Regulation of lipid synthesis from acetate in diploid fibroblast cultures —variation with passage level and stage of cell growth

Regulation of lipid synthesis from acetate in human diploid fibroblast cultures has been studied at various passage levels and at different stages of cell growth. When cultures were transferred to lipid free medium, a stimulation of [¹⁴C]acetate incorporation into lipid occurred within three to six hours after removal of exogenous lipid. In early passage cultures, this stimulation was observed whether cells were transferred to protein-free medium or medium supplemented with delipidized serum protein. However, in late passage cultures the presence of delipidized serum protein was required for the stimulation of lipid synthesis. When logarithmically dividing and stationary phase cultures were compared, the cultures in log phase showed stimulation of acetate incorporation into lipid in the presence or absence of delipidized serum protein, whereas in the stationary cultures the delipidized serum protein was required. When cultures were partially synchronized by a thymidine block, stimulation of acetate incorporation into lipid in the blocked cells only occurred in the presence of delipidized serum protein; in released cells stimulation occurred in protein free medium. When inhibition of lipid synthesis from acetate was compared in young vs. old or dividing vs. stationary cultures, however, no differences were observed. The data indicate the response of diploid fibroblast cultures to change in exogenous lipid is dependent on passage level and state of growth.     

Changes in membrane transport function in G0 and G1 cells

Confluent quiescent monolayers of aneuploid and euploid cells in culture can be stimulated to proliferate by appropriate nutritional changes. In confluent monolayers of WI-38 human diploid fibroblasts the uptake of cycloleucine is increased three hours after these cells are stimulated to proliferate by a change of medium plus 10% serum. No changes in the uptake of cycloleucine are observed in logarithmically-growing WI-38 cells exposed to fresh medium plus 10% serum, or in WI-38 confluent monolayers in which the conditioned medium has been replaced by fresh medium with 0.3% serum (a change that does not cause stimulation of cellular proliferation in WI-38 cells). In 3T6 cells in the stationary phase stimulated to proliferate by nutritional changes, there is a prompt increase in the uptake of cycloleucine, within one hour after stimulation of cell proliferation. Similar results were obtained with stationary 2RA cells which are SV-40 transformed WI-38 fibroblasts. In addition, chromatin template activity which is known to increase in the early stages after stimulation of confluent WI-38 cells, was unchanged in confluent 3T6 or 2RA cells stimulated to proliferate. These results show that at least two of the very early biochemical events occurring in response to stimulation of cell proliferation are different in WI-38 diploid cells and in aneuploid 2RA or 3T6 cells. It is proposed that WI-38 cells in the stationary phase are arrested in the G₀ phase of the cell cycle, while 2RA and 3T6 cells are arrested in the G₁ phase.     

The differential activation of ornithine decarboxylase in synchronized cultures maintained with a salts/glucose medium

Asparagine and cAMP caused differential activation of ornithine decarboxylase activity that was dependent upon the cell cycle phase of Chinese Hamster Ovary cells maintained with a salts/glucose medium. Prior to incubation in the salts/glucose medium, stationary phase cultures were stimulated to proliferate by refeeding with fresh complete growth medium. Induction of ornithine decarboxylase activity was dependent upon two conditions: (1) the salts/ glucose medium required supplementation with an appropriate amino acid and cAMP; (2) cultures had to be in late G1/early S phase when placed in the salts/glucose medium. Cultures in G0 phase had moderate capacity for ornithine decarboxylase enhancement but lost this capacity as they traversed into early G1 or into mid S phase. These results demonstrate the importance of the cell cycle in modulating the activation of ornithine decarboxylase by asparagine and cAMP.     

Morphological changes in the central vacuole ofBlastocystis hominis during in vitro culture

Summary Morphological changes in the central vacuole during the growth in in vitro culture ofBlastocystis hominis were investigated by light and electron microscopy. Most cells in log phase and an early stationary phase showed a positive staining reaction in the central vacuole with PAS or Sudan black B stain, whereas cells in late stationary phase showed few positive reactions. Electron microscopic observations revealed that 95% ofB. hominis cells in log phase and 50% of cells in early stationary phase, had a substantial accumulation of electron-dense material in the central vacuole. In contrast, only 25% of the organisms in late stationary phase had an electron-dense central vacuole, while more than 50% of cells had an electron-lucent central vacuole. These results indicate thatB. hominis accumulated carbohydrates and lipids in the central vacuole during cell growth and that the organism probably consumed these metabolic substances during stationary growth. Therefore, it is strongly suggested that the central vacuole is an important organelle for storage of metabolic substances, such as carbohydrates and lipids, required for cell growth.Abbreviations PBS phosphate-buffered saline - PAS periodic acid-Schiff     

Effect of extracellular polymeric substances on the mechanical properties of Rhodococcus

The mechanical properties of Rhodococcus RC291 were measured using force spectroscopy equipped with a bacterial cell probe. Rhodococcal cells in the late growth stage of development were found to have greater adhesion to a silicon oxide surface than those in the early growth stage. This is because there are more extracellular polymeric substances (EPS) that contain nonspecific binding sites available on the cells of late growth stage. It is found that EPS in the late exponential phase are less densely bound but consist of chains able to extend further into their local environment, while the denser EPS at the late stationary phase act more to sheath the cell. Contraction and extension of the EPS could change the density of the binding sites, and therefore affect the magnitude of the adhesion force between the EPS and the silicon oxide surface. By treating rhodococcal EPS as a surface-grafted polyelectrolyte layer and using scaling theory, the interaction between EPS and a solid substrate was modelled for the cell approaching the surface which revealed that EPS possess a large capacity to store charge. Changing the pH of the surrounding medium acts to change the conformation of EPS chains.     

Behavior in two-phase aqueous polymer systems of L5178Y mouse leukemic cells : II. The lag and exponential phases of growth

The behavior of lag and exponential growth phase L5178Y mouse leukemic cells under normal and prolonged lag phase conditions with respect to partition in aqueous dextran — polyethylene glycol polymer systems has been studied. ‘Backculture’ of early stationary cells into fresh growth medium is accompanied by a decrease in partition ratio from 0.52 to 0.11. The partition ratio remains depressed for a time considerably longer than the duration of lag phase but rises rapidly and returns to its former value as the cells reach late exponential/early stationary phase. If lag phase is prolonged, the time for which the partition ratio remains depressed is also prolonged. In the exponential phase following a prolonged lag phase, the partition ratio rises at a rate slower than during a normal exponential phase and does not reach the same magnitude for the same position in the cycle. Net negative surface charge as measured by particle microelectrophoresis does not change appreciably throughout the growth cycle. The results suggest that the sequence of events at the cell surface on a populational basis which contribute to the partitioning behavior is possibly predetermined or programmed at the time of transfer into fresh medium. The results further substantiate the technique of aqueous polymer partitioning as being the most sensitive method available for monitoring subtle changes in plasma membrane properties during the cell growth cycle.     

Radiation resistance of Deinococcus radiodurans R1 with respect to growth phase

Deinococcus species exhibit an extraordinary ability to withstand ionizing radiation (IR). Most of the studies on radiation resistance have been carried out with exponential phase cells. The studies on radiation resistance of Deinococcus radiodurans R1 with respect to different phases of growth showed that late stationary phase cells of D. radiodurans R1 were fourfold more sensitive to IR and heat as compared with exponential or early stationary phase cells. The increased sensitivity of D. radiodurans R1 to IR in the late stationary phase was not due to a decrease in the intracellular Mn/Fe ratio or an increase in the level of oxidative protein damage. The resistance to IR was restored when late stationary phase cells were incubated for 15 min in fresh medium before irradiation, indicating that replenishment of exhausted nutrients restored the metabolic capability of the cells to repair DNA damage. These observations suggest that stress tolerance mechanisms in D. radiodurans R1 differ from established paradigms.     

Influence of growth environment on coaggregation between freshwater biofilm bacteria

AIM: To characterize the expression of coaggregation between Blastomonas natatoria 2.1 and Micrococcus luteus 2.13 following growth in liquid culture, on agar and in an artificial biofilm matrix composed of poloxamer hydrogel. METHODS AND RESULTS: The ability of B. natatoria 2.1 and M. luteus 2.13 to coaggregate with one another was assessed following growth in liquid culture as colonies on agar or within a poloxamer hydrogel matrix. In all these environments a cycle of gain and loss of coaggregation occurred when the two cell types were aged simultaneously, with optimum expression occurring in early stationary phase. Blastomonas natatoria 2.1 cells only coaggregated maximally after entry into stationary phase. Conversely, M. luteus 2.13 cells only coaggregated in exponential phase and early stationary phase and coaggregation ability was lost in late stationary phase. Maximal coaggregation therefore only occurred between the two strains if both were in early stationary phase, when the surface properties of the two cell types were optimal for coaggregation. CONCLUSION: In addition to occurring between cells grown in liquid culture, coaggregation between aquatic bacteria occurs after growth as a biofilm on agar and in an artificial biofilm matrix in poloxamer. Under all conditions, the B. natatoria 2.1 coaggregation adhesin and complementary receptor on M. luteus 2.13 were only expressed simultaneously during early stationary phase.     

The effect of thioredoxin on the radiosensitivity of bacteria

The ability of Escherichia coli thioredoxin to protect cells from lethal amounts of gamma radiation was tested using bacterial strains engineered to contain different amounts of thioredoxin per cell. Cells grown to late stationary phase demonstrated a decreasing sensitivity to gamma-radiation with increasing amounts of thioredoxin per cell. Exponentially growing cells were equally sensitive to the gamma-radiation regardless of the intracellular concentration of thioredoxin. Cells exhibiting the radiation-resistant phenotype in the stationary phase reverted to the radiation-sensitive phenotype when diluted into fresh growth medium. These results suggest that thioredoxin can protect cells from gamma-radiation under certain metabolic conditions.     

CELL CYCLE COMPARTMENT ANALYSIS OF CHINESE HAMSTER CELLS IN STATIONARY PHASE CULTURES

The distribution of Chinese hamster cells with respect to the compartments of the cell generation cycle was studied in cultures in the stationary phase of growth in two different media. A measure of the state of depletion of the nutrient medium was formulated by defining a quantity termed the nutritive capacity of the medium. This quantity was used to verify that the cessation of cell proliferation is due to nutrient deficiencies and not to density dependent growth inhibition. Cell cultures in stationary phase were diluted into fresh medium and as growth resumed, mitotic index, cumulative mitotic index, label index and viability were measured as a function of time. The distribution of cells with respect to compartments of the cell generation cycle in stationary phase populations was reconstructed from these data. Stationary phase populations of Chinese hamster cells that retained the capacity for renewed growth when diluted into fresh medium were found to be arrested in the G₁ and G₂ portions of the cycle; the relative proportion of these cells in G₁ increased with time in the stationary phase, but the sequence differs in the two media. In early stationary phase, in the less rich medium, more cells are in G₂ than in G₁. Also in this medium a fraction of the population was observed to be synthesizing DNA during stationary phase, but this fraction was not stimulated to renewed growth by dilution into fresh medium.     

The development of a medium supplemented with egg extract for the maintenance of Drosophila cell lines.

A saline extract was prepared from Drosophila eggs. When diluted to a concentration of 1% with Drosophila tissue culture medium, it did not support growth of cells from the Drosophila line D1 during the first few days of subculture as well as medium containing serum. When cells reached a stationary phase, however, the cell density in medium containing extract was greater than in medium containing serum. By altering the concentrations of the extract, and by adding bovine albumin, a medium was obtained in which D1 cells survived initial culturing, and which supported cell growth by day 4 as well as medium plus serum. The initial retardation of growth in medium containing egg extract might be due to the need of the cells to adapt to the new medium. At the present time four Drosophila cell lines have been maintained in this medium for more than 16 passages. Preliminary experiments with primary embryonic Drosophila cells indicate that medium containing 2% extract and bovine albumin retards the differentiation of these cells.     

Cell cycle dependence of cytotoxicity and clastogenicity induced by treatment of synchronized human diploid fibroblasts with sodium fluoride

To study the cell cycle dependence of cytotoxicity and clastogenicity of sodium fluoride (NaF), synchronized human diploid fibroblasts were treated with NaF during different phases of the cell cycle and analyzed. Exponentially growing cells were synchronized by the following two procedures. (1) The cells were synchronized at G₀/G₁ phase by a period of growth in medium containing 1% serum (low serum medium). (2) The cells were synchronized at the G₁/S boundary by growth in low serum medium, followed by hydroxyurea treatment (Tsutsui et al., 1984a). Synchronized cells were treated with NaF for 3 h during the G₁ phase or G₂ phase, and for each of three 3-h periods during the S phase which lasted 9 h. Cytotoxicity, as determined by a decrease in colony-forming ability, was dependent upon the phase of the cell cycle during which NaF treatment was administered. The highest lethality was induced in when the cultures were treated with NaF during the second or third 3 h of S phase (middle or late S phase, respectively), or G₂ phase. Little lethality was observed in cultures in G₁ phase. Inducibility of chromosome aberrations of the cells following treatment with NaF was also dependent upon the phase of the cell cycle. A significant increase in the incidence of chromosome aberrations was observed only in cultures treated with NaF during early and / or middle S phases of cell cycle. These results suggest that cytotoxicity and clastogenicity of NaF to cultured human diploid fibroblasts are cell cycle dependent, and that the cells in early and middle S phases are more sensitive to the effects.     

Evaluation of growth hormone production from GH1 cells in vitro: Effect of culture media and time in culture

Summary Growth hormone production by a rat pituitary tumor cell line (GH1) was measured during lag, exponential, and plateau phases of growth in different culture media. Growth hormone secretion was low during lag and early exponential phase; it increased late in the exponential phase and continued to increase during the plateau phase. This biphasic pattern of growth hormone production was observed in all media and sera utilized. Both the doubling time and growth hormone production were influenced by the choice of media and sera. In addition, the length of time in culture affected the growth fraction with passage level 40 GH1 cells having a 79% growth fraction, whereas the growth fraction of passage level 100 cells was 95%. Using the population doubling time as a criterion for a choice of medium, F-10 medium supplemented with 20% fetal bovine serum consistently yielded the most rapid doubling time (32 hr), whereas Dulbecco's MEM supplemented with 15% horse serum and 2.5% fetal bovine serum yielded the greatest plateau cell density. Growth hormone secretion and the population doubling times were directly related to culture conditions including length of time in culture, choice of tissue culture media, choice of sera, and the phase of cell growth (lag, exponential or plateau).     

Biosynthesis of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant Bacillus subtilis strain

The effect of certain nutrients on the growth and production of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant strain Bacillus subtilis AJ73(pCS9) was studied. Glucose was found to inhibit the synthesis of proteinase in the early (28 h of growth) but not in the late stationary phase (48 h of growth). The inhibitory effect of the other mono- and disaccharides studied was less pronounced. Casamino acids added to the medium at concentrations of 0.1-1% as an additional carbon and nitrogen source stimulated enzyme biosynthesis. Individual amino acids (cysteine, asparagine, glutamine, tryptophan, histidine, and glutamate) also stimulated enzyme biosynthesis in the early stationary phase by 25-30%, whereas other amino acids (valine, leucine, alanine, and aspartate) were ineffective or even slightly inhibitory to enzyme production. The stimulatory effect of the first group of amino acids on the synthesis of proteinase in the late stationary phase was negligible. In contrast, the bivalent ions Ca2+, Mg2+, and Mn2+ stimulated biosynthesis of proteinase in the late stationary phase (by 20-60%) and not in the early stationary phase. The data indicate that there are differences in the biosyntheses of proteinase by the recombinant B. subtilis strain during the early and late periods of the stationary phases.     

Adaptation of Mycobacterium smegmatis to Stationary Phase

Mycobacterium tuberculosis can persist for many years within host lung tissue without causing clinical disease. Little is known about the state in which the bacilli survive, although it is frequently referred to as dormancy. Some evidence suggests that cells survive in nutrient-deprived stationary phase. Therefore, we are studying stationary-phase survival of Mycobacterium smegmatis as a model for mycobacterial persistence. M. smegmatis cultures could survive 650 days of either carbon, nitrogen, or phosphorus starvation. In carbon-limited medium, cells entered stationary phase before the carbon source (glycerol) had been completely depleted and glycerol uptake from the medium continued during the early stages of stationary phase. These results suggest that the cells are able to sense when the glycerol is approaching limiting concentrations and initiate a shutdown into stationary phase, which involves the uptake of the remaining glycerol from the medium. During early stationary phase, cells underwent reductive cell division and became more resistant to osmotic and acid stress and pool mRNA stabilized. Stationary-phase cells were also more resistant to oxidative stress, but this resistance was induced during late exponential phase in a cell-density-dependent manner. Upon recovery in fresh medium, stationary-phase cultures showed an immediate increase in protein synthesis irrespective of culture age. Colony morphology variants accumulated in stationary-phase cultures. A flat colony variant was seen in 75% of all long-term-stationary-phase cultures and frequently took over the whole population. Cryo scanning electron microscopy showed that the colony organization was different in flat colony strains, flat colonies appearing less well organized than wild-type colonies. Competition experiments with an exponential-phase-adapted wild-type strain showed that the flat strain had a competitive advantage in stationary phase, as well a providing evidence that growth and cell division occur in stationary-phase cultures of M. smegmatis. These results argue against stationary-phase M. smegmatis cultures entering a quiescent state akin to dormancy but support the idea that they are a dynamic population of cells.     

Two-stage batch process for the production of ajmalicine by Catharanthus roseus: The link between growth and production stage

The link between the growth stage and the production stage in a two-stage batch process was investigated using (filtered) inocula from different periods of the stationary phase of the growth cycle. In the production stage, ajmalicine production by Catharanthus roseus in a 3-L stirred tank reactor was induced with a high glucose concentration (80 g/L). Ajmalicine production in cultures started with cells from the late stationary phase was five times higher than in cultures started with cells from the early stationary phase. After transfer to the production stage, cells from the early stationary phase showed a transient increase in respiration and enzyme induction, followed by culture browning. In contrast, cells in the late stationary phase showed a typical induction pattern: constant respiration, and permanent enzyme induction. A striking similarity between the geraniol-10-hydroxylase (G10H) activity and the ajmalicine accumulation profile could be observed in all cultures, suggesting that G 10H regulated ajmalicine production in this investigation. The intracellular nitrate concentration was significantly higher in the inoculum showing a high ajmalicine production than in the inoculum with a low production. Consequently, nitrate may act as a marker for the start of the production stage: as soon as the nitrate is depleted in the growth medium secondary metabolism can be induced. (c) 1995 John Wiley & Sons, Inc.     

Kinetics of taxol production, growth, and nutrient uptake in cell suspensions of Taxus cuspidata

Cell culture of Taxus cuspidata may represent an alternative to extraction of bark as a source of taxol and related taxanes. Cell suspensions of a cell line of T. cuspidata were grown for 44 days in shake flasks containing B5C2 medium. Throughout the growth cycle, fresh and dry weight accumulation, taxol yield on a dry weight basis, taxol accumulation in the medium, pH and pigmentation variation in the medium, as well as the uptake of sucrose, glucose, fructose, nitrate, and inorganic phosphate from the culture medium were examined. The results showed that the growth was relatively slow (doubling times of 17 and 20 days for fresh and dry weight, respectively), and taxol accumulation in the cells was non-growth related (higher in the stationary phase) and at relatively low levels (up to 4 mug/g of the extracted dry weight). Taxol concentration in the medium had two peaks: one during the early (0.4mug/mL) and another during the late (0.1-mug/mL) parts of the growth cycle. On a volumetric basis, the average total amount of taxol produced during the stationary phase (day 38) was 0.15 mug/mL, of which approximately 66% was in the medium and 34% was in the cells. Total carbohydrate uptake was closely associated with the increase in dry biomass. Sucrose was apparently extracellularly hydrolyzed after the first 6 days of culture; glucose was used before fructose. Nitrate was assimilated throughout the growth cycle, but phosphate was absorbed within the first week of culture. The pH variation showed an initial drop followed by a trend toward alkalinization for most of the growth period. Dark pigmentation in the medium increased progressively, particularly during the stationary phase. (c) 1994 John Wiley & Sons, Inc.     

A possible role of cellulose-binding protein A (CBPA) in the adhesion of Eubacterium cellulosolvens 5 to cellulose

The cellulose-binding protein A (CBPA) of Eubacterium cellulosolvens 5 is a modular enzyme comprised of a catalytic domain, a cellulose-binding domain and a cell wall-binding domain. Cellobiose-grown cells changed their adhesion ability to cellulose depending on the growth phase. On the other hand, carboxymethyl cellulose (CMC)-grown cells bound to cellulose regardless of their growth phase. The distribution of CBPA in the culture supernatant and cell fractions changed depending on the carbon source contained in the medium and growth phase. The cellobiose-grown cells harvested from the culture of the late stationary growth phase did not bind to cellulose, but their adhesion ability was recovered by treatment with recombinant CBPA. Moreover, cellobiose-grown cells harvested from the culture of an early exponential growth phase bound to cellulose, but their adhesion ability was inhibited by treatment with anti-CBPA antiserum. CBPA rapidly decreased the viscosity of CMC, indicating that CBPA was endoglucanase. The results obtained in this study indicate that CBPA plays an important role in the adhesion of E. cellulosolvens 5 cells to cellulose.     

The development of a medium supplemented with egg extract for the maintenance ofDrosophila cell lines

Summary A saline extract was prepared fromDrosophila eggs. When diluted to a concentration of 1% withDrosophila tissue culture medium, it did not support growth of cells from theDrosophila line D1 during the first few days of subculture as well as medium containing serum. When cells reached a stationary phase, however, the cell density in medium containing extract was greater than in medium containing serum. By altering the concentrations of the extract, and by adding bovine albumin, a medium was obtained in which D1 cells survived initial culturing, and which supported cell growth by day 4 as well as medium plus serum. The initial retardation of growth in medium containing egg extract might be due to the need of the cells to adapt to the new medium. At the present time fourDrosophila cell lines have been maintained in this medium for more than 16 passages. Preliminary experiments with primary embryonicDrosophila cells indicate that medium containing 2% extract and bovine albumin retards the differentiation of these cells. This work was supported by a grant from the Science Research Council of Great Britain.