Activity of membrane ATPase during inhibition and reversion ofEscherichia coli cell division

Activity of Mg²⁺-dependent ATPase from the fraction of cell-free homogenate sedimenting at 35 000×g was studied during the growth and division ofEscherichia coli B. It decreased with the transition to stationary growth phase and after a specific inhibition of cell division. During the reversion of the division of filamentous forms the activity sharply increased; with the end of the reversion it dropped again to the level prior to the inhibition. The possible connection of the activity of Mg²⁺-dependent ATPase with the cell division ofEscherichia coli B is discussed.     

Effect of 12C+5 ion beam irradiation on cell viability and plant regeneration in callus,protoplasts and cell suspensions of Lavatera thuringiaca

Summary The biological effects of irradiation with¹²C⁺⁵ ion beam on plant cells have been analyzed. Protoplasts and cell suspensions ofLavatera thuringiaca, and a somatic hybrid callus (Hibiscus rosa-sinensis +Lavatera thuringiaca), were irradiated with doses from 0.05 to 50 Gy, and the effects on cell growth, cell division, cell viability and embryogenesis rates were analyzed. Irradiation with¹²C⁺⁵ ion beam at relatively very low doses (5.0 Gy) significantly inhibited cell division, yet the survival rate and regeneration capability of the cells through somatic embryogenesis were conserved in more than 70 and 50 %, respectively. These results indicate that cell division is the most sensitive parameter to irradiation, accounting for the inhibition of colony formation and callus growth. The potential use of the¹²C⁺⁵ ion beam in asymmetric protoplast fusion experiments is discussed.     

EFFECTS OF SMALL-SCALE TURBULENCE ON PHOTOSYNTHESIS,PIGMENTATION, CELL DIVISION,AND CELL SIZE IN THE MARINE DINOFLAGELLATE GOMAULAX POLYEDRA (DINOPHYCEAE)1

Several experiments were conducted to understand better the physiological mechanisms underlying growth inhibition of the dinoflagellate Gonyaulax polyedra Stein due to small-scale turbulence shear. To measure photosynthetic ¹⁴C uptake, a “phytoplankton wheel” device for rotating cultures in closed bottles was used. Turbulence was quantified biologically in the bottles by comparing growth inhibition with that in cultures with constant shear between a fixed cylinder and an outer concentric rotating cylinder (a stable Couette flow). At saturating irradiances, particulate photosynthesis (P_sat) or photosynthesis per unit chlorophyll (P^B_sat) were not inhibited completely at the highest turbulence level (26.6 rad.s^?1), and photosynthesis was less sensitive than growth. Photosynthesis per cell (P^C_sat) was increased by turbulence. In three experiments on the effects of turbulence on photosynthesis versus irradiance curves, the slope of the curve, α, for particulate photosynthesis at limiting irradiances did not change. Photosynthesis per unit chlorophyll per unit irradiance (α^B) decreased at high (but not intermediate) turbulence levels. Photosynthesis per cell per unit irradiance, α^C, increased with turbulence, suggesting an increase in photosynthetic efficiency in turbulent cultures. In two of the three experiments, respiration rates increased with turbulence, and in one experiment excretion of photosynthetically fixed ¹⁴C was not affected by motion. Ratios of accessory pigments to chlorophyll a did not change with turbulence, but pigments per cell and per dry weight increased with turbulence. These findings suggest little or no disruption of the photosynthetic apparatus. When turbulence was applied for 1 week, β-carotene increased while peridinin and diadinoxanthin decreased, suggesting inhibition of synthesis of these latter pigments by prolonged turbulence. Since cell numbers did not increase or decreased during turbulent 72–h incubations, cell division was inhibited and also the cells were very much enlarged. Increases in α^C per cell suggest that, in the sea, photo synthetic metabolism can persist efficiently without cell division during turbulent episodes. After turbulence ceases or reaches low levels again, cells can then divide and blooms may form. Thus, blooms can come or go fairly rapidly in the ocean depending on the degree of wave- and wind-induced turbulence.     

Oxygen toxicity in a fission yeast

Continuous exposure of synchronous cultures of Schizosaccharomyces pombe to 2.0 atmospheres oxygen beginning at any point in the first two-thirds of the cell cycle prevented subsequent cell division. Similar exposure during the last one-third of the cell cycle did not prevent cell division. The inhibition of division was totally reversible. Exposure to 2.0 atmospheres oxygen for 2.5 hours did not affect oxygen consumption. Oxygen at 1.0 atmospheres reduced growth rate and protein synthesis by 44%. Similar exposure to 1.0 atmospheres reduced transport of glycine-¹⁴C, L-leucine-¹⁴C, and uracil-¹⁴C by 95%, 73%, and 89% respectively. Analysis of the kinetics of uptake of these materials showed noncompetitive inhibition of transport by oxygen. The primary effect in rapidly appearing oxygen toxicity apparently involved interference with the transport capabilities of the cell membrane.     

DNA SYNTHESIS,CELL DIVISION,AND CELL DIFFERENTIATION DURING LEAF DEVELOPMENT OF XANTHIUM PENNSYLVANICUM

Leaf growth consists of two basic processes, cell division and cell enlargement. DNA synthesis is an integral part of cell division and can be studied with autoradiographic techniques and incorporation of some labeled precursor. Studies were made on the synthesis of nuclear DNA through incorporation of ³H-thymidine in various parts of the lamina during the entire course of leaf development of Xanthium pennsylvanicum. The time course analysis of DNA synthesis was correlated with cell division and rates of cell enlargement. Significant differences in ³H-thymidine incorporation were found in various parts of the lamina. Cell division and DNA synthesis were highest in the early stages of development. Since no ³H-thymidine was incorporated after cessation of cell division (LPI 2.8) in the leaf lamina, it appears that DNA synthesis is not needed for enlargement and differentiation of Xanthium cells. Rates of cell enlargement were negligible in the early development and reached their maximum after cessation of mitoses, between plastochron ages (LPI) 3 and 4. Cells matured between LPI's 5 and 6. Enzymatic activity was correlated with cell division and cell differentiation at various stages of leaf development.     

Independence of Cell Division and DNA Replication in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

IN Escherichia coli the completion of a round of chromosome replication is necessary before cell division can take place^1,2. A normal cell is therefore unable to divide unless it has at least two chromosomes. If DNA synthesis is specifically inhibited, cell division will continue only until each cell contains a single chromosome. Division then ceases but growth continues so that long filamentous cells are formed³. We describe here the consequences of blocking DNA synthesis in Bacillus subtilis. In this case division of the growing cells continues in spite of the inhibition of DNA replication. Eventually, not only are all pre-existing chromosomes segregated into separate cells but large numbers of cells are formed which contain no DNA.     

The Inhibition of Seedling Growth by Streptomycin Sulfate

The action of streptomycin sulfate (SS) on the growth of germinating seeds was investigated. It inhibited root growth more than shoot growth. Chemical analysis of roots indicated that nucleic acids decreased but their elution pattern from a column of methylated albumin kieselghur showed no qualitative differences. SS decreased the number of cells undergoing mitosis per root tip. This number was correlated with root length. One nM N⁵-benzyladenine reversed the effect of SS on both root length and mitosis. Thus the effect of SS appears specific to cell division. SS failed to affect oxidative phosphorylation in isolated mitochondria so its effect on cell division would not be secondary to the inhibition of energy conservation. It s possible inhibition of enzyme activity is selective since it also failed to affect RNase activity in vitro.     

The "Point of no Return" Concept in Cell Division. The Effects of Some Metabolic Inhibitors on Synchronized Chlorella pyrenoidosa

The coarse of growth and cell division in synchronized cultures of Chlorella pyrenoidosa was studied after the addition of metabolic inhibitors at differing times during the cell cycle (14 h light - 10 h darkness with nitrate as nitrogen source. 12 h light: 12 h darkness with urea as nitrogen source). Dinitrophenol (DNP) added to a final concentration of 0.3 mM at any time in the synchronization cycle, the compound remaining in the suspension from the time of addition to the end of the dark period, inhibited spore formation completely. Growth measured as increase in cell volume was less sensitive to the action of the inhibitor. Chloramphenicol (CAP) added dining the 0–5 h interval to a final concentration of 0.1 mM resulted in 80 per cent inhibition of cell division. Similar treatment started at successive times thereafter resulted in a gradual decrease of the inhibition. Treatment at the 14th hour and during the dark period did not affect the sporulation. Similar experiments with 0.9 mM puromycin added at various times during the illumination period gave almost complete inhibition of cell division, while the growth was reduced by only 25 per cent. para-Fluorophenylalanine (p-FPhe) at 3.3 × 10^?2 mM stopped cell division nearly completely irrespective of addition time in the light period. Addition during the dark period also prevented an increase in the number of tree cells. In this case about half of the cells produced spores which were not released. It is concluded that DNP inhibits all stages of preparation for cell division, as well as the division process itself. With CAP a genuine transition point of preparation for cell division was observed, although its interpretation as related to protein synthesis is somewhat uncertain. With puromycin and p-FPhe no transitions were observed.     

COMPARATIVE CYTOHISTOLOGICAL STUDIES ON INHIBITION AND PROMOTION OF STEM GROWTH IN CHRYSANTHEMUM MORIFOLIUM

Sachs , R. M. (U. California, Davis), and A. M. Kopranek . Comparative cytohistological studies on inhibition and promotion of stem growth in Chrysanthemum morifolium. Amer. Jour. Bot. 50(8): 772-779. Illus. 1963.—The present study with Amo, CCC, and Phosfon,³ 3 substances which inhibit stem elongation, shows that all inhibit subapical cell expansion and division in Chrysanthemum morifolium var. ‘Indianapolis Yellow.‘ Furthermore, GA,³ in preventing the inhibition of stem elongation, maintains subapical activity at normal or greater than normal levels. For comparative purposes concentrations of the retardants and GA have been selected which completely prevent or promote the maximum rate of stem elongation. Phosfon causes complete inhibition of root growth and almost completely prevents dry matter accumulation in the tops. However, GA does not prevent such deleterious effects. Thus, GA and the growth retardants are mutually antagonistic only with respect to stem elongation and not to other aspects of growth. Furthermore, none of the retardants inhibits transverse stem growth; on the contrary transverse cell expansion and division in the subapical tissues are stimulated by the retardants, and as a result the stems of such plants are thicker than normal. GA not only prevents the thickening effect of the retardants, but, at the doses applied, GA-treated stems are considerably thinner than those of the controls, having fewer and smaller cells across the pith, cortical, and vascular tissues. Apparently, then, there is a relationship between longitudinal and transverse growth in the subapical tissues such that if one is promoted, the other is inhibited.     

Effects of different sterols on the inhibition of cell culture growth caused by the growth retardant tetcyclacis

Cell division in cell suspension cultures can be completely blocked by the growth retardant tetcyclacis at a concentration of 10^-4 mol l^-1. In rice cells it has been demonstrated that the growth inhibition can be completely overcome by application of cholesterol independent of the duration of pretreatment with tetcyclacis. In suspension cultures of maize and soybean, too, the effect of tetcyclacis on cell division was neutralized by adding cholesterol. Other plant sterols, stigmasterol, campesterol and sitosterol were active in a decreasing order. Modifications in the cholesterol perhydro-cyclopentanophenanthrene-ring system indicate that the hydroxyl group at C-3 and the double bond between C-5 and C-6 in ring B are required for the activity. In contrast, gibberellic acid as well as ent-kaurenoic acid could not compensate retardant effects. Likewise, tetcyclasis did not change the level of gibberellins in rice cells as shown by radioimmunoassay. Thus, it is concluded that in cell suspension cultures sterols play a more important role in cell division than gibberellins.Abbreviation GA_x gibberelin A_x     

Bacterial Cell Division Regulation: Physiological Effects of Crystal Violet on Escherichia coli lon+ and lon− Strains

The Escherichia coli lon⁻ mutants apparently are defective in the ability to recommence cell division after temporary periods of deoxyribonucleic acid (DNA) synthesis inhibition. They are also more susceptible to cell division inhibition by the basic dye, crystal violet (CV), than are lon⁺ strains. In enriched broth, the lon⁺ strain continued to grow and divide in the presence of CV, but lon⁻ cell division was inhibited and filamentous growth resulted. In a supplemented minimal medium containing CV, lon⁻ cell division was only temporarily inhibited. There was no detectable specific effect on DNA synthesis, although CV slowed the rate of mass increase in both media. Trichloroacetic acid-insoluble lipid synthesis was preferentially inhibited in both lon⁺ and lon⁻ strains. In CV-containing enriched broth, diaminopimelic acid incorporation into trichloroacetic acid-insoluble compounds occurred at a rate greater than the rate of mass increase in both lon⁺ and lon⁻ strains. In a CV-containing supplemented minimal medium, diaminopimelic acid was incorporated to a greater extent by lon⁻ cells than by lon⁺ cells.     

Induction of Acetylcholine Esterase Activity in a Mouse Neuroblastoma

TISSUE culture lines of mouse neuroblastoma C1300 contain acetylcholine esterase¹, the specific activity of which depends on the conditions of growth, for the inhibition of cell division leads to an increase in esterase activity^2,3. Although this suggests that the cessation of division is directly responsible for increased enzyme synthesis, it may also be that the increase of specific activity is the result of (1) neurite formation by differentiating neuroblast cells or (2) events accompanying ageing and cell death. This article describes experiments designed to distinguish between these hypotheses and to examine the regulation of esterases in other tissue culture lines.     

Growth ofDunaliella viridis Teodoresco: effect of salinity,temperature and nitrogen concentration

The growth of a strain ofD. viridis has been studied in batch culture under different combinations of temperature, salinity and nitrogen concentrations. Changes in these variables have a significant effect on cell division, biomass production, cell volume and pigment yield. This strain grows optimally at 1 M NaCl and 30 °C. Increasing salinity up to 4 M NaCl leads to a significant decrease of cell division rate and maximal population; growth at lower temperature decreases the rate of division of the cells but increases maximal cell density. Pigment yield decreases with increasing salinity and increases with increasing temperature. Nitrogen concentration has a large effect on total cell biomass and pigment production, but not on cell division rate. Saturation of growth occurs at 5 mM NO ₃ ^? ; higher concentration (e.g. 10 mM) leads to a decrease of maximal cell density and photosynthetic pigment content.     

PHOTOSYNTHESIS AND CELL DIVISION BY ANTARCTIC MICROALGAE: COMPARISON OF BENTHIC,PLANKTONIC AND ICE ALGAE1

Irradiance-dependent rates of photosynthesis and cell division of six species of microalgae isolated from the benthos, plankton and sea ice microbial community in McMurdo Sound, Antarctica were compared. Microalgae isolated from different photic environments had distinct photosynthetic and growth characteristics. For benthic and ice algae, photosynthesis saturated at 6 to 20 μE.m^?2.s^?1 and was photoinhibited at 10 to 80 μE.m^?2.s^?1 while for the planktonic algae, saturation irradiances were up to 13 times higher and photoinhibition was not detected. The slope of the light-limited portion of the P-I relationship was up to 50 times greater for the benthic algae than for either the ice or planktonic algae suggesting that benthic algae used the low irradiances more efficiently for carbon uptake. Cell division was dependent on the incubation irradiance for all but one microalga examined. The dependence of division rates on irradiance was however much smaller than for carbon uptake, suggesting that cell division buffers the influence of short term variations of irradiance on cellular metabolism.     

Succinylacetone, a potent inhibitor of heme biosynthesis: effect on cell growth, heme content and delta-aminolevulinic acid dehydratase activity of malignant murine erythroleukemia cells.

4,6-Dioxoheptanoic acid (succinylacetone, SA) was examined with regard to its ability to a) inhibit the second enzyme of the heme pathway, δ-aminolevulinic acid (ALA) dehydratase, b) lower the heme concentration, and c) inhibit cell growth of murine erythroleukemia (MEL) cells in culture. SA profoundly inhibited ALA dehydratase in broken cell preparations at concentrations as low as 10^?7 M. The stimulation of hemoglobin production by DMSO and butyrate in MEL cells was inhibited by the addition of SA to the cell medium. When 1 mM SA was added to the medium, there was a profound inhibition of ALA dehydratase activity, and the heme concentration of cells declined progressively with each cell division. Cell growth was markedly inhibited after two cell divisions.     

Root growth inhibition by NH4+ in Arabidopsis is mediated by the root tip and is linked to NH4+ efflux and GMPase activity

Root growth in higher plants is sensitive to excess ammonium (NH₄⁺). Our study shows that contact of NH₄⁺ with the primary root tip is both necessary and sufficient to the development of arrested root growth under NH₄⁺ nutrition in Arabidopsis. We show that cell elongation and not cell division is the principal target in the NH₄⁺ inhibition of primary root growth. Mutant and expression analyses using DR5:GUS revealed that the growth inhibition is furthermore independent of auxin and ethylene signalling. NH₄⁺ fluxes along the primary root, measured using the Scanning Ion‐selective Electrode Technique, revealed a significant stimulation of NH₄⁺ efflux at the elongation zone following treatment with elevated NH₄⁺, coincident with the inhibition of root elongation. Stimulation of NH₄⁺ efflux and inhibition of cell expansion were significantly more pronounced in the NH₄⁺‐hypersensitive mutant vtc1‐1, deficient in the enzyme GDP‐mannose pyrophosphorylase (GMPase). We conclude that both restricted transmembrane NH₄⁺ fluxes and proper functioning of GMPase in roots are critical to minimizing the severity of the NH₄⁺ toxicity response in Arabidopsis.     

LIGHT-DEPENDENT INHIBITION OF CELL DIVISION AND RHIZOID ELONGATION IN GEMMAE OF THE FERN,VITTARIA GRAMINIFOLIA

Gametophytes of the shoe-string fern Vittaria graminifolia produce linear, six-celled propagules called gemmae. The terminal cells of each gemma elongate into primary rhizoids in culture, and the inner body cells divide asymmetrically to produce prothallial or rhizoid initials. The initiation of both asymmetric cell division and rhizoid elongation is delayed by light intensities greater than 2 w/m². The maximal rates of cell division and rhizoid elongation are unaltered. A 24-hr pulse of high light intensity delays cell division and rhizoid elongation to the same extent, whenever applied during the first 3 d of culture. The model we propose for cell division hypothesizes the existence of a preparatory phase of finite duration prior to mitosis that is sensitive to light intensity. If a cell is irradiated by light intensities greater than 2 w/m² while in the preparatory phase, its entrance into mitosis is delayed. A similar model is proposed for the initiation of rhizoid elongation. Despite the fact that both cell division and rhizoid elongation are dependent on photosynthesis, direct measurements of CO₂-uptake rates show that the inhibitory effects of high light intensities are not due to an inhibition of photosynthesis.     

SELENIUM: AN ESSENTIAL ELEMENT FOR GROWTH OF THE COASTAL MARINE DIATOM THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE)1,2

An obligate requirement for selenium is demonstrated in axenic culture of the coastal marine diatom Thalassiosira pseudonana (clone 3H) (Hust.) Hasle and Heimdal grown in artificial seawater medium. Selenium deficiency was characterized by a reduction in growth rate and eventually by a cessation of cell division. The addition of 10⁻¹⁰ M Na₂eO₃ to nutrient enriched artifical seawater resulted in excellent growth of T. pseudonana and only a slight inhibition of growth occurred at Na₂SeO₃ concentrations of 10⁻³ and 10_-2 M. By contrast, Na₂SeO₄ failed to support growth of T. pseudonana when supplied at concentrations less than 10⁻⁷ M and the growth rate at this concentration was only one quarter of the maximum growth rate. The addition of 10⁻³ and 10⁻² M Na₂SeO₄ to the culture medium was toxic and cell growth was completely inhibited. Eleven trace elements were tested for their ability to replace the selenium requirement by this alga and all were without effect. In selenium-deficient and selenium-starved cultures of T. pseudonana cell volume increased as much as 10-fold as a result of an increase in cell length (along the pervalvar axis) but cell width was constant. It is concluded that selenium is an indispensable element for the growth of T. pseudonana and it should be included as a nutrient enrichment to artificial seawater medium when culturing this alga.     

Control of protein synthesis in human fibroblasts by intracellular potassium

Intracellular potassium ion (K⁺) in cultured human fibroblasts (HF cells) was maintained at reduced steady-state levels by incubating cells in various ouabain concentrations. Small decreases in cell K⁺ had no effect on protein synthesis and cell growth, but when cell K⁺ fell below 60–80% of control levels, the rate of protein synthesis decreased in proportion to further reductions in K⁺. DNA synthesis was also inhibited, presumably because of its dependence on protein synthesis. On the other hand, RNA synthesis remained uninhibited over a wide range of K⁺ concentrations, an effect characteristic of many specific inhibitors of protein synthesis.In ouabain-treated cells neither levels of ATP nor transport of amino acids was limiting for protein synthesis. Loss of activity of messenger or other species of RNA was not responsible for inhibition of protein synthesis, since in the presence of actinomycin D, the rate of protein synthesis could be decreased or increased solely by adjusting cell K⁺. Release from ouabain inhibition restored K⁺ levels, macromolecular synthesis, and cell growth, but there was no resulting synchrony of cell division. In cell populations partially synchronized by serum starvation and refeeding protein synthesis was sensitive to reduction in K⁺ levels throughout the cell cycle.Our quantitative results show that cell K⁺ levels, when sufficiently reduced, can determine the rate of protein synthesis and hence the rate of cell growth.