Calculation of Steel's cell loss factor and of the parameters of cellular kinetics for a population with an exponential growth of the cell number and cell death at G0 phase with a probability equal to 1

The method of calculation of three cell kinetics parameters (the Steel's cell loss factor phi, the proliferative pool Pc, and the mean number m of the proliferating cells after mitotic division of one cell) was shown to be the same for the exponential growth state of cell number with cell death at the G0-phase, and for the exponential growth state with cell death occurring immediately after mitosis. The value of the mean number delta of non-proliferating cells that appeared after mitotic division of one cell is different for these two models of the exponential growth state with the equal values of the other three parameters (phi, Pc, and m). A method is proposed for calculating the parameter delta on the data of the percentage of labeled cells obtained in the experiments with continuous cultivation of cells in the nutrient medium containing 3H-thymidine. The kinetics of cell line HL-60 (the experimental data of Foa et al., 1982) can be described at the first approximation, by a model of the exponential growth state with the cell death at the G0-phase, with Pc = 0.80, phi = 0.24, m = 1.61, delta = 0.39, and the life time of the non-proliferating cells tQ = 24 hours.     

Viability and proliferative activity of a Chinese hamster cell culture exposed to nitrosoureas in exponential and stationary growth phases

The lethal effect of antitumor nitrosourea chloroethyl derivatives on proliferating (exponential phase of growth) and non-proliferating (stationary phase of growth) cells is observed at a concentration 5-fold less than that of methyl derivatives revealed by the colony-formation technique. 1,3-bis(2-chlororoethyl)-1-nitrosourea is equally effective towards proliferating and non-proliferating cells, but chlorozotocin exerts a primary cytotoxic effect on proliferating cells. 1-methyl-1-nitrosourea at low concentration causes death more readily of proliferating cells than non-proliferating ones. However, studies on proliferative activity during the first hours after treatment with 1-methyl-1-nitrosourea revealed drug sensitivity in cells being at the early stationary phase of growth.     

KINETIC ANALYSIS OF CELL SIZE AND DNA CONTENT DISTRIBUTIONS DURING TUMOR CELL PROLIFERATION: EHRLICH ASCITES TUMOR STUDY

In order to study the growth dynamics of proliferating and non-proliferating cells utilizing discrete-time state equations, the cell cycle was divided into a finite number of age compartments. In analysing tumor growth, the kinetic parameters associated with a retardation in the growth rate of tumors were characterized by computer simulation in which the simulated results of the growth curve, the growth fraction, and the mean generation time were adjusted to fit the experimental data. The cell age distribution during the period of growth was obtained and by a linear transformation of the state transition matrices, was employed to specify the cell size and DNA content distributions. In an application of the model, the time-course behavior of cell cycle parameters of Ehrlich ascites tumor is illustrated, and the parameters important for the transition of cells in the proliferating compartment to the non-proliferating compartment are discussed, particularly in relation to the G₁-G₀ and G₂-G₀ transitions of non-cycling cells as revealed by the variation of cell size distribution.     

Kinetic analysis of cell size and DNA content distributions during tumor cell proliferation: Ehrlich ascites tumor study.

In order to study the growth dynamics of proliferating and non-proliferating cells utilizing discrete-time state equations, the cell cycle was divided into a finite number of age compartments. In analysing tumor growth, the kinetic parameters associated with a retardation in the growth rate of tumors were characterized by computer simulation in which the simulated results of the growth curve, the growth fraction, and the mean generation time were adjusted to fit the experimental data. The cell age distibution during the period of growth was obtained and by a linear transformation of the state transition matrices, was employed to specify the cell size and DNA content distributions. In an application of the model, the time-course behavior of cell cycle parameters of Ehrlich ascites tumor is illustrated, and the parameters important for the transition of cells in the proliferating compartment to the non-proliferating compartment are discussed, particularly in relation to the G1-G0 and G2-G0 transitions of non-cycling cells as revealed by the variation of cell size distribution.     

Heat Shock–Induced Changes in the Respiration of the Yeast Saccharomyces cerevisiae

The incubation of Saccharomyces cerevisiaeat elevated temperature (45°C) stimulated the respiration of yeast cells and decreased their survival rate. The respiration-deficient mutant of this yeast was found to be more tolerant to the elevated temperature than the wild-type strain. At the same time, the cultivation of the wild-type strain in an ethanol-containing medium enhanced the respiration, catalase activity, and thermotolerance of yeast cells, as compared with their growth in a glucose-containing medium. It is suggested that the enhanced respiration of yeast cells at 45°C leads to an intense accumulation of reactive oxygen species, which may be one of the reasons for the heat shock–induced cell death.     

Temperature and nutrient availability control growth rate and fatty acid composition of facultatively psychrophilic <Emphasis Type="Italic">Cobetia marina</Emphasis> strain L-2

A facultative psychrophilic bacterium, strain L-2, that grows at 0 and 5°C as minimum growth temperatures in complex and defined media, respectively, was isolated. On the basis of taxonomic studies, strain L-2 was identified as Cobetia marina. The adaptability of strain L-2 to cold temperature was higher than that of the type strain and of other reported strains of the same species. When the bacterium was grown at 5–15°C in a defined medium, it produced a high amount of trans-unsaturated fatty acids. By contrast, in a complex medium in the same temperature range it produced a low amount of trans-unsaturated fatty acids. In the complex medium at 5°C, the bacterium exhibited a three-fold higher growth rate than that obtained in the defined medium. Following a temperature shift from 11 to 5°C, strain L-2 grew better in complex than in defined medium. Furthermore, when the growth temperature was shifted from 0 to 5°C both the growth rate and the yield of strain L-2 growing in complex medium was markedly enhanced. These phenomena suggest that an upshift of the growth temperature had a positive effect on metabolism. The effects of adding complex medium components to the defined medium on bacterial growth rate and fatty acid composition at 5°C were also studied. The addition of yeast extract followed by peptone was effective in promoting rapid growth, while glutamate addition was less effective, resulting in a cis-unsaturated fatty acid ratio similar to that of cells grown in the complex medium. These results suggest that the rapid growth of strain L-2 at low temperatures requires a high content of various amino acids rather than the presence of a high ratio of cis-unsaturated fatty acids in the cell membrane.     

Effect of glycine betaine on the sucrose catabolism of an l-lysine producing mutant of Brevibacterium lactofermentum

Summary Effect of exogenous betaine on the growth of an l-lysine-producing mutant of Brevibacterium lactofermentum was examined in a medium containing different carbon sources such as glucose, fructose, or sucrose. The growth rate decreased significantly with a rise in temperature when sucrose was the carbon source. Both the specific sucrose consumption rate and the invertase activity of the mutant decreased with the culture period when the cultivation temperature was 35°C. The addition of betaine restored both growth and invertase activity on medium containing sucrose as the carbon source at 35°C. Betaine protected the invertase activity against the inactivating effects of high temperature in vitro. Furthermore, the addition of exogenous invertase into production medium at 35°C restored the growth rate to that at 32°C. These results indicated that growth decreased on medium containing sucrose at 35°C due to a decrease in invertase activity, and that addition of betaine was an effective way to enhance growth on this medium at a higher temperature. Offprint requests to: Y. Kawahara     

Study of physicochemical factors limiting the growth ofKluyveromyces marxianus

Summary The effects of various physicochemical parameters on the growth of twoKluyveromyces marxianus strains were investigated, including: pH values, sodium chloride, water activity in the medium and temperature. Both yeast strains were unaffected by pH changes. Optimal pH for growth was found to be 4 with both strains, but they were able to develop within the pH 3–8 range. Suitable growth was obtained at temperatures of 4–44°C and the optimal temperature for growth was 36°C for both strains. Modelling of this latter parameter is described. Growth of both microorganisms was considerably modified by increased NaCl or decreased water activity in the medium.     

Inactivation of Candida albicans by ultraviolet radiation

Summary Inactivation of Candida albicans by ultraviolet (uv) light is markedly dependent upon (a) the cell division stage and (b) the nutrition and growth temperatures of cells both before and after irradiation. Cells grown at 37°C after irradiation show lower survivals than those grown at 25°C. At either recovery temperature, cells which had been cultured before irradiation at 37°C are able to sustain less uv damage prior to inactivation than those cultured at 25°C. The radiosensitivities of budding and non-budding cells are the same when survivals are scored at 25°C; at low uv dosages, cells show slightly poorer recoveries on enriched medium than on minimal medium whereas at higher dosages, their recoveries on both kinds of media are equivalent. In contrast, at 37°C, uv treated non-budding cells are much more susceptible to inactivation than budding cells; non-budding cells also express much poorer recovery on enriched medium than on minimal medium at 37°C whereas budding cells survive equally well on either medium. Though non-budding cells grown for irradiation on minimal or enriched media exhibit the same radiosensitivites, budding cells grown for irradiation on enriched medium are more susceptible to inactivation at 37°C than those grown on minimal medium.The particularly poor recovery by irradiated non-budding cells at 37°C is correlated with their unique tendency to undergo a transitory filamentation when initiating growth at that temperature. Evidence is presented that neither the filamentous growth per se nor the temporary inhibition of cell division associated with filamentation causes the poor recovery. Furthermore, while irradiated non-budding cells at 37°C exhibit singular susceptibility to inhibition of recovery by metabolic antagonists which disturb protein synthesis, the course of their filamentous growth is not affected by such agents. It is concluded that recovery from irradiation and the instigation of cytokinesis by non-budding cells of C. albicans result from different metabolic processes which may be related through a common temperature sensitive step. C. albicans does not photoreactivate and observations on recovery by cells prevented from undergoing immediate postirradiation replication do not indicate the existence of a system for dark repair of DNA damage comparable to that occurring in bacteria. Difficulties attending a valid demonstration of DNA dark repair in yeasts are discussed.     

Low-temperature effects on cyanobacterial membranes

The effect of change in ambient temperature on fatty acid unsaturation has been studied in the cyanobacteriumAnabaena variabilis. When cells isothermally grown at 22°C are compared with those grown at 38°C, the relative content of oleic acid decreases and that of linolenic acid increases in all of the lipid classes. After a temperature shift from 38 to 22°C, palmitic acid is rapidly desaturated in monogalactocyldiacylglycerol, but in no other lipids, and oleic acid is slowly desaturated in most lipid classes. When cells ofAnacystis nidulans are exposed to low temperature such as 0°C, they lose physiological activities and finally die. This low-temperature damage is initiated by the phase transition of lipids in the plasma membrane. The phase transition of thylakoid membrane that occurs at intermediate temperature produces loss of activity related to photosynthesis. This is, however, recovered when the cells are rewarmed to growth temperature. A model for the mechanism of the low-temperature damage in the cyanobacterial cells is proposed.     

Geobacillus uralicus,a New Species of Thermophilic Bacteria

The K^T ₂ strain of thermophilic spore-forming bacteria was isolated from a biofilm on the surface of a corroded pipeline in an extremely deep well (4680 m, 40–72°C) in the Urals. The cells are rod-shaped, motile, gram-variable. They grow on a complex medium with tryptone and yeast extract and on a synthetic medium with glucose and mineral salts without additional growth factors. The cells use a wide range of organic substances as carbon and energy sources. They exhibit a respiratory metabolism but are also capable of anaerobic growth on a nitrate-containing medium. Growth occurs within the 40–75°C temperature range (with an optimum of 65°C) and at pH 5–9. The minimum generation time (15 min) was observed at pH 7.5. Ammonium salts, nitrates, and arginine are used as nitrogen sources. The G+C content of the DNA is 54.5 mol %. From the morphological, physiological, and biochemical properties and the nucleotide sequence of the 16S rRNA gene, it was concluded that the isolate K^T ₂ represents a new species of the genus Geobacillus, Geobacillus uralicus.     

Effect of growth rate on ethanol tolerance ofSaccharomyces cerevisiae

Δ^5,7 Saccharomyces cerevisiae cells growing in chemostat at a specific growth rate of 0.075/h exhibited higher ethanol tolerance measured as ethanol-induced death and anaerobic growth inhibition than the cells growing at 0.2/h, the difference being dependent on the carbon-to-nitrogen molar proportion in the medium. The observed difference in sensitivity to ethanol of anaerobic growth between the slowly and rapidly-growing cells was completely reversed as a result of a block in sterol synthesis causing a negligible synthesis of Δ^5,7. Two physiological parameters, budding frequency and membrane composition, evidently affected ethanol tolerance. Differences between the Δ^5,7 and deficient strains documented a profound effect of the quality of the sterol present on the physiological state of the cell.     

Lipid metabolism in large T47D human breast cancer spheroids: 31P- and 13C-NMR studies of choline and ethanolamine uptake

³¹P- and ¹³C-NMR were used to determine the kinetics of choline and ethanolamine incorporation in T47D clone 11 human breast cancer cells grown as large (300 μm) spheroids. Spheroids were perfused inside the spectrometer with 1,2-¹³C-labelled choline or ethanolamine (0.028 mM) and the buildup of labeled phosphorylcholine (PC) or phosphorylethanolamine (PE) was monitored. To analyze the NMR kinetic data, it was assumed that each signal represents a weighted average of signal from the proliferating and non-proliferating compartments of the large spheroid. The average ATP pool size was 4±1 fmol/cell compared to 8±1 fmol/cell in small (150 μm) proliferating spheroids (P < 0.0002). The average PC pool size at steady state was reduced to 11±6 fmol/cell compared to 22±8 (P < 0.007). This could be correlated with an overall reduction of choline uptake in the non-proliferating spheroid fraction. The rate of the enzyme choline kinase was 0.3 fmol/(cell h) compared to 1.0 fmol/(cell h) (P < 0.0001) for proliferating cells. The rate constant of CTP: phosphocholine cytidyltransferase (0.05 h^?1) was not significantly altered, but the rate of the enzyme was reduced from 1.3 to 0.2–0.5 fmol/(cell h). The pool size of PE in medium containing serum ethanolamine (1.7 μM) was approximately the same (15 fmol/cell) in small and large spheroids. In the presence of high ethanolamine (0.028 mM) the average PE level decreased slightly (11 fmol/cell) and the rate of the enzyme ethanolamine kinase in the non-proliferating fraction was 0.7 fmol/(cell h) versus 1.0 fmol/(cell h) in the proliferating cells (P < 0.07). The rate constant of CTP: phosphoethanolamine cytidyltransferase (0.07 h^?) was not significantly altered but the corresponding reaction rate was reduced from 1.4 to 0.2–0.8 fmol/(cell h). The kinetics of choline incorporation did not alter in the presence of 0.028 mM ethanolamine.     

Studies on the growth and encystment ofPolytomella agilis

Summary Changes in cell population density, cell protein and cell carbohydrate levels of the flagellatePolytomella agilis during growth in batch cultures on a complex medium at 25° C, 18° C and 9° C were examined. At 25° C, cell protein and carbohydrate levels fell markedly during exponential population growth. At 18° C, cell protein values remained fairly constant, while cell carbohydrate increased. During growth at 9° C, both cell protein and carbohydrate increased. Changes in these cellular parameters were related to marked differences in the rates of population growth at these temperatures. The cellular locus of changes observed at 25° C was examined by centrifugal fractionation of disrupted cells on density gradients.Encystment was studied in cultures grown at 25° C. The maximum number of cysts produced was 5–10 per cent of the peak population density; the rate and degree of encystment were not increased by growth on conditioned medium or starvation. Cysts isolated from mixed populations by a centrifugal procedure remained viable, even after storage at —5° C for several weeks. The soluble proteins of separately disrupted motile and encysted forms ofP. agilis were examined and compared by disc electrophoresis.This investigation was supported by U.S.P.H.S. Grant AI-07926-02.     

Effect of in vitro storage at 4 °C on survival and proliferation of poplar shoots

Shoot explants of in vitro proliferating cultures of Populus tremula (L.) x Populus tremuloides (L.) were stored for three months at 4°C, in dark or light, in basal culture medium with or without 2-isopentenyladenine (2iP), and in rooting medium with naphthalene acetic acid. They were transferred to cold at different times after subculturing. One hundred percent of shoots survived all tested conditions, in spite of leaf browing and necrosis. After transfer to 24°C for 2 weeks and a normal multiplication cycle, the shoots proliferated at a rate similar to controls or at a higher rate in the case of shoots introduced into the cold 7 or 14 days after subculture and stored in dark on medium containing 2iP.Abbreviations 2iP 2-isopentenyladenine - NAA naphthaleneacetic acid - MS Murashige & Skoog (1962) medium     

Ambient temperature: a factor affecting performance and physiological response of broiler chickens

An experiment was conducted to elucidate the influence of four constant ambient temperatures (20°, 25°, 30° and 35°C) on the performance and physiological reactions of male commercial broiler chicks from 3 to 7 weeks of age. A 12 h light-dark cycle was operated, while relative humidity and air circulation were not controlled. Exposure of broiler chickens to the 20°, 25°, 30° and 35°C treatments showed highly significant (P<0.0001) depression in growth rate, food intake and efficiency of food utilization, and a significant increase in water consumption for the 30° and 35°C groups. Mortality was, however, not affected by the temperature treatments. Changes in physiological status, such as increased rectal temperatures, decreased concentration of red blood cells, haemoglobin, haematocrit, and total plasma protein were observed in birds housed in the higher temperature (30° and 35°C) environments. Moreover, in these broiler chickens, there was an increased blood glucose concentration and a decreased thyroid gland weight. These results indicate that continuous exposure of broiler chickens to high ambient temperatures markedly affects their performance and physiological response.     

A unique minute-rough colonial variant of Candida albicans

Summary A heretofore undescribed minute-rough colonial variant has been isolated from cultures of a red, adenine auxotroph ofCandida albicans, strain WC—7. The variant has been detected only in WC—7 populations which are propagated at 37° C on very low concentrations of adenine. It produces colonies much smaller than those of WC—7 at both 25° C and 37° C on defined media containing either ammonium ion or casein hydrolysate as nitrogen sources. On ammonium nitrogen, young variant colonies are smooth and contain only typical yeast cells. While colonies which grow at 37° C retain these characteristics upon extended incubation, those growing at 25° C progressively roughen due to extensive development of pseudohyphae as the glucose levels in their vicinities decline. Under comparable conditions, colonies of the parental strain WC—7 remain smooth and free of pseudohyphae, Supplementing the medium with large amounts of glucose, intermediates of the tricarboxylic acid cycle or any one of several amino acids biosynthetically derived from intermediates of oxidative respiration prevents formation of pseudohyphae by the variant without significantly affecting its growth rate. Genetically, the variant is unstable and reverts frequently to a stable, rapidly growing form apparently identical to strain WC—7. Evidence is presented indicating that, under certain circumstances, variant cells can exercise a contact-inhibition of the growth of their revertants. Possible physiological bases of the variriant's cultural properties are discussed.     

Regulation of histidine biosynthetic enzymes in a mutant of Escherichia coli with an altered histidyl-tRNA synthetase

Summary A mutant of Escherichia coli was isolated that grew at a normal rate in minimal medium at 26°C, grew at a normal rate in minimal medium at 37°C only if exogenous histidine was supplied, and grew more slowly than normal at 42°C even in the presence of histidine. In very rich media the growth rate of the mutant was normal at 26°C and 30°C, but not at 37°C or 42°C. It may be described as a temperature-conditional histidine bradytroph with a decreased ceiling to its growth rate.The histidyl-tRNA synthetase of the mutant was found to be abnormal; in crude extracts the enzyme activity was less stable and had approximately a tenfold higher apparent K _Mfor histidine than normal.Under many growth conditions the histidine biosynthetic enzymes in the mutant were derepressed several hundred fold compared to the wild strain, even in the presence of exogenous genous histidine. In general, the degree of derepression in the mutant was proportional to the difference in growth rate between the mutant and normal strains; this relationship, however, did not hold below 30°C or above 37°C.The properties of the mutant could be related to the properties of its histidyl-tRNA synthetase by assuming that the enzyme participates both in protein synthesis and in histidine biosynthetic enzyme regulation and that at low temperature it functions relatively more effectively in protein synthesis than in repression, while at high temperature it functions relatively more effectively in repression.Abbreviations used tRNA transfer RNA - AICAR aminoimidazole carboxamide ribose-5-phosphate     

Effects of ethanol on thermal death and on the maximum temperature for growth of the yeast Kluiveromyces fragilis

Summary The maximum temperature for growth of a strain of Kluyveromyces fragilis was depressed by increasing ethanol concentration from its normal value around 45 °C to 26.5 °C at 6.4% (w/v) of ethanol. Ethanol enhanced thermal death by increasing S, the entropy of activation of thermal death. Entropy coefficients of 7.6 entropy units per mol of ethanol per liter of medium for unadapted cells and of 5.7 for adapted cells ware obtained.     

Correlation between histone phosphorylation and tumor ageing in Ehrlich ascites tumor cells

The histone fraction F1 can be divided into subfractions by gel electrophoresis. The microheterogeneity of F1 histone has been investigated in EAT cells in mice between 3 and 16 days after inoculation. The cell number per mouse increases during the first 8 days (proliferation phase); thereafter it remains constant (non-proliferating phase). We could demonstrate that the number of F1 subfractions is reduced from 5 in proliferating cells to 3 in non-proliferating ones. In short term experiments using [³²P]phosphate the label was only found in F1 histone from proliferating cells but not in that from resting cells. However, F2a1 histone, which is the other phosphorylated histone in interphase cells, was labelled in young and old cell populations. When ³²P-labelled F1 histone was treated with alkaline phosphatase not only was the label split off but also the number of subfractions was reduced from 5 to 3. These results lend additional evidence to the hypothesis that at least some of the F1 subfractions are derived from the same protein by phosphorylation.