Physiological and morphological characteristics of stationary phase vibrio cells able to support phase growth

Growth of phase alpha 3a on stationary phase Vibrio cultures requires micro-aerophilic conditions and is inhibited by aeration. Since pre-conditioning of the bacteria by allowing them to stand for 24 h after shaking for 3 d is an important aspect of the stationary phase phage growth system, various physiological and morphological characteristics of the stationary phase cells during the transition from shaking to standing were investigated. Shaken stationary phase cells were less viable and more sensitive to ultraviolet irradiation and heat than standing stationary phase cells. During pre-conditioning the small, non-flagellated cells present in shaken stationary phase cultures underwent morphological changes and became large, flagellated rods which resembled exponential phase cells. The transition of stationary phase cells from shaking to standing was associated with a marked increase in total RNA synthesis but a rapid and large decrease in total protein synthesis. Intracellular concentrations of ATP in shaken stationary phase cells were 53% lower than those in standing stationary phase cells. Studies on leucine uptake indicated that its transport was inhibited by isoleucine and that the major part (90%) of the total leucine uptake was due to a shared system for uptake of both amino acids. Shaken stationary phase cells transported less leucine than standing stationary phase cells. Inhibition of phage growth in aerated stationary phase cultures was not due to the prevention of phase absorption by shaking. It is suggested that the observed differences between shaken and standing stationary phase cells could be due to aeration affecting the template specificity of the Vibrio RNA polymerase.     

REGULATION OF INITIATION OF DNA SYNTHESIS IN CHINESE HAMSTER CELLS : I. Production of Stable, Reversible G1-Arrested Populations in Suspension Culture

Suspension cultures of Chinese hamster cells (line CHO) were grown to stationary phase (approximately 8–9 x 10⁵ cells/ml) in F-10 medium. Cells remained viable (95%) for at least 80 hr in stationary phase, and essentially all of the cells were in G₁ Upon resuspension or dilution with fresh medium, the cells were induced to resume traverse of the life cycle in in synchrony, and the patterns of DNA synthesis and division were similar to those observed in cultures prepared by mitotic selection. Immediately after dilution, the rates of synthesis of RNA and protein increased threefold. This system provides a simple technique for production of large quantities of highly synchronized cells and may ultimately provide information on the biochemical mechanisms regulating cell-cycle traverse.     

ROLE OF THE LIGHT-DARK CYCLE AND MEDIUM COMPOSITION ON THE PRODUCTION OF COCCOLITHS BY EMILIANIA HUXLEYI (HAPTOPHYCEAE)1

Production of coccoliths by cells of Emiliania huxleyi (Lohmann) Hay and Mohler was measured during exposure of the cells to two diel light-dark cycles (16:8 h). During the light period about eight coccoliths per cell were formed at a constant rate of one coccolith per 2 h. Cells divided during the first half of the dark period. No coccolith production took place during the dark period. With electron microscopy we found early-stage, coccolith-production compartments in cells after mitosis while still in the dark. No calcification was observed in these compartments. Cells grown on enriched seawater (Eppley's medium) tended to produce enough coccoliths to cover the cell in a single layer. When these cells reached the stationary phase coccolith production stopped. Coccolith production was induced by removal of extracellular coccoliths. Cells grown on medium containing 2% of the nitrate and phosphate of Eppley's medium tended to produce coccoliths in the stationary phase. This resulted in the formation of multiple layers of coccoliths. The multiple covering was restored after decalcification of stationary cells. Formation of multiple layers of coccoliths may help the cells reach deeper, nutrient-rich water by increasing the sinking rate of the cells.     

EFFECTS OF HARVEST STAGE AND LIGHT ON THE BIOCHEMICAL COMPOSITION OF THE DIATOM THALASSIOSIRA PSEUDONANA1

The marine diatom Thalassiosira pseudonana (Hustedt, clone 3H) Hasle and Heimdal was cultured under three different light regimes: 100 μmol photon · m^?2· s^?1 on 12:12 h light : dark (L:D) cycles; 50 μmol photon · m^?2· s^?2 on 24:0 h L:D; and 100 μmol photon · m^?2· s^?1 on 24:0 h L:D. It was harvested during logarithmic and stationary phases for analysis of biochemical composition. Across the different light regimes, protein (as % of organic weight) was highest in cells during logarithmic phase, whereas carbohydrate and lipid were highest during stationary phase. Carbohydrate concentrations were most affected by the different light regimes; cells grown under 12:12 h L:D contained 37–44% of the carbohydrate of cells grown under 24:0 h L:D. Cells in logarithmic phase had high proportions of polar lipids (79 to 89% of total lipid) and low triacylglycerol (≤10% of total lipid). Cells in stationary phase contained less polar lipid (48 to 57% of total lipid) and more triacylglycerol (22 to 45% of total lipid). The fatty acid composition of logarithmic phase cells grown under 24:0 h L:D were similar, but the 100 μmol photon · m^?2· s^?1 (12:12 h L:D) cells at the same stage contained a higher proportion of polyunsaturated fatty acids (PUFAs) and a lower proportion of saturated and monounsaturated fatty acids due to different levels of 16:0, 16:1(n-7), 16:4(n-1), 18:4(n-3), and 20:5(n-3). With the onset of stationary phase, cells grown at 100 μmol photon · m^?2· s^?1 (both 12:12 and 24:0 h L:D) increased in proportions of saturated and monounsaturated fatty adds and decreased in PUFAs. Concentrations (% organic or dry weight) of 14:0, 16:0, 16:1(n-7), 20:5(n-3), and 22:6(n-3) increased in cells of all cultures during stationary phase. The amino acid compositions of cells were similar irrespective of harvest stage and light regime. For mariculture, the recommended light regime for culturing T. pseudonana will depend on the nutritional requirements of the animal to which the alga is fed. For rapidly growing bivalve mollusc larvae, stationary-phase cultures grown under a 24:0 h L:D regime may provide more energy by virtue of their higher percentage of carbohydrate and high proportions and concentrations of energy-rich saturated fatty acids.     

Protein turnover in exponential and stationary phase Vibrio cells

Vibrio strain 14 supports phage alpha 3a growth in standing stationary phase cells but not in shaking (aerated) stationary phase cells. In exponential cells, protein was turned over at 1.8% h-1, and the rate was increased by starvation or inhibition of protein synthesis. In shaking stationary phase cells the rate of protein turnover was low (1.0% h-1) for proteins synthesised during growth but high (20% h-1) for recently synthesised proteins. In contrast recently synthesised proteins in standing stationary phase cells were stable over 60 min and proteins synthesised during growth were turned over at 2.9% h-1. ppGpp and pppGpp were detected in exponential cells, but were not detected in stationary phase cells.     

Growth-phase-dependent immunodeterminants of Rhizobium trifolii lipopolysaccharide which bind trifoliin A, a white clover lectin.

The lipopolysaccharide (LPS) from Rhizobium trifolii 0403 was isolated at different stages of growth and was examined for its (i) ability to bind a white clover lectin (trifoliin A), (ii) immunochemical properties, and (iii) composition. There was significantly more binding of trifoliin A to purified LPS and cells in the early stationary phase than to cells in the exponential phase. Immunofluorescence and enzyme-linked immunosorbent assays indicated that new antigenic determinants of the LPS appeared for brief periods on cells at the end of the lag phase and again at the beginning of the stationary phase. These new antigens were not detected on cells in midexponential or late stationary phase. Monovalent fragments of immunoglobulin G antibodies raised against the unique antigenic determinants in the LPS competitively blocked the binding of trifoliin A to cells in the early stationary phase. Gas chromatographic analysis showed that the relative quantity of several glycosyl components in the LPS increased as the culture advanced from the midexponential to the early stationary phase. In addition, LPS from cells in the early stationary phase had a higher aggregate molecular weight. Quinovosamine (2-amino-2,6-dideoxyglucose) was identified by combined gas chromatography-mass spectrometry as a sugar component of the LPS which had not been previously reported. D-Quinovosamine, N-acetyl-D-quinovosamine, and its n-propyl-beta-glycoside were effective hapten sugars which inhibited the binding of trifoliin A, anti-clover root antibody, and homologous antibody to these new determinants in the LPS. White clover plants had more infected root hairs after incubation with an inoculum of cells in the early stationary phase than after incubation with cells in the midexponential phase. The profound influence of the growth phase on the composition of lectin-binding polysaccharides of Rhizobium may be a major underlying cause of conflicting data among laboratories testing the lectin-recognition hypothesis. In addition, these chemical modifications may reflect mechanisms which regulate Rhizobium-root hair recognition in this nitrogen-fixing symbiosis.     

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.     

Relation between the damaging effect of surface-active compounds on Escherichia coli cells and the phase of culture growth

The techniques of cell electrophoresis and electro-orientation spectroscopy were used to study the effect of sodium dodecyl sulfate (SDS) and cetyl trimethyl ammonium bromide (CTAB) on Escherichia coli K-12 cells from the culture at the exponential and stationary growth phases. SDS (2 x 10(-4) M) considerably damaged cells at the exponential phase, particularly at pH less than 6.0, whereas cells at the stationary phase were damaged to a less degree and only at pH less than 5.3 or after their treatment with Trilon B. The damaging effect of SDS decreased in an isotonic medium (0.25 M sucrose) as compared to a hypotonic medium (distilled water). CTAB also damaged cells at the exponential phase more than those at the stationary phase, and its damaging action decreased with pH. Mg2+, Ca2+, and Sr2+ cations diminished the degree of cell damage with CTAB, but did not exert any noticeable protection in the case of SDS. The different sensitivity of cells at the exponential and stationary growth phases may be associated with changes in their surface electric charge and with the existence of hydrophobic regions on the cell surface. The higher electric charge of cells at the stationary growth phase is presumed to stem from a rise in the amount of surface lipopolysaccharides which bear a negative electric charge.     

DOPAMINERGIC PROPERTIES OF A SOMATIC CELL HYBRID LINE OF MOUSE NEUROBLASTOMA X SYMPATHETIC GANGLION CELLS

Abstract— Studies were made on the regulation of dopamine metabolism in a cell line derived by hydridization of a non-tyrosine-hydroxylase-containing line of murine neuroblastoma cells with a neur-onally-enriched population of murine embryonic sympathetic ganglion cells. Hybrid subclones with tyrosine hydroxylase activity were selected by exposure to tyrosine-free medium. The cells also exhibited DOPA decarboxylase activity and the subclone (named T28) with the highest specific activities of both enzymes was further characterized. The hybrid T28 line did not contain dopamine-β-hydroxylase activity. The specific activity of tyrosine hydroxylase as well as of DOPA decarboxylase increased significantly in T28 cultures when the cells entered the stationary phase of growth. Both of these enzymes were also induced after several days of exposure to 1 m m -dibutyryl cyclic AMP in culture medium containing either 5% or 0.8% serum. However, maintenance in medium containing 0.8% serum alone, which inhibited cell multiplication, did not induce either enzyme. The dopamine content of T28 cells was also regulated as a function of cell density. High density (stationary phase) cultures of T28 cells contained about 300 pmol dopamine per mg protein and at least half of this endogenous amine appeared to he stored in vesicles or granules (as judged by depletion with reserpine or α-methyl- m -tyramine). The T28 and other neuronal hybrid lines appear to be useful model systems for neuro-chemical studies.     

HearNPV在生长对数期与平台期宿主细胞中的复制差异分析

使用实时荧光定量PCR技术对HearNPV在生长对数期和平台期HzAM1细胞的复制差异进行分析。结果表明,HzAM1细胞生长对数期的倍增时间为22 h,生长对数期的细胞以S期细胞为主(48.6%),而平台期细胞中以G2/M期细胞为主(72.6%)。在这两种不同状态的细胞中,病毒的复制主要在感染后60 h内完成,在感染后14~20 h,病毒复制倍增时间分别为1.8 h和1.9 h,几乎没有差别。但是感染生长对数期细胞时,吸附侵入细胞内的BV数量、BV释放的数量、最终的病毒产量以及病毒表达的蛋白产量明显高于被病毒感染的生长平台期细胞。如生长对数期细胞内复制合成的病毒DNA总量的25%装配形成BV病毒粒子出芽释放到细胞外,而对于平台期细胞,病毒DNA仅有13%装配形成BV病毒粒子出芽释放到细胞外。病毒感染两种生长状态的细胞,病毒DNA均从感染后7~8 h开始复制,没有明显差别;而生长对数期细胞从被感染后18~20 h释放子代病毒BV,生长平台期细胞则在感染后22~25 h开始释放病毒BV。在感染后30~60 h,在生长对数期被感染的细胞释放BV的速度约为483 copies/cell/h,而平台期细胞约为100 copies/cell/h。最初吸附侵入到生长对数期细胞内的BV粒子数量明显多于侵入到生长平台期细胞内的BV数量。实验证实,生长对数期与平台期的细胞膜的流动性有很大差别,推测健康细胞表面有活性的病毒受体数量可能决定了侵入细胞内的BV的数量。     

THE CORRELATION OF GOLGI ACTIVITY AND POLYSACCHARIDE SECRETION IN PORPHYRIDIUM12

Quantitative morphometric measurements were made of the area occupied by the Golgi complex in log and stationary phase cells. The technique involved the cutting out and weighing of cellular compartments from electron micrographs of median cell sections. Data from the morphometric measurements combined with physiological measurements of secretory activity yielded a simple model for the involvement of the Golgi complex in the biogenesis of cell surface polysaccharides. Golgi were larger and more numerous in log phase, perhaps indicating a higher rate of polysaccharide synthesis than in stationary phase. In log phase, polysaccharide-containing vesicles accumulate adjacent to the cell membrane. In stationary phase, the polysaccharide is secreted to the cell surface by exocytosis, giving rise to a capsule. Thus the shift from log to stationary phase results in structural and functional cellular differentiation, eg, from a thin to a thick capsule. The ecological implications of capsule formation are discussed.     

Melanin content and hydroperoxide metabolism in human melanoma cells

Human melanoma cells were grown to exponential and stationary phases showing melanin contents of 4.2 +/- 0.3 and 11.3 +/- 0.6 micrograms/10(6) cells, respectively. The cells were separated in four subpopulations by a Percoll gradient; the subpopulation of density 1.07 (g/ml) was the most enriched in pigmented cells and produced 28 and 58% of the cells in exponential and stationary phases, respectively. Melanoma cells had similar superoxide dismutase and glutathione peroxidase activities in exponential and stationary phases. Moreover melanoma cells exhibited a higher catalase activity in the stationary phase: whole homogenate and cytosol activities were 7.0 +/- 0.3 and 10.8 +/- 0.6 U/mg protein, whereas in exponential phase the activities were 4.9 +/- 0.1 and 7.6 +/- 0.3 U/mg protein for whole homogenate and cytosol, respectively. The intracellular H2O2 steady-state concentration was 3.3 +/- 0.2 and 2.1 +/- 0.2 microM H2O2 for exponential and stationary phases, respectively. The spontaneous chemiluminescence of the two culture phases was 169 +/- 27 cps/10(6) cells (exponential) and 78 +/- 24 cps/10(6) cells (stationary). The cytotoxicity of H2O2 generated extracellularly by glucose oxidase was determined after 60 min of exposure. IC50 values for exponential and stationary cell cultures were 0.9 and 2.4 mU/ml of glucose oxidase, respectively. The increased catalase activities in the stationary phase as compared with the exponential phase are consistent with the decreased intracellular H2O2, with the decreased spontaneous chemiluminescence, and with the increased resistance to exogenous H2O2.     

Carbon dynamics of logarithmetic and stationary phase phytoplankton as determined by track autoradiography

Track autoradiographic analysis of photosynthetic radiocarbon incorporation at the cellular level indicated that the carbon uptake rate and carbon pool size of exponentially growing (log phase) Scenedesmus cells was threefold that of stationary phase cells, while carbon turnover rates were similar. Carbon fixation was uncoupled from growth and cell division in the stationary phase cells, which were larger and contained less chlorophyll per unit volume than log phase cells. Changes in the temporal pattern of isotope incorporation were evident at the cell level prior to the cessation of division and transition to stationary phase, while bulk carbon fixation responded only the second day after cell division ceased. The carbon uptake patterns of a marine nanoplankter from a nutrient-enriched natural sample resembled that of log phase cells while the control population pattern resembled that of stationary cells. The physical, biochemical, and metabolic differences between log and stationary phase cells are potentially measurable by flow cytometry procedures currently in use and under development. The use of flow cytometry to sort cell types for analysis by track autoradiography and subsequent correlation of metabolic characteristics with flow cytometry signatures is a feasible means of investigating the heterogeneity of phytoplankton metabolic state in the marine environment.     

Regulation of maltose metabolism in stationary phase cultures of an asporogenous mutant of Bacillus licheniformis

An asporogenous strain of Bacillus licheniformis accumulated maltose by an energy dependent transport mechanism during an extended stationary phase. Maltose transport was sensitive to the effects of the uncoupler tetrachlorosalicylanide (TCS), and was also inhibited by glucose. Maltose stimulated synthesis of a p -nitrophenyl-α- D -glucoside-hydrolysing enzyme ( p NPGase) in log phase and in stationary phase cells. In the presence of glucose this induction was inhibited. Glucose was used preferentially to maltose in stationary phase cells. The uptake of maltose from the medium, and the synthesis of p NPGase, were immediately and completely inhibited in the presence of glucose. These results are consistent with a mechanism of inducer exclusion mediating the repressive effect of glucose upon p NPGase synthesis in stationary phase cells. Catabolite repression of α-amylase synthesis by glucose was also demonstrated in late stationary phase mutant cells.     

Cultured human tumour cells may be arrested in all stages of the cycle during stationary phase: demonstration of quiescent cells in G1, S and G2 phase

Six human colon carcinoma cell lines were induced to enter stationary phase of growth by nutrient deprivation and cell crowding. Growth kinetics parameters (cell number, flow cytometric analysis of DNA distribution, and labelling and mitotic indices) were measured sequentially for all lines during the various stages of in vitro growth. Our results demonstrated that a substantial fraction of cells (9-18%) were located in G2 phase when they changed from an exponential to a stationary mode of growth. Moreover, a large number of cells in stationary phase of growth had an S-phase DNA content, as determined by flow cytometry, but failed to incorporate radioactive DNA precursors (up to 15-fold difference). To substantiate these findings, cells in stationary phase of growth were induced to enter exponential growth by re-seeding in fresh medium at a lower density. Subsequently observed changes in DNA-compartment distribution, and in labelling and mitotic indices were those expected from cells that had been arrested at different stages of the cycle during their previous stationary phase. Thus, the non-proliferating quiescent state (Q), traditionally located 'somewhere' in G1 phase, appears to be composed also of cells that can be arrested at other stages of the cycle (Qs and QG2). Although the proportion of such cells is rather small, their contribution to the growth kinetics behaviour of human in vivo tumours will become apparent following 'recruiting' or 'synchronizing' clinical manoeuvres and will prevent the formation of a clear-cut wave of synchronized cells.     

Mutagenicity of methyl methanesulfonate and cyclophosphamide in resting and growing Saccharomyces cerevisiae D7 cells.

In order to evaluate the optimal experimental conditions and to identify the best growth phase for yeast genotoxicity studies, comparative experiments were performed with stationary and growing cells. Methyl methanesulfonate (MMS) and cyclophosphamide (CP) were used as chemical mutagens and strain D7 of Saccharomyces cerevisiae as detector of induced mitotic gene conversion (trp+ convertants) and point reverse mutation (ilv+ revertants) in log or stationary phase cells after either 4 or 16 h of treatment. The highest MMS-induced toxicity and genotoxicity were observed after 16 h of exposure in a suspension test with log phase cells, which is consistent with the greater permeability and sensitivity of growing yeast cells. The maximal induction of genetic effects and toxicity by CP was conversely obtained after 16 h of treatment in stationary phase cells. This may be ascribed to the greater ability of detoxication of growing cells as compared to resting cells. Our results suggest that in evaluating the mutagenicity of chemicals in yeast systems it is important to consider factors such as growth phase and exposure time.     

STATIONARY PHASE OF CULTURED MAMMALIAN CELLS (L5178Y)

The stationary phase of the mammalian cells L5178Y in culture can be divided into two stages: (a) an early phase characterized by the decline of mitotic index, followed by a stabilization of the cell number, and (b) a late stage, occurring several hours after the flattening of the growth curve, during which dead or dying cells appear in the cultures. The estimates of rates of cell progress showed that the rates from G₁ to S and from G₂ to M were affected in the early stationary phase. The main cause of cessation of increase in cell number in the early stationary phase is resulted from the decline in mitotic index, which is caused by prolongation of the G₂ period. The importance of the G₂ stage in regulating the cell growth is discussed in relation to other known situations in the literature.     

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.     

NUCLEOLAR AND BIOCHEMICAL CHANGES DURING UNBALANCED GROWTH OF TETRAHYMENA PYRIFORMIS

Numerous nucleoli can be observed in the macronucleus of the logarithmically growing ciliated protozoan Tetrahymena pyriformis; at late log phase the nucleoli aggregate and fuse. In stationary phase this fusion process continues, leaving a very few large vacuolated nuclear fusion bodies in the nucleus. When these stationary phase cells are placed into fresh enriched proteose peptone medium, the large fusion bodies begin to disaggregate during the 2.5-hour lag phase before cell division is initiated. By 3 to 6 hours after inoculation the appearance of the nucleoli in many cells returns to what it was in logarithmic cells. In view of the possible role of nucleoli in ribosome synthesis, attempts were made to correlate the morphological changes to changes in RNA and protein metabolism. The beginning of an increased RNA synthesis was concomitant with the beginning of disaggregation of the large fusion bodies into nucleoli, which was noticed in some cells by 1 hour after the return to fresh enriched proteose peptone medium. Increased protein synthesis then followed the increased RNA synthesis by 1 hour. The supply of RNA precursors (essential pyrimidines) were removed from cultures which were grown on a chemically defined synthetic medium, in order to study the relation between nucleolar fusion and synthesis of RNA and protein. Pyrimidine deprivation drastically curtailed RNA and protein synthesis, but did not cause fusion of nucleoli. When pyrimidines were added back to this culture medium, RNA synthesis was immediately stimulated and again preceded an increased protein synthesis by 1 hour. These studies suggest the involvement of unfused nucleoli in RNA and protein synthesis and demonstrate the extreme plasticity of nucleoli with respect to changes in their environment.