Physiological changes accompanying the recovery of cell division in "giant" cells of Chlorella vulgaris (EMERSON strain)

A homogeneous population of "giant" cells of the EMERSON strainof Chlorella vulgaris, produced following culture under carefullycontrolled conditions in a glucose medium in the dark, recoversits capacity to undergo cell division when returned to autotrophicconditions. A similar recovery also occurs after a prolongedperiod of culture in the dark. The division of the giant cellsis accompanied, in each case, by marked pigment synthesis anda consequent recovery of photosynthetic capacity. Under autotrophicconditions the recovery of cell division and restoration ofthe full pigment concentration are complete within a 24 hr period.The recovery which takes place in a glucose medium in the darkoccurs only after a period of 10–14 days growth. (Received May 9, 1970; )     

Regeneration of Spheroplasts in a N2-Fixed Filamentous Blue-Green Alga Anabaena cylindrica

Over 90% of cells of Anabaena cylindrica growing in the medium containing 0. 1 mol/L KC1 for 7～9 d transformed into spheroplasts or semispheroplasts which were either sensitive or not sensitive to hypotonic condition. After treating the materials with 0. 1% lysozyme at 28 ℃ for 3～4 h the transformed spheroplasts were almost 100% sensitive to the hypotonic condition. The spheroplasts then regenerated and divided through culture in the inorganic medium containing 0.15 mol/L CaCl2 with a rate over 25 %. The regeneration of different spheroplasts was not synchronous, the fastest division being after 3 d. Cell division was mainly equational but also irregular division or budding.     

Bicarbonate enhances synchronous division of the giant nuclei of sporophytes in<Emphasis Type="Italic"> Bryopsis plumosa</Emphasis>

A mature sporophyte of Bryopsis plumosa (Hudson) C. Agardh forms a huge number of zoospores in its cell continuum. Zoospore formation starts with the division of a single giant nucleus and subsequent repeated mitosis. We found that an elevation of photosynthetic activity triggered the division of a mature giant nucleus. Transfer to short-day conditions was not necessary. Giant nuclei did not divide in darkness or in the presence of 1 μM DCMU. Giant nuclei of as many as 90% of sporophtyes started to divide following the addition of 5 mM NaHCO₃ to the growth medium under continuous white light (6–12 W m⁻²). Frequency of nuclear division increased with increased light intensity. By combining those parameters that promoted the division of giant nuclei, we developed the "two-step culture method" which is composed of preliminary and main cultures. This new method guarantees that giant nuclei of more than 90% of all sporophytes synchronously divide between 72 and 96 h after the transfer to the main culture (continuous white light of 12 W m⁻² in PES medium supplemented with 5 mM NaHCO₃).     

Effect of serotonin on proliferating and resting cells in culture

The influence of exogenous serotonin on cell division of L929 and L-41 cell strains has been investigated under various conditions of cell growth in culture (the incubation either in 10% serum medium without changing the medium, or in the medium with 0.5% serum). The data obtained show that serotonin in physiological concentration (10(-7) M) stimulates proliferation of resting cells. In proliferating cells, compared to resting ones, the sensitivity to exogenous amine appeared statistically non-significant. Exogenous serotonin is suggested to be a proliferating stimulus for resting cells.     

Giant Cells of Escherichia coli: a Morphological Study

Bacterial growth without division was observed in a giant cell-producing strain of Escherichia coli K-12. Giant cell production is controlled by the lon(-) (failure of cell division after irradiation) and mon(-) (formation of irregularly shaped cells) genes. Irradiation of a lon(-)mon(-) strain (P678-A(4)) with low doses of ultraviolet or ionizing radiation results in the production of large, amorphous giant cells with 500 to 1,000 times the volume of the nonirradiated parents. The concentration of NaCl in the growth medium was found to influence irradiated-cell morphology. Low concentrations (0.2% NaCl) resulted in elongated cells, whereas spherical giant cells were produced in the presence of high salt (1% NaCl) concentrations.Thin-section electron microscopy revealed an extensive network of intracellular membranes forming vacuoles, vesicles, and cisternae. These structures bear a striking resemblance to the rough and smooth membranes (endoplasmic reticulum, Golgi complex, vacuoles, etc.) found in eucaryotic cells.     

Ultrastructure of the cell surface of heteromorphic Nostoc muscorum CALU 304 cocultured with Rauwolfia tissue]

The ultrastructure of the heteromorphic cells (HMCs) of the cyanobacterium Nostoc muscorum CALU 304 grown in pure culture, monoculture, and a mixed culture with the Rauwolfia callus tissue was studied. The comparative analysis of the cell surface of HMCs, the frequency of the generation of cell forms with defective cell walls (DCWFs), including protoplasts and spheroplasts, and the peculiarities of the cell surface ultrastructure under different growth conditions showed that, in the early terms of mixed incubation, the callus tissue acts to preserve the existing cyanobacterial DCWFs, but begins to promote their formation in the later incubation terms. DCWFs exhibited an integrity of their protoplasm and were metabolically active. It is suggested that structural alterations in the rigid layer of the cell wall may be due to the activation of the murolytic enzymes of cyanobacteria and the profound rearrangement of their peptidoglycan metabolism caused by the Rauwolfia metabolites diffused through the medium. These metabolites may also interfere with the functioning of the universal cell division protein of bacteria, FtsZ. In general, the Rauwolfia callus tissue promoted the unbalanced growth of the cyanobacterium N. muscorum CALU 304 and favored its viability in the mixed culture. The long-term incubation of the Rauwolfia tissue with the N. muscorum CALU 304 cells led to their transformation to L-forms.     

Biological properties and ultrastructure of brucellae during L-transformation and reversion]

There was shown a difference in the biological properties and the ultrastructure of two strains of brucellae, spheroplasts obtained from them under the action of penicillin, L-form and revertants obtained from the L-form. Spheroplasts formation was characterized by a change of brucellae into R-form and some virulence reduction. The cells had an outer and a cytoplasmic membranes, and usually lost their capacity to binary division. L-forms were obtained during the 9th and the 35th passage on a medium with penicillin; their formation was accompanied by the change in serological properties of the culture and significant reduction of the virulence; the cells were characterized by a marked polymorphism and the capacity to budding; they had 2 membranes on the cell surface and an intensively developed system of intracytoplasmic membranes. The revertants formed on the medium without penicillin during the 16th-30th passage or spontaneously on the medium with penicillin. They differed from the initial strains of brucella culture by a marked increase in penicillin-resistance, by the changes in serological properties, and also by polymorphism of cells, capable, however, of binary division.     

Effect of penicillin on the morphology and reproduction ofAzotobacter chroococcum

The effect of penicillin on the morphology and reproduction of some strains ofA. chroococcum was studied on a number of solid media. When the growth was not entirely suppressed by the penicillin, filamentous cells and spheroplasts were formed. The formation of spheroplasts was stimulated by peptone. Gonidia were sometimes formed inside the spheroplasts and also inside giant cells. They were released from the cell after disruption or after lysis of the cell wall. In some cases they produced dwarf cells. Under certain conditions groups of gonidia present in a cell fused and formed one or more normal-looking cells inside the mother cell. Sometimes one or moreAzotobacter cells developed inside a spheroplast or at the site of a spheroplast with a lysed cell wall. Microcolonies consisting of small cocci representing gonidia and dwarf cells were also observed occasionally at the sites of spheroplasts with lysed cell walls. Occasionally tiny groups of small elements with a less marked structure were found at such sites, probably representing debris of lysed cells. The production of normal-looking cells inside filamentous cells was greatly stimulated on a medium containing 10 percent horse serum, with a drop of sterile water containing 200 or 250 I.U. penicillin added in the centre of the plate. The growth ofA. chroococcum was greatly retarded when the medium contained 10 U/ml penicillin and seemed to be checked entirely at concentrations of 20 U/ml penicillin or higher. Occasionally, however, even at concentrations of 100 and 300 U/ml penicillin, a few filamentous cells were found and also a few microcolonies, visible only through the microscope, consisting of gonidia or regenerative rods. By repeated exposure ofAzotobacter to penicillin populations could be obtained that were adapted to high concentrations of this antibiotic.     

Chlorella vulgaris (Chlorellaceae) does not secrete autoinhibitors at high cell densities

Filtrates (conditioned medium) from high-density Chlorella vulgaris cultures in photobioreactors were obtained and tested for autoinhibitory activity under different conditions. Exponentially growing cells were inoculated at low initial cell concentration (2 × 10⁵ cells/ml) in 90% conditioned medium (CM) supplemented with 10% fresh medium (FM) at low (atmospheric) CO₂ levels. The time sequence of DNA histograms of cells in CM cultures showed that there is an accumulation of cells with two and four DNA equivalents in the culture over a period of time, signifying a blockage of cells at the division stage of the cell cycle. Examination of the chemical composition of CM showed the presence of high concentrations (> 10 mM) of bicarbonate. Adding similar bicarbonate concentrations to FM were found to have similar effects as CM cultures, causing blockage of cell division, though the intensity of the blocking effect was lower. The bicarbonate-free CM did not show any cell cycle modulating or inhibitory activity. The growth of cells cultivated at high (5%) CO₂ levels in 90% CM supplemented with 10% FM was comparable to 10% FM cultures, indicating nutrient limitation in 90% CM culture. When the 90% CM culture was supplemented with 100% nutrients, the growth rate and final cell concentration was similar to 100% FM culture. Based on these results we conclude that C. vulgaris does not secrete any autoinhibitor(s) or cell cycle modulating compound(s) under the conditions from which the CM was obtained.     

Molybdenum accumulation in chlD mutants of Escherichia coli.

The content of molybdenum in wild-type and chlD cells was measured under a variety of growth conditions to determine if cells with a defective chlD gene were able to accumulate molybdenum. The chlD cells accumulated less molybdenum than wild-type cells did but concentrated molybdenum to a level at least 20-fold higher than the concentration in the culture medium. Molybdenum was present within spheroplasts of chlD cells and was not dialyzable. The chlD cells accumulated as much molybdenum as wild-type cells did when grown in medium containing 0.1 mM molybdate; thus, the capability of incorporation of molybdenum into cellular component(s) was equivalent to that of the wild type under these conditions.     

Extracellular regulation of fibroblast multiplication: A direct kinetic approach to analysis of role of low molecular weight nutrients and serum growth factors

The principles of enzyme kinetic analysis were applied to quantitate the relationships among serum-derived growth factors, nutrients, and the rate of survival and multiplication of human fibroblasts in culture. The survival or multiplication rate of a population of cells plotted against an increasing concentration of a growth factor or nutrient in the medium exhibited a hyperbolic pattern that is characteristic of a dissociable, saturable interaction between cells and the ligands. Parameters equivalent to the K_m and V_max of enzyme kinetics were assigned to nutrients and growth factors. When all nutrient concentrations were optimized and in steady state, serum factors accelerated the rate of multiplication of a normal cell population. The same set of nutrients that supported a maximal rate of multiplication in the presence of serum factors supported the maintenance of non-proliferating cells in the absence of serum factors. Therefore, under this condition, serum factors are required for cell division and play a purely regulatory iole in multiplication of the cell population. The quantitative requirement for 18 nutrients of 29 that were examined was significantly higher (P < 0.001) for cell multiplication in the presence of serum factors than for cell maintenance in the absence of serum factors. This indicated specific nutrients that may be quantitatively important in cell division processes as well as in cell maintenance. The quantitative requirement for Ca²⁺, Mg²⁺, K⁺, Pi, and 2-oxocarboxylic acid for cell multiplication was modified by serum factors and other purified growth factors. The requirement for over 30 other nutrients could not clearly be related to the level of serum factors in the medium. Serum factors also determined the Ca²⁺, K⁺, and 2-oxocarboxylic acid requirement for maintenance of non-proliferating cells. Therefore, when either Ca²⁺, K⁺, or 2-oxocarboxylic acid concentration was limiting, factors in serum played a role as cell “survival or maintenance” factors in addition to their role in cell division as “growth regulatory” factors. However, with equivalent levels of serum factors in the medium, the requirement for Ca²⁺, K⁺, and 2-oxocarboxylic acids was still much higher for multiplication than for maintenance. Kinetic analysis revealed that the concentrations of individual nutrients modify the quantitative requirement for others for cell multiplication in a specific pattern. Thus, specific quantitative relationships among different nutrients in the medium are important in the control of the multiplication rate of the cell population. When all nutrient concentrations were optimal for multiplication of normal cells, the multiplication response of SV40-virus-transformed cells to serum factors was similar to that of normal cells. When serum factors were held constant, transformed cells required significantly less (P < 0.001) of 12 of the 26 nutrients examined. Therefore, the transformed cells only have a growth advantage when the external concentration of specific nutrients limits the multiplication rate of normal cells. Taken together, the results suggest that the control of cell multiplication is intimately related to external concentrations of nutrients. Specific growth regulatory factors may stimulate cell proliferation by modification of the response of normal cells to nutrients. Transforming agents may confer a selective growth advantage on cells by a constitutive alteration of their response to extracellular nutrients.     

Involvement of a cell size control mechanism in the induction and maintenance of oscillations in continuous cultures of budding yeast

Spontaneous oscillations occur in glucose-limited continuous cultures of Saccharomyces cerevisiae under aerobic conditions. The oscillatory behavior is detectable as a periodic change of many bioparameters such as dissolved oxygen, ethanol production, biomass concentration, as well as cellular content of storage carbohydrates and is associated to a marked synchronization of the yeast population. These oscillations may be related to a periodic accumulation of ethanol produced by yeast in the culture medium.The addition of ethanol to oscillating yeast cultures supports this hypothesis: indeed, no effect was observed if ethanol was added when already present in the medium, while a marked phase oscillation shift was obtained when ethanol was added at any other time. Moreover, the addition of ethanol to a nonoscillating culture triggers new oscillations. An accurate analysis performed at the level of nonoscillating yeast populations perturbed by addition of ethanol showed that both the growth rate and the protein content required for cell division increased in the presence of mixed substrate (i.e., ethanol plus limiting glucose). A marked synchronization of the yeast population occurred when the added ethanol was exhausted and the culture resumed growth only on limiting glucose. A decrease of protein content required for cell division was also apparent. These experimental findings support a new model for spontaneous oscillations in yeast cultures in which the alternative growth on limiting glucose and limiting glucose plus ethanol modifies the critical protein content required for cell division.     

Formation of the population composition of a stationary cell culture and the participation of nonproliferating cells with a doubled DNA content in regulating its density

The conditions of the cultivation of chick embryo diploid cells were alternated (prolonged maintenance with or without medium replacement, with or without consequent cell replating in fresh medium). In different times of culture growth, the cell DNA content was assessed by cytophotometry; the percentage of non-labeled mitoses after incubating the cells with 3H-thymidine and colcemide, as well as the cell density were determined. The phenomenon, detected earlier, of the accumulation of cells containing 4c DNA during the transition of the culture from logarithmic into the stationary phase of growth, was confirmed. These cells were shown to differ in their ability to survive in conditions of stationary culture and by proliferative potential. The fraction of cells reversibly arrested in G2-period was described, by which fraction the change of the cell population size is occurring after the decrease of its proliferation rate. The transitional stage is distinguished at the beginning of the stationary phase of culture growth. During this stage the stabilization of structural and numerical composition of the population is taking place.     

Croissance et synthese des proteines de suspensions cellulaires de Tabac sensibles à la kinétine

Culture conditions, allowing the unlimited growth and maintenance in shaken suspensions of a kinetin dependent strain of tobacco pith cells, were determined. Cell clusters from 1 to 25 cells were selected from the cultures to study the cell multiplication after addition of specific growth factors to the basal medium. Cell division was found to be strictly dependent upon the presence in the medium of both kinetin and auxin. In complete medium the generation period was 45 to 49 hours. Kinetics of the total protein synthesis in the cell suspensions have been measured either by estimation of protein nitrogen or by ³⁵S incorporation into the proteins. After 3 days of culture, growth was exponential, as expressed by cell number or protein synthesis, which varied proportionally. Evidence was also found that the initial incorporation rate of radioactivity into the proteins was more rapid than expected from the increment of the protein net weight. It seems therefore reasonable to assume that a significant amount of protein turn over does exist during the initial period of growth. This phenomenon was observed even when no cell division occurred. Kinetin significantly activated protein synthesis, whether or not auxin was present in the medium.     

Positive selection of allelic exchange mutants in Mycobacterium bovis BCG

Abstract A resistant mutant with vancomycin MIC of 100 μg/ml was isolated relatively easily through step pressure in the laboratory from a Staphylococcus aureus strain with initial MIC of 1.5 μg/ml for the antibiotic. Upon addition of vancomycin (50 μg/ml) to the growth medium mass increase of the culture and peptidoglycan synthesis continued but cell division (daughter cell separation), cell wall turnover and autolysis were inhibited, resulting in the production of multicellular clumps of bacteria. Parallel with the increase of culture density, the concentration of vancomycin measured both by biological activity and by HPLC gradually declined in the culture medium. Cell division and wall turnover of the culture resumed with the production of cells of normal morphology at the time when the concentration of the drug in the medium decreased below 0.5–1.0 μg/ml. There was no detectable change in the antibiotic concentration in the culture medium during growth of a vancomycin-resistant ( vanA -positive) strain of Enterococcus faecium and an intrinsically vancomycin-resistant strain of Leuconostoc . The vancomycin-resistant staphylococcal mutant gave no signal with the vanA or vanB DNA probes and contained no detectable d-lactate terminating cell wall precursors. The biochemical mechanism and clinical significance of such glycopeptide-resistant mutants remains to be established.     

The role of vanadium in green plants

Vanadium, although essential for growth and chlorophyll formation in unicellular green algae, reveals toxic influences on cell division of Chlorella pyrenoidosa, these disturbances arising in the same range of V-concentrations as the known positive effects of the trace metal. In permanent light, as documented by cell volume statistics, vanadium (4-10(-7) g-at/1 as NH4VO3) causes a significant shift of the distribution maxima to higher values of the algal cell volume, the shift having its optimum at 10(-5) g-at V/1. It is documented in pH-constant liquid culture that this effect is not due to a change of pH in the nutrient medium. Under synchronous conditions of algal cultivation (16:8h), vanadium causes a total arrest of cell division after 3 periods; this stop lasts for the next 3 cycles. Afterwards, asynchronous divisions newly occur and lead to generally larger autospores. Staining of algal cell nuclei revealed an inhibitory V-effect on nuclear division, yielding giant nuclei with multiple sets of chromosomes, and thereby limiting cell division. Under these conditions, Chlorella pyrenoidosa is not synchronizable in presence of vanadium.     

Surface Ultrastructure of the Heteromorphic Cells of Nostoc muscorumCALU 304 in a Mixed Culture with the RauwolfiaCallus Tissue

The ultrastructure of the heteromorphic cells (HMCs) of the cyanobacterium Nostoc muscorumCALU 304 grown in pure culture, monoculture, and a mixed culture with the Rauwolfiacallus tissue was studied. The comparative analysis of the cell surface of HMCs, the frequency of the generation of cell forms with defective cell walls (DCWFs), including protoplasts and spheroplasts, and the peculiarities of their ultrastructure under different growth conditions showed that, in the early terms of mixed incubation, the callus tissue acts to preserve the existing cyanobacterial DCWFs, but begins to promote their formation in the later incubation terms. DCWFs exhibited an integrity of their protoplasm and were metabolically active. It is suggested that structural alterations in the rigid layer of the cell wall may be due to the activation of the murolytic enzymes of cyanobacteria and the profound rearrangement of their peptidoglycan metabolism caused by the Rauwolfiametabolites diffused through the medium. These metabolites may also interfere with the functioning of the universal cell division protein of bacteria, FtsZ. In general, the Rauwolfiacallus tissue promoted the unbalanced growth of the cyanobacterium N. muscorumCALU 304 and favored its viability in the mixed culture. The long-term mixed cultivation substantially augmented the probability of the formation of L-forms of N. muscorumCALU 304.     

Inorganic polyphosphate in mitochondria of Saccharomyces cerevisiae at phosphate limitation and phosphate excess

Isolated mitochondria of Saccharomyces cerevisiae cells grown on glucose possess acid-soluble inorganic polyphosphate (polyP). Its level strongly depends on phosphate (P(i)) concentration in the culture medium. The polyP level in mitochondria showed 11-fold decrease under 0.8 mM P(i) as compared with 19.3 mM P(i). When spheroplasts isolated from P(i)-starved cells were incubated in the P(i)-complete medium, they accumulated polyP and exhibited a phosphate overplus effect. Under phosphate overplus the polyP level in mitochondria was two times higher than in the complete medium without preliminary P(i) starvation. The average chain length of polyP in mitochondria was of <15 phosphate residues at 19.3 mM P(i) in the culture medium and increased at phosphate overplus. Deoxyglucose inhibited polyP accumulation in spheroplasts, but had no effect on polyP accumulation in mitochondria. Uncouplers (FCCP, dinitrophenol) and ionophores (monensin, nigericin) inhibited polyP accumulation in mitochondria more efficiently than in spheroplasts. Fast hydrolysis of polyP was observed after sonication of isolated mitochondria. Probably, the accumulation of polyP in mitochondria depended on the proton-motive force of their membranes.     

Preparation and characteristics of protoplasts of Streptomyces kanamyceticus

To prepare actively regenerating protoplasts of S. kanamyceticus, the influence of the conditions of the mycelium cultivation, the culture age, lytic conditions, composition of the regeneration medium, the procedure of the culture inoculation to the regeneration medium and other parameters were studied. The study resulted in development of optimal conditions for preparation of S. kanamyceticus protoplasts in a number of 1.10(9) protoplasts per ml. The cultivation on the ST medium with 10 to 15% sucrose and addition of glycine up to 1% for 30 hours (the stationary growth phase) followed by treatment of the culture with lysozyme in an amount of 2 mg/ml for 1 hour at 32 degrees C provided preparation of up to 100% of actively regenerating protoplasts free of mycelium fragments. The size of the protoplasts increased up to 1.5 micron against the usually observed size of 0.7 to 1.0 micron with using modified lyzing buffer with 20% of sucrose according to the method recommended for S. erythreus. However, 50 to 70% of the protoplasts had point of linear regions in the cell walls, which suggested that spheroplasts were mainly forming and the phenomenon was associated with the characteristic properties of the strain cell wall structure.     

Growth and cell division ofEscherichia coli 15 TAU after transfer to deficient media with different sources of carbon and energy

We studied growth and cell division ofEscherichia coli 15 TAU after transfer to thymine-free medium with different sources of carbon and energy or to the same medium in which not only thymine but also arginine and/or uracil were omitted. After transfer to thymine-free medium only a fraction of cells divides once. The size of the dividing fraction is predetermined particularly by conditions of balanced growth before inhibition of DNA synthesis and only slightly affected by conditions after transfer, while the growth rate after shift to medium with different source of carbon and energy changes abruptly. Following transfer to arginine-deficient media cell division proceeds much more slowly than in other cases tested. The fraction of cells which causes a deviation of rate maintenance after shift-up and shift-down (Cooper, 1969) seems to be the same as the cell fraction dividing after transfer to thymine-free medium.