteinase C. Activities of proteinases A and B showed no correlation with acquisition of flocculating ability.     

Anthranilic acid and 3-indolyl-propan-1,2-diol as precursors of photorubin in Saccharomyces

Summary The formation of photorubin, the orange-red pigment produced by Saccharomyces, is due to a photochemical reaction between anthranilic acid and an indole derivative identified with 3-indolyl-propan-1,2-diol. These two compounds are excreted into the medium by strains partly or completely requiring pyridoxine which are grown in the presence of a suboptimal amount of this vitamin. If pyridoxine is added to the growth medium in amounts either sufficient or exceeding the needs of the cells (over 50 g per liter), or if the strain is auxo-autotrophic, a secretion of hypoxanthine occurs.     

Two distinct pathways for trehalose assimilation in the yeast Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae can synthesize trehalose and also use this disaccharide as a carbon source for growth. However, the molecular mechanism by which extracellular trehalose can be transported to the vacuole and degraded by the acid trehalase Ath1p is not clear. By using an adaptation of the assay of invertase on whole cells with NaF, we showed that more than 90% of the activity of Ath1p is extracellular, splitting of the disaccharide into glucose. We also found that Agt1p-mediated trehalose transport and the hydrolysis of the disaccharide by the cytosolic neutral trehalase Nth1p are coupled and represent a second, independent pathway, although there are several constraints on this alternative route. First, the AGT1/MAL11 gene is controlled by the MAL system, and Agt1p was active in neither non-maltose-fermenting nor maltose-inducible strains. Second, Agt1p rapidly lost activity during growth on trehalose, by a mechanism similar to the sugar-induced inactivation of the maltose permease. Finally, both pathways are highly pH sensitive and effective growth on trehalose occurred only when the medium was buffered at around pH 5.0. The catabolism of trehalose was purely oxidative, and since levels of Ath1p limit the glucose flux in the cells, batch cultures on trehalose may provide a useful alternative to glucose-limited chemostat cultures for investigation of metabolic responses in yeast.     

Activity of protein kinase C during the differentiation of chick limb bud mesenchymal cells

Abstract. To investigate the relationship between protein kinase C (PKC) and chondrogenesis, PKC activity was assayed in cultures of stage 23/24 chick limb bud mesenchymal cells under various conditions. PKC activities of cytosolic and particulate fractions were low in 1 day cultured cells. As chondrogenesis proceeds, cytosolic PKC activity increased more than twofold, while that of the particulate fraction increased only slightly. Three days' treatment of cultures with phorbol-12-myristate-13-acetate (PMA, 5 × 10⁻⁸ M ) inhibited chondrogenesis judged by the accumulation of Alcian blue bound to the extracellular matrix and depressed PKC activity in cytosolic fraction. When cells were grown for 3 days in control medium after 3 days' treatment with PMA, chondrogenesis resumed and PKC activity recovered to normal values. PKC activity in cultures plated at low density (2 × 10⁶ cells/ml) where chondrogenesis is reduced was as low as that in 1 day cultured cells plated at high density (2 × 10⁷ cells/ml) or that in PMA treated cells. On the other hand, staurosporine promoted chondrogenesis without affecting PKC activity. Furthermore, reversal of PMA's inhibitory effect on chondrogenesis by staurosporine was not accompanied by recovery of PKC activity. These data indicate that increases in PKC activity is closely related to chondrogenesis and that PMA inhibits chondrogenesis by depressing PKC. However, staurosporine's enhancing effect on chondrogenesis is not related to PKC activity.     

Relationship between active oxygen species and cardenolide production in cell cultures of Digitalis thapsi: effect of calcium restriction

Elimination of calcium ions from the medium of undifferentiated cell cultures of Digitalis thapsi increased cardenolide production and induced extracellular H₂O₂ accumulation, as measured by the quenching of pyranine fluorescence. The addition of catalase reduced the response and the inclusion of superoxide dismutase enhanced the loss of fluorescence. This suggested that, besides H₂O₂, the superoxide anion was also formed before dismutating to H₂O₂. Additionally, exogenous H₂O₂ or superoxide dismutase stimulated cardenolide production whereas the addition of catalase markedly reduced it. These results point to a connection between H₂O₂ and cardenolide formation. The absence of calcium did not alter the levels of lipid peroxidation products; however, changes in the antioxidant system of D. thapsi cells were observed. Catalase activity was extremely low in control cultures and remained unaltered upon calcium elimination. Ascorbate peroxidase activity was not modified in calcium-free cultures. By contrast, calcium deprivation stimulated superoxide dismutase activity and strongly inhibited glutathione reductase activity. Also, a significant decrease in reduced glutathione was observed. These responses were emulated by treatment of the cultures with the glutathione biosynthesis inhibitor buthionine sulfoximine and by ethyleneglycol-bis-β-aminoethyl ether and LaCl₃. All these results indicate that the depletion of extracellular calcium induces changes in the redox state of cells and suggest that this alteration stimulates cardenolide formation in D. thapsi cultures.     

Dependence of Clostridium botulinum gas and protease production on culture conditions

Reports that Clostridium botulinum toxin can sometimes be detected in the absence of indicators of overt spoilage led to a systematic study of this phenomenon in a model system. Media with various combinations of pH (5.0 to 7.0) and glucose (0.0 to 1.0%) were inoculated with vegetative cells of C. botulinum 62A and incubated anaerobically at 35 degrees C. Although growth and toxin production occurred at all pH and glucose combinations, accumulation of gas was delayed or absent in media with low pH, low glucose levels, or both. Other proteolytic C. botulinum strains gave similar results. Trypsin activation was required to detect toxin in some low pH cultures. The trypsinization requirement correlated with low proteolytic activity in the cultures. Proteolytic activity of the strains examined was 5- to 500-fold lower in botulinal assay medium than in cooked meat medium. The results indicate that the absence of gas accumulation does not preclude the presence of botulinal toxin and that proteolytic cultures grown under adverse conditions may require trypsinization for the detection of toxin.     

Studies on the regulation of insulin receptors in cultured BALB/3T3 fibroblasts

The level of [¹²⁵I]insulin binding to BALB/ 3T3 fibroblasts was low in growing cells and high in stationary cells. Since frequent changes of medium (every 2 h) did not modify the hormone binding of the stationary cells, it is unlikely that serum factors directly regulate the number of insulin receptors. Cells were grown to different densities by plating them in different concentrations of serum. Insulin binding was low in dense cultures maintained actively growing by high serum concentration, while binding was high in sparse cultures which were growth-arrested due to serum depletion. Thus, cell density does not directly regulate the insulin receptors. The growth status of the cells is the only factor that explains consistently the variations of insulin binding in these and previous [1, 2] experiments. Synchronization of the cells by two different methods did not show a reproducible cellcycle dependence for the insulin receptors.