Metabolism of alpha-factor by a mating type cells of Saccharomyces cerevisiae.

When a mating type cells of Saccharomyces cerevisiae are exposed to the mating pheromone alpha-factor in liquid cultures, there is a time-dependent loss of alpha-factor activity from the culture fluid. This loss of biological activity can be directly correlated with the proteolysis of the pheromone by a mating type cells. The metabolism of alpha-factor by a mating type cells may be measured by using either in vitro 125I-labeled or in vivo 35S-labeled pheromone. Addition of chloroquine to growing cultures of a mating type cells at concentrations which cause no detectable alterations in cell growth produces a potentiation of alpha-factor mediated cell cycle arrest. This potentiation of alpha-factor activity is directly correlated with the inhibition of alpha-factor proteolysis. Thus, while proteolytic digestion of alpha-factor appears to be related to the mechanism whereby a mating type cells "detoxify" alpha-factor and recover from cell cycle arrest, proteolysis of the mating factor is not necessary for alpha-factor mediated cell cycle arrest.     

Mechanism of α Factor Biosynthesis in Saccharomyces cerevisiae

The biosynthesis of alpha factor, a mating-type-specific regulatory oligopeptide which is secreted by Saccharomyces cerevisiae cells of alpha mating type, was studied. In batch cultures only small amounts of the peptide were synthesized during the exponential growth phase. During the stationary phase, alpha factor was produced at a constant rate and accumulated in the culture medium. Inhibition of translation in wild-type cells by cycloheximide, or in mutant strains under conditions which blocked protein or ribonucleic acid (RNA) synthesis completely inhibited the production of alpha factor. These results indicate that the factor is produced by ribosomal translation of a specific messenger RNA and not by an extraribosomal mechanism of peptide synthesis.     

Effect of alpha factor pheromone on the activity of enzymes which catalyze the synthesis and hydrolysis of cell wall structural polymers from Saccharomyces cerevisiae

Cells of Saccharomyces cerevisiae of the a mating type treated with alpha factor contain an increased amount of structural polymers (beta-glucans and chitin) in their cell walls and, consequently, exhibit higher glucan synthetase and chitin synthetase activities than untreated cells. However, alpha factor has no detectable effect on the activities of these enzymes when they are assayed, "in vitro", in the presence of the pheromone. On the other hand, the activity of beta-glucanases remains constant during the time that growth of a cells is kept arrested by alpha factor at the G1 phase of the cell division cycle and starts to increase when budding of the cells is reinitiated.     

Cytofluorometry and fluorescence confocal laser scanning microscopy (CLSM) in the study of neutral lipid dynamics in Paramecium primaurelia mating types during cell line development

BACKGROUND: In Paramecium primaurelia, an exconjugant cell can produce two lines with different mating capacities. Mating type II cells can form a higher food vacuole number and digest the nutrient taken up in a shorter time; thus, mating type II cells grow at a faster rate than do mating type I cells. The present study was done to determine whether cells that ingest more nutrients also have a larger amount of storage lipids. METHODS: Quantitative and qualitative determinations of neutral lipids were obtained by means of cytofluorometry and fluorescence confocal laser scanning microscopy (CLSM), respectively, by using nile red on cells in different physiologic states. RESULTS: Lipid droplet number and neutral lipid content were higher in mating type II cells than in mating type I cells in the early logarithmic growth phase (i.e., immature well-fed cells). These values were reversed during the middle and the late logarithmic phases and became equal in the stationary phase (i.e., mature starved cells). In well-fed cells maintained with food excess, differences in neutral lipid content between the two mating types also were present in mature cells. CONCLUSIONS: Although differences between mating type I and mating type II lines were not correlated to cell size, a relation was found between lipid content and food ingestion capacity. A depletion of bacteria in the culture medium could be responsible for the lack of differences in mature starved cells. CLSM allowed us to gather volume information about the lipid droplet distribution within the cell.     

On-line determination of optimum time for switching from growth phase to production phase in tissue plasminogen activator production in a microcarrier cell culture

To maximize the productivity of tissue plasminogen activator (TPA) by a mammalian cell culture, on-line determination of the optimum time to switch from the cell growth phase to the TPA production phase was investigated. By measuring the TPA production activity of the cells during the cell growth culture, it was shown that this optimum time was not necessarily the same as the time at which the cell concentration was maximized, and that the optimum time varied with growth culture batch. The TPA production activity of the cells during the growth culture could be estimated by on-line regression analysis using physiological data of the current state, including the oxygen consumption rate (Io₂) and cell concentration, as well as data from past batches. Applying this on-line estimation, the optimum switching time was determined to be the time at which the TPA production activity of the cells in the growth culture became highest, or higher than a certain value according to determined criteria.     

Dehydroascorbate uptake activity correlates with cell growth and cell division of tobacco bright yellow-2 cell cultures

Recently, ascorbate (ASC) concentration and the activity of a number of enzymes from the ASC metabolism have been proven to correlate with differences in growth or cell cycle progression. Here, a possible correlation between growth and the activity of a plasma membrane dehydroascorbate (DHA) transporter was investigated. Protoplasts were isolated from a tobacco (Nicotiana tabacum) Bright Yellow-2 cell culture at different intervals after inoculation and the activity of DHA transport was tested with (14)C-labeled ASC. Ferricyanide (1 mM) or dithiothreitol (1 mM) was included in the test to keep the external (14)C-ASC in its oxidized respectively reduced form. Differential uptake activity was observed, correlating with growth phases of the cell culture. Uptake of DHA in cells showed a peak in exponential growth phase, whereas uptake in the presence of dithiothreitol did not. The enhanced DHA uptake was not due to higher endogenous ASC levels that are normally present in exponential phase because preloading of protoplasts of different ages did not affect DHA uptake. Preloading was achieved by incubating cells before protoplastation for 4 h in a medium supplemented with 1 mM DHA. In addition to testing cells at different growth phases, uptake of DHA into the cells was also followed during the cell cycle. An increase in uptake activity was observed during M phase and the M/G1 transition. These experiments are the first to show that DHA transport activity into plant cells differs with cell growth. The relevance of the data to the action of DHA and ASC in cell growth will be discussed.     

Estimating the rate of plasmid transfer: an end-point method

We describe a method for determining the rate parameter of conjugative plasmid transfer that is based on single estimates of donor, recipient and transconjugant densities and the growth rate in exponential phase of the mating culture. The formula for estimating the plasmid transfer rate, gamma, was derived from a mathematical model describing cell growth and plasmid transfer in batch culture. Computer simulations were used to explore the sensitivity of this method to the realities of bacterial life, such as growth rate differences, plasmid segregation and transitory derepression of pilus synthesis. As predicted by the theory, mating experiments with the plasmid R1 in Escherichia coli K12 demonstrated that the estimate gamma is unaffected by cell density, donor:recipient ratio and mating time. Unlike previous techniques, our method allows us to investigate the effect of environmental factors on plasmid transfer rates when these factors also influence population growth rates. To illustrate this, we examined the effect of temperature on the rate of plasmid transfer.     

The exchange factor Cdc24 is required for cell fusion during yeast mating

During Saccharomyces cerevisiae mating, chemotropic growth and cell fusion are critical for zygote formation. Cdc24p, the guanine nucleotide exchange factor for the Cdc42 G protein, is necessary for oriented growth along a pheromone gradient during mating. To understand the functions of this critical Cdc42p activator, we identified additional cdc24 mating mutants. Two mating-specific mutants, the cdc24-m5 and cdc24-m6 mutants, each were isolated with a mutated residue in the conserved catalytic domain. The cdc24-m6 mutant responds normally to pheromone and orients its growth towards a mating partner yet accumulates prezygotes during mating. cdc24-m6 prezygotes have two apposed intact cell walls and do not correctly localize proteins required for cell fusion, despite normal exocytosis. Our results indicate that the exchange factor Cdc24p is necessary for maintaining or restricting specific proteins required for cell fusion to the cell contact region during mating.     

Biosynthesis of endochitinase of Serratia marcescens.

The growth of a mutant strain of Serratia marcescens with high chitinase activity and the biosynthesis of endochitinase by this strain were investigated. The study was carried out using semisynthetic culture medium without inducers and culture medium containing colloidal chitin as a sole nitrogen and carbon source, with and without mitomycin C. The mutant strain, unlike the native one, was shown to produce endochitinase and to secrete the enzyme into the medium during the growth on culture medium without the inducers, chitin and mitomycin C. During growth on the medium with chitin the mutant strain differed from the native one with a short lag-phase of growth, the early appearance of endochitinase in the culture liquid and a high level of endochitinase activity. The difference between the strains disappeared after the addition of mitomycin C, an inducer of the cell SOS-response, to the culture medium containing chitin. Specific endochitinase activity of S. marcescens mutant strain grown on various culture media had two maxima, namely at the beginning and at the end of the stationary phase. Mitomycin C increased the specific activity in a second peak of endochitinase activity during the growth of the mutant strain.     

Characterisation of growth enhancing factor production in different phases of in vitro fish macrophage development

We previously described the release of macrophage growth factor(s) (MGF) into culture supernatants (CCM) by a goldfish macrophage cell line (GMCL) and in vitro derived kidney macrophages (IVDKM). In this study, we report that IVDKM growth can be subdivided into three developmental phases, defined using both morphological and flow cytometric characteristics: a lag phase, a proliferative phase, and a senescence phase. Analysis of the growth inducing capabilities of CCM indicated that maximum activity was consistently found in supernatants isolated from IVDKM cultures during the proliferative phase of development. In contrast, CCM from the senescence phase proved to be poor inducers of macrophage growth. Overall, we identify a link between the seeding-CCM composition, the extent of IVDKM growth and the rate of entrance into a senescent state characterised by IVDKM apoptotic cell death. Use of IVDKM CCM obtained at the peak of macrophage growth maximised macrophage growth factor (MGF) activity, and prevented the introduction of negative regulators of IVDKM proliferation, which will contribute significantly to our MGF purification efforts. Furthermore, the collection of IVDKM, prior to their commitment into apoptotic pathways, will prove to be essential in the selection of specific cell subsets for studies of antimicrobial mechanisms of macrophages.     

Variation of (1 leads to 3)-beta-glucanases in Saccharomyces cerevisiae during vegetative growth, conjugation, and sporulation.

The total (1 leads to 3)-beta-glucanase activities associated with cell extracts and cell walls of Saccharomyces cerevisiae were measured during vegetative growth, conjugation, and sporulation. Using a system of column chromatography, we resolved (1 leads to 3)-beta-glucanase activity into six different enzymes (namely, glucanases I, II, IIIA, IIIB, IV, and V). The contributions of the individual enzymes to the total activity at the different stages of the life cycle were determined. Total glucanase activity increased during exponential growth and decreased in stationary resting-phase cells. Glucanase IIIA was the predominant enzyme in stationary resting-phase cells. Glucanases I, II, IIIB, and IV were either absent or present at low levels in stationary phase cells, but their individual activities (in particular, glucanase IIIB activity) increased substantially during exponential growth. Total (1 leads to 3)-beta-glucanase activity did not change significantly during conjugation of two haploid mating strains, S. cerevisiae 2180A and 2180B, and no notable changes were detected in the activities of the individual enzymes. Sporulation was accompanied by a rapid increase and then a decrease in total glucanase activity. Most of the increase was due to a dramatic rise in the activity of glucanase V, which appeared to be a sporulation-specific enzyme. Glucanase activity was not derepressed by lowering the glucose concentration in the growth medium.     

Fungal melanin inhibitor and related compounds from Penicillium decumbens

In Silene vulgaris (M.) G. cell culture three growth phases were distinguished, namely, a lag phase, an exponential phase and a stationary phase. Pectin termed silenan and an acidic arabinogalactan were isolated as cell wall polysaccharides of S. vulgaris callus at the different growth phases during culture. Production of silenan as the galacturonan (or rhamnogalacturonan) core was observed at the beginning of the exponential phase and at the stationary phase of the callus growth. Arabinogalactan, containing the galacturonic acid residues, is formed at the exponential phase followed by attachment to the core of silenan in the middle of the exponential phase. The arabinogalactan constituent of silenan appeared to be destroyed gradually at the stationary growth phase. The monosaccharide compositions of silenan and arabinogalactan were determined at various phases of the callus growth. Silenan was found to be formed in maximum amounts at the exponential phase of the cell growth. Insignificant alterations of the yields of acidic arabinogalactan were found during culture while total productivity per litre of medium and rate of production per day of arabinogalactan were found to be maximal at the exponential phase of growth.     

Inositol is specifically involved in the sexual program of the fission yeast Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe is a natural auxotroph for inositol and fails to grow in the complete absence of it. It was previously reported that a small concentration of inositol in the culture medium supports vegetative growth, but not mating and sporulation, and a tenfold of that concentration also supports mating and sporulation. The purpose of the present work was to investigate whether a moderate inositol starvation specifically affected events of the sexual program of development. A homothallic culture grown to the stationary phase in medium with a small inositol concentration was sterile but cells in the stationary phase of growth synchronously entered and completed the sexual cycle when inositol was added, without need of previous cell divisions. This suggests the involvement of inositol in a mechanism (or mechanisms) of the sexual program. The events of the program that were affected by inositol starvation were investigated. Commitment to mating and production of pheromone M were shown not to be inositol-dependent. A diploid strain homozygous at the mating-type locus and carrying a pat1-114 temperature-sensitive mutation in homozygous configuration sporulated under inositol starvation at the restrictive temperature; therefore starvation did not directly affect meiosis or sporulation. In contrast, production of pheromone P and the response of cells to pheromones were found to be inositol-dependent. The possibility that inositol or one of its derivative compounds is involved in pheromone P secretion and in pheromone signal reception is discussed.     

Cell cycle,cell size and mitochondrial activity of hybridoma cells during batch cultivation

Cell cycle, cell size and rhodamine 123 fluorescence in cell populations of two batch cultures were analysed and quantified with a fluorescence-activated cell sorter (FACS). Two cultures derived from either exponential or stationary phase innocula were investigated in order to demonstrate the dependency of the subsequent cell growth on innoculum condition. The results demonstrated that the level of activity of cells in the innoculum culture could have a significant effect on cellular activity during the initial phase of the inoculated culture, as it advances through its growth cycle. Positive correlation was found between the cell size and mitochondrial activity (as measured by rhodamine 123 uptake) with S and G₂ fractions as the cell progressed through the cell cycle. The enumeration of the fractions of cell cycle phases has helped in prediction of the changes in cell numbers following perturbation of the culture condition.     

Production, secretion, and stability of human secreted alkaline phosphatase in tobacco NT1 cell suspension cultures

Tobacco NT1 cell suspension cultures secreting active human secreted alkaline phosphatase (SEAP) were generated for the first time as a model system to study recombinant protein production, secretion, and stability in plant cell cultures. The SEAP gene encodes a secreted form of the human placental alkaline phosphatase (PLAP). During batch culture, the highest level of active SEAP in the culture medium (0.4 U/mL, corresponding to approximately 27 mg/L) was observed at the end of the exponential growth phase. Although the level of active SEAP decreased during the stationary phase, the activity loss did not appear to be due to SEAP degradation (based on Western blots) but due to SEAP denaturation. The protein-stabilizing agents polyvinylpirrolidone (PVP) and bacitracin were added extracellularly to test for their ability to reduce the loss of SEAP activity during the stationary phase. Bacitracin (100 mg/L) was the most effective treatment at sustaining activity levels for up to 17 days post-subculture. Commercially available human placental alkaline phosphatase (PLAP) was used to probe the mechanism of SEAP deactivation. Experiments with PLAP in sterile and conditioned medium corroborated the denaturation of SEAP by factors generated by cell growth and not due to simple proteolysis. We also show for the first time that the factors promoting activity loss are heat labile at 95 degrees C but not at 70 degrees C, and they are not inactivated after a 5 day incubation period under normal culture conditions (27 degrees C). In addition, there were no significant changes in pH or redox potential when comparing sterile and cell-free conditioned medium during PLAP incubation, indicating that these factors were unimportant.     

Light Conditions for Sexual Reproduction in Heterothallic Strains of Closterium

Sexual reproduction in heterothallic strains of Closterium peracerosum-strigostan-littorale,KAS-4-29 (mating type minus) and KAS-4-30 (mating type plus),depended on a light intensity higher than 3,000 lux. Two kindsof cell division occurred in the mating medium: 1) one kindoccurred at low light intensity (less than 1,000 lux) and wasnot linked to conjugation, and 2) a sexual one occurred at highlight intensity (higher than 3,000 lux) and was linked to conjugation.3-(4-Chlorophenyl)-l,l-dimethylurea at 1 µM added at thebeginning of the mating culture completely inhibited conjugationbut not cell division. Chloramphenicol at 20 to 40 µg/mladded at the beginning of the culture inhibited conjugationcompletely and cell division partly. The drugs added after 12h of the mating culture did not inhibit the division processes.Varying light intensity after 12 h of culture did not affectsexual activity. The photosynthetic activity was highest inthe cells at the beginning of the mating culture, and then markedlydecreased. The data indicate that the early period of the matingprocess depends strictly on the light conditions. (Received March 12, 1982; Accepted November 30, 1982)     

Cytokinin Production in Relation to the Growth of Pea-root Callus Tissue

The natural occurrence of free cytokinins was examined in relationto the growth of serially propagated pea-root callus tissuecultures. The relatively slow-growing pea-root callus was harvestedat regular intervals throughout a 12-week period and fresh weight,dry weight, cell number, and cytokinin activity were determined.At the end of the culture period the fresh weight had increasedabout 34 times, the cell number about 100-fold, and the dryweight approximately 19 times over that found on inoculation.Purified ethanol extracts from pea-root callus were tested forcytokinin activity by the soybean callus bioassay. Cytokininactivity was detected in extracts made at all stages of growthtested. There was a sharp rise in cytokinin content during theearly period of culture. The peak in cytokinin activity establishedat the beginning of the phase of growth was associated witha high frequency in mitoses. As growth proceeded there was adecline in both cytokinin content and in the mitotic index.     

Expression of differentiated function by hepatocytes in primary culture: variable effects of glucagon and insulin on gluconeogenesis during cell growth

Hormonal effects on gluconeogenesis from lactate were studied during the growth cycle of adult rat parenchyma liver cells using a primary monolayer culture system previously described [25]. Basal and glucagon-stimulated gluconeogenic ability were found to decline rapidly during log phase, insulin-stimulated growth. A progressive recovery of gluconeogenesis activity was observed after cell division subsided. Rates of lactate-gluconeogenesis were found also to decline in the absence of prior insulin exposure. This decline was not as rapid as the loss observed in cells cultured with insulin. However, in insulin-deficient cultures gluconeogenesis was completely abolished after 12 days and did not reelevate with further incubation unless cells were washed and exposed to glucagon. Decreasing growth rates of insulin-supplemented cultures by decreasing serum concentrations resulted in comparatively higher gluconeogenic activity. The results presented here are consistent with previous observations of hepatic parenchymal expression of 'differentiated function' during cellular growth phases in culture (i.e., differentiated functions are generally lost during rapid growth and regained as cells become quiescent). The present study, however, presents unexpected effects of insulin on the apparent growth-state dependent gluconeogenic recovery. Our data imply that although insulin has long been known to inhibit gluconeogenesis, its presence in culture may facilitate long-term basal maintenance of gluconeogenic enzyme activity. Insulin also functions as a growth factor whose initial mitogenic effect correlates with decreased gluconeogenic function. These changes show no simple or predictive correlation with cyclic nucleotide metabolism.     

High-level expression and secretion of recombinant mouse endostatin by Escherichia coli

The expression of murine endostatin was achieved by placing its gene downstream of an alkaline phosphatase gene (phoA) promoter. To ensure proper folding and secretion of the recombinant protein, the mouse endostatin was fused with alkaline phosphatase signal peptide. SDS/polyacrylamide gel electrophoresis analysis of the culture medium of recombinant Escherichia coli cells revealed that endostatin was efficiently secreted. The signal peptide was efficiently cleaved during secretion as demonstrated by N-terminal amino acid sequencing. The maximum yield of secreted endostatin during fermentation was 40 mg/liter. Up to 28 mg of endostatin was purified from 1 liter of cell culture broth. The biological activity of recombinant protein was tested in a bovine aortic endothelial (BAE) cell proliferation assay. The recombinant endostatin inhibited the growth of BAE cells stimulated by basic fibroblast growth factor, and its ED50 was comparable to that from a previous report. Flow cytometric measurements of BAE cells cultivated in medium with endostatin demonstrated a cell cycle arrest mainly in the G0/G1 phase and a decrease in the S phase.     

Flocculation of lag phase yeast cells ofCandida albicans

Flocculation of the yeast form ofCandida albicans occurs during the early or lag phase of growth. Once a developing culture has entered the logarithmic phase of growth, the cells are no longer capable of flocculating. The flocculation during these early stages in the growth of the culture was studied and appeared to be similar in many physical respects to agglutination of mating strains ofHansenula wingei. Variations in the carbohydrate content, nitrogen content, pH, and temperature of the growth medium did not alter the amount of flocculation. Studies with electrical fields indicated that an electrostatic charge is not involved. Treatment of the surface of the cells with solvents showed that lipid solvents do not inhibit flocculation, but phenol, which removes both carbohydrate and protein material, does inhibit flocculation. Lipases also do not affect flocculation but enzymes that break down carbohydrates or protein such as trypsin, papain, and pancreatin do affect flocculation. Phenol extracts from sonicated, washed cell walls were analyzed by electrophoresis and paper chromatography for proteins, amino acids, and polysaccharides. These analyses confirm earlier reports of constituents of the cell wall but do not show a difference in the types of compounds present in the cell walls of flocculant cells as compared with the cell walls of older non-flocculant cells.