Sensitization of three strains of chlorococcal algae for UV- effects by 5- bromodeoxyuridine

The sensitization of chlorococcal algae by 5-BdU for the purpose of UV-light mutagenesis was studied. The results obtained were compared with our earlier findings on the sensitization of the same algal strains by 5-BU. No shielding effect of the 5-BdU molecules against UV-light was observed. Probably, the uptake of them from the liquid medium did not result in such excess as compared with the treatment by 5-BU, even if the cells were long enough (24 h) exposed to the concentration of 5-BdU. Likewise, neither stimulating nor inhibiting growth effects on the growing cell colonies were observed after treatment with 5-BdU. The sensitization of the algal cells for UV-light effects was effective in all the experiments. An increased damage of the algal cells by UV-light after sensitization was proved in all the parameters recorded. The frequencies of permanent changes of the cells or their colonies were also increased, but their spectrum did not change significantly. A suitable combination of the 5-BdU sensitization of the cells before their influencing by UV-light and the induction of their repair mechanisms by visible light may decrease the frequencies of the lethal or sublethal damage and increase the frequencies of the useful permanent changes in the characteristics of the chlorococcal algae. The results obtained are discussed from the viewpoint of the regulated mutation process in the breeding of algae.     

Comparison of N-ethyl-N-nitrosourea Effects in three strains of chlorococcal algae

Three strains of three different species of chlorococcal algae(Chlorella kesslerii Scenedesmus quadricauda andScenedesmus obliquus) were compared in their physiological and genetical responses to N-ethyl-N-nitrosourea (NEU). The mutagen was applied in the basic doses corresponding to their specific tolerance to it. Exponentially-graded doses derived from the basic ones acted for the same period of time and single basic doses acted at linearly-increased periods of time in parallel experiments. The influence of the mutagen on the cell cycle just in progress was characterized by the increase in the frequency of cell divisions which yielded a lower number of autospores. The conditions of the cells during the first three days after treatment were determined according to the average increase of their lag phase. The relative toxicity of the mutagen was expressed as the survival frequencies. The mutation effects were evaluated according to the frequency of permanent changes observed in the growing cell colonies : lethal, morphological and pigmentation. NEU was found to be a relatively suitable mutagen for the chlorococcal algae studied.     

Growth of Variants of Myxococcus virescens

Some observations on variant strains of Myxococcus virescens B2 with special emphasis on characteristics associated with the ability to grow in dispersion are reported. The isolated strains were divided into two major classes according to their mode of growth in shaken and static liquid cultures based on casitone and casamino acids media. Strains growing in dispersion were designated D⁺-strains and those growing in aggregates or as films, D^?-strains. Colony morphology, cell morphology, growth in liquid and on solid medium and morphogenesis were compared. The ability to grow in dispersion shown by D⁺-strains seemed to be associated with a smooth colony on casitone agar, inability to form typical fruiting bodies and a low linear growth rate of colonies on solid medium as compared with the D^?-strains. In contrast D^?-strains produced rough colonies on casitone agar, were able to fruit and evidently formed an adhesive slime in the form of fibrils extending from the cell surface. It is suggested that the observed differences depend on different envelopes of the cells in the two classes.     

Influence of low doses of mutagens on growth of algae on a solid medium

The effects of low doses of two mutagens (UV-light and ethyl methansulphonate) on the growth of algae on a solid minimal (anorganic) medium were studied. It was supposed that low doses of mutagens will be more suitable for the induction of growth mutations. The UV-light had an inhibitory effect in the whole range of the applied doses on the growth of colonies of algae from the cells inoculated on the solid medium immediately after irradiation. Ethyl methansulphonate produced growth stimulation if applied in the lowest doses. The growth was inhibited in a further range of doses and then there appeared the range of lethal doses. The growth responses to the influence of these mutagens were different in all the three algal species used and to the previous cultivation conditions before their exposure to the mutagens. It is certain that most of these growth responses are only modifications. The influence of ethyl methansulphonate differed according to the method of application. If it affected the algae for a long time (it means if they were inoculated directly on the solid medium containing mutagens), or, if the algae were exposed to its influence immediately before their inoculation on the solid medium, the growth responses of colonies were quite different. Growth responses with the single studied strains differed quantitatively only.     

A self-renewing, bipotential erythroid/mast cell progenitor in continuous cultures of normal murine bone marrow

We have established permanent lines of nonadherent cells from fresh normal mouse bone marrow in media containing pokeweed mitogen-stimulated spleen cell conditioned medium (PWSCM). These lines continuously produced erythropoietic progenitor cells (detected by their ability to form erythroid bursts in semi-solid medium containing erythropoietin) together with cells having characteristics of the mast cell lineage (as demonstrated by metachromatic staining with toluidine blue, histamine content and membrane receptors for IgE). Sixteen such cell lines have been established in sixteen attempts. Cloning experiments were carried out to determine the nature of the progenitor cell(s) responsible for the permanence of these cultures. When cells were cultured in methylcellulose medium containing PWSCM, colonies were observed which reached macroscopic size after 4 weeks of incubation. Replating of individual primary colonies resulted in secondary colony formation, indicating the presence of progenitor cells with self-renewal potential. Forty-seven primary colonies were picked and their cells were suspended in liquid culture medium containing PWSCM. Of these, twenty-one could be expanded to establish permanently growing sublines. Sixteen of these sublines were found to be composed of both erythroid progenitors and mast cells. In five sublines only mast cells could be seen; none of the sublines appeared to be purely erythroid. Karyotypic analysis of mast cells and of erythroid cells of seven sublines derived from individual colonies which arose in cocultures of male and female cells revealed that the mast cells and erythroid cells were both of the same sex in each of the seven sublines; this demonstrates the single cell origin of each colony and of the two lineages derived from it. We conclude that these nonadherent, factor-dependent cell lines are maintained by self-renewal and differentiation of bipotential progenitor cells apparently restricted to the erythroid and mast cell lineages.     

Colony morphology on agar is a sensitive indicator for growth effectors

Colonies of Chinese hamster ovary (CHO) cells growing on agar exhibit changes in colony morphology and size in response to extremely low doses of hormones and growth factors. Computer-aided densitometric scanning of photographs of these colonies allows the quantitative measurement of these morphological changes. Correlation of these changes with dosage for a variety of growth effectors is a sensitive assay for the effects of these factors, both alone and in combination, and should be useful in comparing effects of agents with similar biological activities and in the search for variant cell types with altered responsiveness. Cells growing attached to solid substrates are often responsive to these low dosages and do not seem to mimic in vivo growth effector phenomena as well as colonies growing in three dimensions on agar.     

Isolation ofThiobacillus ferrooxidans from various habitats and their growth pattern on solid medium

Different strains of iron-oxidizingThiobacillus ferrooxidans were grown and purified on solid medium containing Bapco agar, agarose, and carrageenan (Type 1). These strains produced easily countable isolated colonies that could be transferred after 7 days of incubation at 30°C. Increase in viable cell number in relation to growth and iron oxidation was studied by both microscopic count and direct plating method. Colony morphology of different strains growing on solid medium helped in differentiating the colony types.     

Reversible and permanent effects of the carbon sources and various antibiotics on the morphology and metabolic properties of Ustilago cynodontis cells

The effects of various carbon sources and of antibiotics on the morphology of hypha cells of the fungus Ustilago cynodontis is described. Nonfermentable substrates promote readily reversible yeastlike colonies from hypha cells: all the hypha cells spread on these substrates give rise to yeastlike colonies that revert to the mycelial phenotype when transferred to glucose medium. Among the antibiotics tested, chloramphenicol (CAP) is found to promote, under certain circumstances, a long-lasting, even permanent modification on the morphology of the colonies: the colonies developed on CAP-glucose media are yeastlike, and a percentage of them give rise to colonies whose morphology remains yeastlike even on drug-free media: this effect is also obtained with cells cultivated in liquid medium. This permanent morphological modification is accompanied by a change of metabolic properties. Similar permanent effects are obtained with ethidium bromide, suggesting that mitochondrial functioning is involved in these modifications.     

Effect of certain factors on the variability of strains of pertussis microbes

The population of degraded cells having stable changes in some phenotypical properties were isolated after subculturing some laboratory Bordetella pertussis strains in Bordet-Gengou culture medium and casein-charcoal agar with blood, treated with mitomycin C and allowed to proliferate in the spleen of mice injected intravenously with microbial suspensions. The characteristics indicative of cell degradation were the growth of large yellowish-white colonies appearing in 24 hours, the destruction of the agglutinogenic complex and toxic substances causing the atrophy of the spleen in mice, the increased capacity for active proliferation in the spleen. Electron-microscopic study revealed that the variants obtained by subculturing in culture media had the damaged membrane with the formation of cell-wall invaginations having rounded membrane-like formations on their surface, disappearing after treatment with mitomycin C; the treatment of the initial strains with mitomycin C resulted in cytoplasmic damage with the coagulation of the nucleoid.     

Genotype versus phenotype variability in Chlorella and Micractinium (Chlorophyta, Trebouxiophyceae)

The most recent revision of the genus Chlorella, based on biochemical and SSU rDNA analyses, suggested a reduction to a set of four "true" spherical Chlorella species, while a growing number of morphologically different species such as Micractinium (formerly Micractiniaceae) were found to cluster within the clade of "true"Chlorella. In this study, the generic concept in Chlorellaceae to Chlorella and Micractinium was evaluated by means of combined SSU and ITS-2 rDNA sequence analyses and biotests to induce development of bristles on the cell wall. Molecular phylogenetic analyses of Chlorella and Micractinium strains confirmed their separation into two different genera. In addition, non-homoplasious synapomorphies (NHS) and compensatory base changes (CBC) in the secondary structures of SSU and ITS-2 rDNA sequences were found for both genera using this approach. The Micractinium clade can be differentiated into three different genotypes. Using culture medium of the rotifer Brachionus calyciflorus, phenotypic plasticity in Chlorella and Micractinium was studied. Non-bristled Micractinium cells developed bristles during incubation with Brachionus culture medium, whereas Chlorella did not produce bristles. Grazing experiments with Brachionus showed the rotifer preferred to feed on non-bristled cells. The dominance of colonies versus solitary cells in the Micractinium culture was not correlated with the "Brachionus factor". These results suggest that morphological characteristics like formation of bristles represent phenotypic adaptations to the conditions in the ecosystem.     

The influence of sodium nitrite on the osmoresistance of Chinese hamster cells

We have previously demonstrated that incubation of V-79 cells in the medium containing the nitric oxide donor, NaNO2, increases cell resistance to damaging effect of gamma-rays, UV radiation and hyperthermia. In the present study, we investigated the effects of the nitric oxide donor on the sensitivity of V-79 cells to changes in osmomolarity of the medium by adding different amounts of sodium chloride or water. We found that pretreatment of the cells with NaNO2 resulted in a significant increase in the number of growing cells in 48 h after the treatment. The osmomolarity-dependent morphological changes in cultured cells were also substantially diminished following NaNO2 treatment. This effect could be observed under both hyper- and hypoosmosis, and was dependent on concentration of sodium chloride in hypertonic medium (being maximal under 0.17 M NaCl) and on the amount of water in hypotonic medium (being maximal under 1.1 times the dilution with water). In the experiments with increased osmomolarity, we found that the observed increase in the number of growing cells following NaNO2 treatment was accompanied with a significant increase of the mitotic index. These findings indicate that nitric oxide increases cell resistance to the damaging effects of osmotic shock in the way which is similar to the protective effect of these molecules against radiation and hyperthermia. Similarities in the effects of NaNO2 under different conditions leading to cell damage suggest that nitric oxide might serve as the universal factor participating in recovery of damaged cells and mediating increased cellular resistance to the damaging conditions.     

The Effect of Rhamnolipid Biosurfactant Produced by <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> on Model Bacterial Strains and Isolates from Industrial Wastewater

In this study, the effect of rhamnolipid biosurfactant produced by Pseudomonas fluorescens on bacterial strains, laboratory strains, and isolates from industrial wastewater was investigated. It was shown that biosurfactant, depending on the concentration, has a neutral or detrimental effect on the growth and protein release of model Gram (+) strain Bacillus subtilis 168. The growth and protein release of model Gram (−) strain Pseudomonas aeruginosa 1390 was not influenced by the presence of biosurfactant in the medium. Rhamnolipid biosurfactant at the used concentrations supported the growth of some slow growing on hexadecane bacterial isolates, members of the microbial community. Changes in cell surface hydrophobicity and permeability of some Gram (+) and Gram (−) isolates in the presence of rhamnolipid biosurfactant were followed in experiments in vitro. It was found that bacterial cells treated with biosurfactant became more or less hydrophobic than untreated cells depending on individual characteristics and abilities of the strains. For all treated strains, an increase in the amount of released protein was observed with increasing the amount of biosurfactant, probably due to increased cell permeability as a result of changes in the organization of cell surface structures. The results obtained could contribute to clarify the relationships between members of the microbial community as well as suggest the efficiency of surface properties of rhamnolipid biosurfactant from Pseudomonas fluorescens making it potentially applicable in bioremediation of hydrocarbon-polluted environments.     

Global analysis of endothelial cell line proliferation patterns based on nutrient-depletion models: implications for a standardization of cell proliferation assays

It is known that cell populations growing in different environmental conditions may exhibit different proliferation patterns. However, it is not clear if, despite the diversity of the so-observed patterns, inherent cellular growth characteristics of the population can nevertheless be determined. This study quantifies the proliferative behaviour of the permanent endothelial human cell line, Eahy926, and establishes to which extent the estimation of the cell proliferation rate depends on variations of the experimental protocols. Cell proliferation curves were obtained for cells cultured over 16 days and the influences of cell seeding densities, foetal bovine serum content and frequency of culture medium changes were investigated. Quantitative dynamic modelling was conducted to evaluate the kinetic characteristics of this cell population. We proposed successive models and retained a nutrient-depletion toxicity dependant model, which takes into account the progressive depletion of nutrients, as well as the increase of toxicity in the cell culture medium. This model is shown to provide a very good and robust prediction of the experimental proliferation curves, whatever are the considered frequency of culture medium changes and serum concentrations. Thus, the model enables an intrinsic quantification of the parameters driving in vitro EAhy926 proliferation, including proliferation, nutrient consumption and toxicity increase rates, rather independently of the experiments design. We therefore propose that such models could provide a basis for a standardized quantification of intrinsic cell proliferation kinetics.     

Enrichment and characterization of clonogenic epithelial cells from adult rat liver and initiation of epithelial cell strains

Summary A highly efficient method is described for obtaining prolifertive epithelial cells from adult rat livers for the reproducible establishment of liver epithelial cell strains. When cells were isolated from livers of 10-to 15-wk-old male Fischer 344 rats by a collagenase-perfusion method, collected by centrifugation at 50×g for 5 min, and cultured in Williams' medium E containing fetal bovine serum and dexamethasone, colonies of epithelial cells different in size and morphology from hepatocytes were obtained. Sequential perfusion with collagenase and dispase yielded numerous epithelial cell colonies. When isolated cells were fractionated by differential centrifugation, the great majority of hepatocytes were sedimented at 50 ×g for 1 min, whereas many non-hepatocytic cells remiined in the supernatant and could be sedimented by a second centrifugation at 50×g for 5 min. Culture of the two fractions revealed that almost all the epithelial cell colonies were derived from cells in the non-hepatocytic cell fraction. The epithelial cells were cytochemically negative for γ-glutamyl transpeptidase activity, whereas an increase in the activity was detected in hepatocytes with duration in culture. Ultrastructural characteristics of hepatocytes were not found in the cells of newly established cell strains. These results suggest that adult rat liver epithelial cells propagable in culture were derived from a cell type other than the hepatocyte.     

Growth of Gram-negative bacteria in the presence of organic solvents

The growth behavior of Gram-negative bacteria when exposed to high concentrations (50% v/v) of water-insoluble organic solvents was investigated. The solvents were chosen according to their polarity values as denoted by a logarithmically expressed parameter log P, where P is the partition coefficient of a given solvent in an equimolar mixture of octanol and water. The cell growth was measured by the number of colonies developed on a solid agar medium in direct contact with the solvents. All 31 strains tested showed characteristic growth patterns. The survival and subsequent growth of bacteria increased with the increase in the log P value and was found to be strain specific. For all the strains, 100% cell growth was reached from 0% within 0.1–0.4 log P units. Log P₅₀ values, defined as the log P values at which 50% of the cells form colonies, were determined for each bacterial strain. On the whole, Pseudomonas strains were found to be more resistant to apolar solvents than all other bacteria tested. This resistance was dependent not only on the polarities but also on the toxic nature of different organic solvents, the cell membrane components, and to a limited extent, the growth medium. A tenfold increase in the Mg²⁺ concentration in the growth medium enhanced the solvent resistance of E. coli but had no such effect on Pseudomonads. In general, different growth temperatures had no impact on the solvent resistance of the Gram-negative bacteria tested.     

Iron-induced pigment formation and ionizing radiation resistance in three strains of Micrococcus violagabriellae

Three strains of Micrococcus violagabriellae-Littlepig, Pig (Parental), and Superpig--were studied. The pulcherrimin pigment (which imparts a violet-red color to the colonies of the Parental and Superpig strains) is produced only when the cells are grown in the presence of excess iron and provides a unique system for the study of the radioprotection conferred by a pigment. Cell suspensions of each of the three strains (initially grown in the presence and in the absence of excess iron) were exposed to gamma rays and plated on media containing an excess of iron and on media without iron. The survival curves of the treated strains indicate a correlation between the color intensity of the pigment (pulcherrimin), and the ability of the organisms to survive moderate doses of ionizing radiation. Studies using radioactive iron (59Fe) showed that a correlation also exists between the intensity of the pigment and the amount of iron that the cells of each strain can remove from the medium. The effect of gamma irradiation on postirradiation cell growth is discussed for each strain irradiated during logarithmic growth and during a stationary state.     

Do MCF7 cells cope with metformin treatment under energetic stress in low glucose conditions?

There is a growing body of evidence about metformin being effective in cancer therapy. Despite controversies about the ways of its effectiveness, several ongoing clinical trials are evaluating the drug when used as an adjuvant or a neo-adjuvant agent. We aimed to investigate metformin’s effects on proliferation, metastasis, and hormone receptor expressions in breast cancer cell line MCF-7 incubated in two different glucose conditions. MCF-7 cells were incubated in high or low glucose media and treated with various doses of metformin. The cell viability was studied using MTT test. The Ki-67, estrogen and progesterone receptor expression were evaluated by ICC and galectin-3 expression was evaluated by ELISA or spectrophotometrically. The cell viability following consecutive metformin doses in either glucose condition for 24 and 48 h represented a significant decrease when compared to control. The proliferation detected in low glucose medium following metformin at doses < 20 mM was found significantly decreased when compared to high glucose medium at 48 h. In terms of galectin-3 levels, the increase in high glucose medium treated with metformin and the decrease in low glucose medium were found statistically significant when compared to control. Progesterone receptor staining demonstrated a significant increase in low glucose medium. Our findings represent better outcomes for cancer lines incubated in low glucose medium treated with metformin in terms of viability, receptor expression and metastatic activity, and highlight the potential benefit of metformin especially in restraining the cancer cell’s ability to cope energetic stress in low glucose conditions.     

Transfection protocol for antisense oligonucleotides affects uniformity of transfection in cell culture and efficiency of mRNA target reduction

In an effort to optimize the transfection of cell lines with antisense oligonucleotides, we examined cellular accumulation of a labeled oligonucleotide by flow cytometry. We were surprised to observe that a routinely used transfection protocol, a fixed lipid/oligonucleotide ratio, resulted in variable transfection efficiency depending on the concentration of oligonucleotide used. A significant population of cells, especially at lower doses of oligonucleotide and cationic lipid, were untransfected. We investigated lipid/oligonucleotide ratios, different lipid preparations, and different cell types and found that these variables did not alter the percentage of cells transfected at these lower doses of oligonucleotide. However, when lipid-oligonucleotide complexes were formed at the high dose and then diluted into a solution of lipid or a complex of lipid and unlabeled, negative control oligonucleotide, a constant percentage of cells was transfected. Under these conditions, mRNA target reduction dose-response curves were also shifted to lower doses. We hypothesize that poor transfection observed at a low concentration of lipid-oligonucleotide complex when diluted in medium is due to loss of active complexes, either by adsorption to the substrate or by changes in physical characteristics of complexes. By maintaining a constant lipid concentration, more consistent transfection was achieved.     

Longevity of U cells of differentiated yeast colonies grown on respiratory medium depends on active glycolysis

Colonies of Saccharomyces cerevisiae laboratory strains pass through specific developmental phases when growing on solid respiratory medium. During entry into the so-called alkali phase, in which ammonia signaling is initiated, 2 prominent cell types are formed within the colonies: U cells in upper colony regions, which have a longevity phenotype and activate the expression of a large number of metabolic genes, and L cells in lower regions, which die more quickly and exhibit a starvation phenotype. Here, we performed a detailed analysis of the activities of enzymes of central carbon metabolism in lysates of both cell types and determined several fermentation end products, showing that previously reported expression differences are reflected in the different enzymatic capabilities of each cell type. Hence, U cells, despite being grown on respiratory medium, behave as fermenting cells, whereas L cells rely on respiratory metabolism and possess active gluconeogenesis. Using a spectrum of different inhibitors, we showed that glycolysis is essential for the formation, and particularly, the survival of U cells. We also showed that β-1,3-glucans that are released from the cell walls of L cells are the most likely source of carbohydrates for U cells.