Cell size, macromolecular composition, and O2 consumption during agitated cultivation of Naegleria gruberi.

Cell size, macromolecular composition, carbohydrate utilization patterns, and O2 concentrations were measured throughout the growth stages of Naegleria gruberi in agitated culture in a complex medium. Biphasic logarithmic growth occurred during the intial 83 hr of growth and the mean generation time was 7.0 hr and 19 hr during initial and secondary log growth stages, respectively. The maximum yield was 5 X 10(6) amebae/ml. The pH rose rapidly (1 pH unit) during the secondary log growth phase (52-83 hr) and continued into the stationary growth phase (83-120 hr). Dry weight, total protein, carbohydrate, and RNA per ameba increased just before the secondary log growth phase. RNA increase 31% to 35% per ameba at the end of each phase of log growth. DNA increased approximately 2-fold throughout the different growth phases. Average cell size increased 90% during biphasic log growth then decreased during stationary phase. O2 tension decreased from 100% to 18% of saturation during the biphasic growth phase, then increased during stationary growth to near 100% saturation. Glucose and total carbohydrate assays showed little utilization of those substrates throughout the growth stages. Naegleria gruberi presumably has a predominantly aerobic metabolism, also its metabolism may change during the different growth phases.     

Preparation and Properties of Mitochondria from Naegleria gruberi*

Whole cell respiration rates were measured polarographically for Naegleria gruberi during growth in agitated cultures. Log growth phase amebae consumed 80 ng atoms O/min/mg cell protein. At stationary phase, respiration rate decreased 4–fold. Intact mitochondria were isolated from N. gruberi and their oxidative and phosphorylative capacities were studied polarographically. As with the mammalian system, the mitochondria oxidized succinate and NAD-linked substrates, but unlike rat liver mitochondria, those from the protozoan rapidly oxidized citrate and NADH. The rates of substrate oxidation were ADP-dependent, with ADP:O ratios equalling ? 2.8 for NAD-linked substrates and ? 2.2 for succinate. The respiratory control ratios. 2 to 4 for 11 substrates, were dependent on Pi, Mg²⁺, and serum albumin. Potassium cyanide, azide, malonale, and rotenone inhibited electron transport the same way as that of the mammalian system: however, amytal inhibited both glutamate and succinate respiration. Pentachlorophenol, DNP, and bilirubin uncoupled oxidation from phosphorylation. Difference spectra of oxidized and dithionite-reduced mitochondria had distinct absorption bands of flavins and of c-, b-, and α-type cytochromes.     

Amebostomes of Naegleria fowleri1

The strain of ameba, culture incubation temperature, and phase of ameba growth affected the number of amebostomes present on amebae of Naegleria fowleri. Serial passage of N. fowleri through mice decreased the average number of amebostomes. Amebostomes were shown to be functional by their ability to engulf yeast cells.     

CHLOROPHYLL,NITROGEN, AND PHOTOSYNTHETIC PATTERNS DURING GROWTH AND SENESCENCE OF TWO BLUE-GREEN ALGAE1,2

A standardized, multiflask, batch culture system was developed to study the processes of algal senescence in Anacystis nidulans and Phormidium molle Gom, var. tenuior W. et G. West. Growth data over a 3-year period gave reproducible and comparable time-course curves. Although A. nidulans is unicellular and P. molle filamentous, the patterns of change with age were similar. Mean logarithmic doubling times and carbon yields were, respectively, 6.9 hr and 390 mg C/liter for A. nidulans and 7.2 hr and 710 mg C/liter for P. molle. Chlorophyll concentration and photo-synthetic capacity per unit carbon rose rapidly during the logarithmic phase to maximum levels in either late log phase (P. molle) or early linear phase (A. nidulans), then fell throughout the declining growth phase to low levels in the stationary phase. Nitrate was rapidly exhausted from the medium during the period of logarithmic growth and stoichiometrically converted to particulate organic form; very little subsequent fixation of molecular nitrogen occurred. The phycocyanins were rapidly destroyed during the logarithmic phase while the carotenoids remained relatively constant throughout the whole growth period and then slowly declined. Preliminary electron micrographs showed a progressive deterioration in cellular ultrastructure, especially a reduction in the number of photosynthetic thylakoids, commenting in the linear growth phase. Analysis of the results suggests that occurrence of linear growth kinetics and termination of culture growth were caused by exhaustion of nitrate. The observed decreases in chlorophylls, phycocyanins, and photosynthetic capacity during active culture growth show that senescence effects may not be, as assumed, restricted to the stationary phase of growth.     

Conditions for Maximum Enflagellation in Naegleria fowleri1

ABSTRACT. Ameba to flagellate transformation in Naegleria fowleri (Lovell strain) was affected by growth temperature, phase of growth, strain of ameba, culture agitation, enflagellation temperature, enflagellation diluent, and cell concentration. Amebae transformed best when they were grown without agitation and enflagellated with agitation. Regardless of growth temperature (23°, 30°, 37°, and 42°C were tested), amebae transformed best at 37°C. Enflagellation was greatest for cells harvested between 24 h (mid-exponential) and 84 h (late stationary) of growth.     

Modification of low density lipoprotein metabolism by growth factors in cultured vascular cells and human skin fibroblasts. Dependence upon duration of exposure

Phospholipase A, sphingomyelinase and lysophospholipase activities were examined in cell homogenates and cell-free culture media of virulent and virulent-attenuated Naegleria fowleri and nonpathogenic Naegleria gruberi. Homogenates of virulent N. fowleri contained from 3 to 250 times the lipolytic activity of virulent-attenuated and non-pathogenic Naegleria spp. Similarly, the cell-free media of virulent N. fowleri cultures contained large quantities of phospholipase A, lysophospholipase and sphingomyelinase while comparable activities in the cell-free media of virulent-attenuated and nonpathogenic Naegleria spp. were only slightly, if at all, detectable. Lipolytic enzymes accumulated in the media of virulent N. fowleri cultures at various stages during growth but not in virulent-attenuated and nonpathogenic Naegleria cultures. In general, phospholipase A and sphingomyelinase accumulated during the log phase of growth while lysophospholipase appeared only in the late stationary phase. We conclude that pathogenic Naegleria contain potent lipolytic enzymes that are released selectively into the media during growth. These enzymes could contribute to the pathogenesis of Naegleria-induced primary amoebic meningoencephalitis.     

Enzyme patterns in mitochondria of eggs, liver, and skeletal muscle during larval development of Xenopus

The amebo-flagellate Naegleria gruberi contains three major RNA polymerase activities separable from whole cell extracts by DEAE-Sephadex column chromatography. One is resistant to the durg α-amanitin and the other two are sensitive to it. These separated activities were characterized and then examined during the differentiation of ameba to flagellate, which is dependent on RNA synthesis and which exhibits gross changes in RNA metabolism under the conditions employed. It was found that no detectable changes occurred qualitatively or quantitatively in these polymerases during differentiation. Care was taken to account for total enzyme activity in every fraction of the extraction procedure. It is concluded from the above observation that gross changes in RNA synthesis as well as differential gene activity can occur with absolutely no major fluctuations in the DNA-dependent RNA polymerases.     

Comparison of Naegleria fowleri and Naegleria gruberi Cultivated in the Same Nutrient Medium1

The human pathogenic amoeboflagellate Naegleria fowleri and the nonpathogenic species N. gruberi can be cultivated axenically but usually in different media. Naegleria fowleri 6088 has been adapted to grow in Balamuth H-4 medium, usually used to propagate N. gruberi nB81. and nB81 has been adapted to grow in supplemented Nelson's medium, usually used to propagate N. fowleri. N. gruberi nB81. grown in either medium, enflagellated 135 to 150 min after subculture to non-nutrient amoeba saline, whereas 6088 required 225 min. Naegleria gruberi nB81 grown in either medium was agglutinated by 100 ug concanavalin A/ml, whereas N. fowleri 6088 was not. Naegleria fowleri and N. gruberi grown in Nelson's medium became rounded to a greater extent upon chilling at 5° C and remained rounded longer than Naegleria grown in Balamuth medium. The specificity of the surface antigens was an inherent characteristic of each species and not dependent upon the propagating medium. but Naegleria grown in Nelson's medium was agglutinated more reproducibly and more effectively by antiserum. N. gruberi was somewhat more resistant to acriflavine, actinomycin D, cycloheximide, or tetracycline than N. fowleri, regardless of the culture medium. Naegleria fowleri 6088 grown in Nelson's medium, however, was more resistant to actinomycin D, daunomycin. mithramycin. sulfamethoxazole, or tyrocidine than 6088 grown in Balamuth medium. There are limitations on the validity of comparisons of N. fowleri and N. gruberi based upon cultures grown in different media.     

The production of extracellular carbohydrates by estuarine benthic diatoms: the effects of growth phase and light and dark treatment

Epipelic diatoms are important constituents of estuarine microphytobenthic biofilms. Field‐based investigations have shown that the production of carbohydrates by such taxa is ecologically important. However, limited information exists on the dynamics of carbohydrate production by individual species of epipelic diatoms. The production of low and high molecular weight extracellular carbohydrates in axenic cultures of five species of benthic estuarine diatoms, Cylindrotheca closterium (Ehrenberg), Navicula perminuta (Grun.) in Van Heurck, Nitzschia frustulum (Kütz.) Grunow, Nitzschia sigma (Kütz.) Grunow, and Surirella ovata (Kütz.) Grunow, were investigated. All species produced colloidal (water‐soluble) carbohydrates during growth, with maximal production occurring during stationary phase. During logarithmic growth, approximately 20% of extracellular carbohydrates consisted of polymeric material (extracellular polymeric substances [EPS]), but during stationary phase, EPS content increased to 34%–50%. Pyrolysis–mass spectrophotometry analysis showed differences in the composition of EPS produced during logarithmic and stationary phase. All species synthesized glucan as a storage carbohydrate, with maximum glucan accumulation during the transition from log to stationary phase. Short‐term labeling with ¹⁴C‐bicarbonate found that between 30 and 60% of photoassimilates were released as colloidal carbohydrate, with EPS consisting of approximately 16% of this colloidal fraction. When cells were placed in darkness, EPS production increased, and between 85 and 99% of extracellular carbohydrate produced was polymeric. Glucan reserves were utilized in dark conditions, with significant negative correlations between EPS and glucan for N. perminuta and S. ovata. Under dark conditions, cells continued to produce EPS for up to 3 days, although release of low molecular weight carbohydrates rapidly ceased when cells were dark treated. Three aspects of EPS production have been identified during this investigation: (1) production during rapid growth, which differs in composition from (2) EPS directly produced as a result of photosynthetic overflow during growth limiting conditions and (3) EPS produced for up to 3 days in the dark using intracellular storage reserves (glucans). The ecological implications of these patterns of production and utilization are discussed.     

Axenic cultivation of Naegleria gruberi : Requirement for methionine

A simplified axenic medium for Naegleria gruberi strain NEG-M contains -methionine, dextrose, yeast extract, a macromolecular fraction of fetal calf serum, and phosphate buffer. Amoebae cultured in suspension in this medium grow with doubling times of 8–10 h (at 32 °C) to yield 2–4 × 10⁶ cells/ml. Amoebae from growing or early stationary phase cultures, transferred to nonnutrient buffer, differentiate synchronously into flagellates. Differentiation occurs reproducibly 80 min after initiation (time for 50% flagellates at 25 °C) if amoebae are taken from a culture maintained at pH 6.6.     

The Utilization of Glucose and Proline by Culture Forms of Trypanosoma brucei*

Exponentially dividing culture forms of Trypanosoma brucei did not utilize glucose provided in the culture medium. The inclusion of 2-deoxyglucose in the medium had no effect on the growth of the trypanosomes. Glucose could be replaced by proline in the liquid phase of biphasic medium without affecting the doubling time of the organisms. Proline added to the culture medium in this way disappeared during the log phase of growth. Glucose in the culture medium was used by the trypanosomes only when the stationary growth phase had been reached. Lipid accumulated in stationary phase trypanosomes grown in glucose-containing medium, but there was no lipid accumulation in log phase organisms or in those which had been grown in proline-containing medium. Bloodstream trypanosomes transferred to liquid medium rapidly utilized glucose over the first 12 hr of culture, and this was accompanied by an accumulation of free pyruvate in the medium. The rate of glucose utilization fell off over the next 36 hr; this was accompanied by a lowering of free pyruvate in the medium and a rise in the proline oxidase activity of the trypanosomes. The possible biologic significance of proline to trypanosomes developing in the midgut of the tsetse vector is discussed.     

Polyhydric alcohol production and intracellular amino acid pool in relation to halotolerance of the yeast Debaryomyces hansenii

Changes in polyol production and the intracellular amino acid pool were followed during the growth cycle of Debaryomyces hansenii in 4 mM and 2.7 M NaCl media. The intracellular levels of polyols were markedly enhanced by high salinity, the dominant solutes being glycerol in log phase cells and arabinitol in stationary phase cells. At low salinity arabinitol was the most prominent intracellular solute throughout the growth cycle. There were no major changes in the composition of the total amino acid pool with changes in cultural salinity. The amount of total free amino acids related to cell dry weight was 15–50% lower in cells cultured in 2.7 M NaCl as compared to 4 mM NaCl media.After subtraction of contributions from intracellular polyols the calculated cellular C/N ratio was found to be unaffected by cultural age and salinity during the late log and early stationary phase. On prolonged incubation of stationary phase cells, this ratio decreased, particularly at high salinity. The sensitivity of cells towards exposure to high salinity was measured in terms of the length of the lag phase after transference to 2.7 M NaCl media. This lag phase decreased with increasing intracellular polyol concentrations. At a given polyol content, stationary phase cells were considerably less sensitive than were log phase cells.When cultured at high salinity the mutant strain, 26-2b, grew more slowly and retained less of the total polyol produced during the early growth stages than did the wildtype. Exogenously supplied mannitol, arabinitol, and glycerol stimulated the growth of the mutant in saline media. Erythritol was without effect.Abbreviations GLC gas-liquid chromatography - TCA trichloroacetic acid     

Respiration and cyanogenesis inPseudomonas aeruginosa

The respiratory ability of batch cultures ofPseudomonas aeruginosa strain 9-D2 peaks during midlog phase at 3.8 nmol O₂/min/10⁸ cells. This ability declines in late log phase, just prior to the time the culture begins to produce cyanide. The respiration of this organism is particularly sensitive to cyanide inhibition during midlog-phase growth, but is extremely resistant to this compound in stationary phase. These inhibition patterns are biphasic for each of these situations and indicate several respiratory responses to HCN. Addition of cyanide to midlog-phase cells resulted in the production of a stationary-phase type of cyanide respiration pattern in 2 h. A non-cyanideproducing mutant of this organism produced significantly less of the cyanide-resistant respiration components.     

Changes in cytochrome levels during growth of the yeast-like fungusAureobasidium pullulans

Cytochromes ofAureobasidium pullulans have been identified and partially characterized using low-temperature and carbon-monoxide-difference spectroscopy. The presence ofa-,b-, andc-type cytochromes is demonstrated, as are other unidentified redox components. During exponential growth in batch culture, cytochrome levels showed complex changes. Changes in respiration rates and in the levels of cytochromea+a ₃ closely paralleled cellular growth: both increased exponentially until stationary phase, when no further increase occurred. Theb- andc-type cytochromes showed biphasic increases, initially doubling every, generation time and then increasing more slowly during the stationary phase. Sensitivity of respiration to 100M potassium cyanide gradually decreased during exponential growth, falling from virtually 100% inhibition after about 20 h growth to 30% inhibition in the stationary phase. The results suggest that in stationary-phase cultures, an alternative cyanide-insensitive but salicylhydroxamic-acid-sensitive terminal oxidase also operates.     

A functional glycogen biosynthesis pathway in Lactobacillus acidophilus: expression and analysis of the glg operon

Glycogen metabolism contributes to energy storage and various physiological functions in some prokaryotes, including colonization persistence. A role for glycogen metabolism is proposed on the survival and fitness of Lactobacillus acidophilus, a probiotic microbe, in the human gastrointestinal environment. L. acidophilus NCFM possesses a glycogen metabolism (glg) operon consisting of glgBCDAP‐amy‐pgm genes. Expression of the glg operon and glycogen accumulation were carbon source‐ and growth phase‐dependent, and were repressed by glucose. The highest intracellular glycogen content was observed in early log‐phase cells grown on trehalose, which was followed by a drastic decrease of glycogen content prior to entering stationary phase. In raffinose‐grown cells, however, glycogen accumulation gradually declined following early log phase and was maintained at stable levels throughout stationary phase. Raffinose also induced an overall higher temporal glg expression throughout growth compared with trehalose. Isogenic ΔglgA (glycogen synthase) and ΔglgB (glycogen‐branching enzyme) mutants are glycogen‐deficient and exhibited growth defects on raffinose. The latter observation suggests a reciprocal relationship between glycogen synthesis and raffinose metabolism. Deletion of glgB or glgP (glycogen phosphorylase) resulted in defective growth and increased bile sensitivity. The data indicate that glycogen metabolism is involved in growth maintenance, bile tolerance and complex carbohydrate utilization in L. acidophilus.     

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 (Q_s, and Q_G). 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.     

Carbohydrate Metabolism and Activity of Pyrophosphate: Fructose-6-Phosphate Phosphotransferase in Photosynthetic Soybean (Glycine max, Merr.) Suspension Cells

Activity of pyrophosphate:fructose-6-phosphate phosphotransferase (PFP) was investigated in relation to carbohydrate metabolism and physiological growth stage in mixotrophic soybean (Glycine max Merr.) suspension cells. In the presence of exogenous sugars, log phase growth occurred and the cells displayed mixotrophic metabolism. During this stage, photosynthetic oxygen evolution was depressed and sugars were assimilated from the medium. Upon depletion of medium sugar, oxygen evolution and chlorophyll content increased, and cells entered stationary phase. Activities of various enzymes of glycolysis and sucrose metabolism, including PFP, sucrose synthase, fructokinase, glucokinase, UDP-glucose pyrophosphorylase, and fructose-1,6-bisphosphatase, changed as the cells went from log to stationary phases of growth. The largest change occurred in the activity of PFP, which was three-fold higher in log phase cells. PFP activity increased in cells grown on media initially containing sucrose, glucose, or fructose and began to decline when sugar in the medium was depleted. Western blots probed with antibody specific to the -subunit of potato PFP revealed a single 56 kilodalton immunoreactive band that changed in intensity during the growth cycle in association with changes in total PFP activity. The level of fructose-2,6-bisphosphate, an activator of the soybean PFP, increased during the first 24 hours after cell transfer and returned to the stationary phase level prior to the increase in PFP activity. Throughout the growth cycle, the calculated in vivo cytosolic concentration of fructose-2,6-bisphosphate exceeded by more than two orders of magnitude the previously reported activation coefficient (K_a) for soybean PFP. These results indicate that metabolism of exogenously supplied sugars by these cells involves a PFP-dependent step that is not coupled directly to sucrose utilization. Activity of this pathway appears to be controlled by changes in the level of PFP, rather than changes in the total cytosolic level of fructose-2,6-bisphosphate.     

Cell Size,Macromolecule Composition,Nuclear Number,Oxygen Consumption and Cyst Formation During Two Growth Phases in Unagitated Cultures of Acanthamoeba castellanii1

SYNOPSIS. The size, composition and physiology of average cells have been studied in cultures of Acanthamoeba castellanii during the phases of logarithmic growth and population growth deceleration (PGD). Most of the features examined were relatively constant during log phase, but had significant changes during PGD. Average cell volume increased about 60% and total dry mass about 15–20% during the latter period. Total protein content remained constant thruout both growth phases, but cytochrome oxidase doubled during PGD. DNA, RNA and glycogen levels began to change during late log phase. DNA decreased about 50% and RNA increased about 75%. Glycogen decreased 50% during the RNA build-up and then increased to a plateau above the log phase level. A final decrease in glycogen followed an increase in the relative numbers of cysts in late PGD. It was found that PGD begins when O₂ becomes limiting and evidence that the subsequent changes in macromolecule composition are related to encystation is discussed.     

Ultrastructure of Cysts of Naegleria spp: A Comparative Study*

SYNOPSIS. Ultrastructure of cysts of Naegleria gruberi, Naegleria fowleri, and Naegleria jadini was compared by transmission electron microscopy. Pores in the cyst wall were observed in all 3 species. In N. gruberi they were surrounded by a collar and sealed with a relatively large mucoid plug; no such collar was seen around the pores in the other 2 species, in which the plug was smaller than that in N. gruberi. An electron-dense plaque serving as an additional pore closure was present in all 3 species. In N. gruberi, the cyst wall consisted of an inner thick and an outer thin layer; however, only the inner component was present in cysts of N. fowleri and N. jadini, which had a smooth appearance. At the ultrastructural level, excystment of N. fowleri involved digestion of the mucoid plug and emergence of the trophozoite through the pore. Some digestion of the cyst wall also appeared to take place during excystment.     

Variation in the latency of acid phosphatase and ribonuclease throughout the growth cycle of Tetrahymena pyriformis

The latency of both acid phosphatase and ribonuclease was studied throughout the growth cycle of the ciliate protozoan Tetrahymena pyriformis and was found to be remarkably low in cells in the logarithmic phase of growth. The latency increased progressively throughout the log phase until it reached a maximum just after the cells had entered the stationary phase. The specific activity of acid phosphatase remained constant throughout the whole of the growth cycle while that of ribonuclease decreased as the cells began to leave log phase. The possibility is discussed that all rapidly dividing cells have a high proportion of their acid hydrolases outside the lysosomes and that these free hydrolases may have a function in cell division.