Buoyant Density Of Cells Research Articles

Intracellular Potassium in Cell Growth and Proliferation of Human Mesenchymal Stem Cells and Blood Lymphocytes

Many data show that K+ ions are essential for cell proliferation. In this brief review, we summarize our own studies and literature data that characterize the relationship between modulations of intracellular K+ content and the intensity of cell proliferation. Using flame emission assay we compared the transport of monovalent cations in transformed cells and in human mesenchymal stem cells in growing cultures, as well as during stress-induced cell cycle arrest and transition from monolayer (2D) to three-dimensional (3D) spheroids. A decrease in the intracellular content of K+ (evaluated as the ratio of the content of K+ in cells to the mass of cellular protein) associated with the accumulation of G1 cells in the population and accompanied by a stop in proliferation was revealed. The relationship between intracellular K+ and initiation of cell proliferation was further analyzed in human blood lymphocytes (HBL) as a model for the transition of cells from quiescence to proliferation. In HBL stimulated to proliferate, the content of K+ in cells increases during the transition G0/G1/S, accompanying the growth of small lymphocytes into blasts. Cellular water content, calculated from buoyant cell density, is higher in proliferating HBLs and in Jurkat leukemic T cells than in resting HBL. Available data suggest that intracellular K+ is important for successful cell proliferation as the main intracellular ion involved in the regulation of cell volume during the transition from quiescence to proliferation, and high K+ content and associated high water content in the cell are a characteristic feature of cell proliferation and transformation.

Variances in cellular sedimentation behavior as an effective enrichment method of hydrocarbon-overproducing Micrococcus luteus strains

BackgroundAliphatic hydrocarbons of microbial origin are highly interesting candidate biofuels because these molecules are identical or very similar to the main components of petroleum-based gasoline and diesel fuels. The high-GC Gram-positive bacterium Micrococcus luteus is capable of naturally synthesizing long-chain, iso- and anteiso-branched alkenes which are formed via the head-to-head condensation of fatty acid thioesters by a dedicated enzyme system. The present study describes the relation we observed between olefin production and cell buoyancy in Micrococcus luteus and the use of this phenotype to simply and efficiently separate cells from a mixture based on their hydrocarbon content.MethodsWe generated M. luteus mutants producing different amounts of olefins and used them in mixing and sedimentation experiments, olefin content analysis by GC-MS and in equilibrium centrifugation in Percoll gradients.ResultsWe found well-detectable differences in the buoyant densities of the examined strains, which correlated with the amounts of hydrocarbons produced by the cells. We also demonstrate how our observations can be used to simply and efficiently fractionate cells based on their hydrocarbon content.ConclusionsIn summary, we show that cultures of M. luteus cells sediment at distinct rates depending on the amounts of alkenes produced. Our results indicate that buoyant cell density is the primary cause for the observed differences in sedimentation behaviour. The simple separation strategy described here can be a valuable tool in various mutagenesis and enrichment protocols, aimed at generating and isolating strains with increased olefin productivity.

Reduction of selenite and detoxification of elemental selenium by the phototrophic bacterium Rhodospirillum rubrum.

The effect of selenite on growth kinetics, the ability of cultures to reduce selenite, and the mechanism of detoxification of selenium were investigated by using Rhodospirillum rubrum. Anoxic photosynthetic cultures were able to completely reduce as much as 1. 5 mM selenite, whereas in aerobic cultures a 0.5 mM selenite concentration was only reduced to about 0.375 mM. The presence of selenite in the culture medium strongly affected cell division. In the presence of a selenite concentration of 1.5 mM cultures reached final cell densities that were only about 15% of the control final cell density. The cell density remained nearly constant during the stationary phase for all of the selenite concentrations tested, showing that the cells were not severely damaged by the presence of selenite or elemental selenium. Particles containing elemental selenium were observed in the cytoplasm, which led to an increase in the buoyant density of the cells. Interestingly, the change in the buoyant density was reversed after selenite reduction was complete; the buoyant density of the cells returned to the buoyant density of the control cells. This demonstrated that R. rubrum expels elemental selenium across the plasma membrane and the cell wall. Accordingly, electron-dense particles were more numerous in the cells during the reduction phase than after the reduction phase.

Buoyant density of Escherichia coli is determined solely by the osmolarity of the culture medium

In previous studies, we had shown that the buoyant density of Escherichia coli is determined by the osmolarity of the growth medium by varying the osmolarity of the medium with NaCl or sucrose. However, the buoyant density of the cells always exceeded that of the growth medium. Here we determined the effect of medium with a buoyant density greater than the expected buoyant density of cells by adding Nycodenz to Luria broth. Percoll gradients of cells were analyzed by laser light scattering. The buoyant density for 125- and 375-mOsM-grown cells was 0.002 g/ml and 0.003 g/ml more, respectively, for cells grown in the presence of Nycodenz than those grown without Nycodenz, while the buoyant density of 250-mOsM-grown cells was 0.005 g/ml less for cells grown in the presence of Nycodenz than those grown without Nycodenz. Cells grown in 500-mOsM medium with or without Nycodenz had the same buoyant density. The buoyant density of cultures grown in defined medium was the same as those grown in rich medium, with only the medium osmolarity correlating to buoyant density. We conclude from these experiments that neither buoyant density nor chemical make-up of the medium determines the buoyant density of cells grown in that medium. Only the medium osmolarity determines cell buoyant density, suggesting that E. coli has no mechanisms to sense buoyant density.

Co-ordination between elongation and division in Escherichia coli mediated by the wee gene product.

A procedure to construct strains of Escherichia coli containing conditional lethal mutations in two different genes was used to construct a ftsA-3(ts) wee(Am) supF(ts) strain. This strain, OV-25-7, was used to ascertain whether the wee gene product (Wee) acts at the level of regulation of cell elongation or at the co-ordination of elongation and division. The mass per unit length and the buoyant density of cells in the absence of Wee increased only if division was allowed, as in the case of strain OV-25 (wee(Am) supF(ts)), but not when it was inhibited, as in strain OV-25-7. These results suggested that in E. coli the wee gene product was acting at the level of coordination between elongation and cell division.

Evidence for osmoregulation of cell growth and buoyant density in Escherichia coli.

The buoyant density of cells of Escherichia coli B/r NC32 increased with the osmolarity of the growth medium. Growth rate and its variability were also dependent upon the osmolarity of the medium. Maximum growth rates and minimum variability of these rates were obtained in Luria broth by addition of NaCl to a concentration of about 0.23 M.

Interrelations among Na and K content, cell volume, and buoyant density in human red blood cell populations.

This study establishes a method for determining the concentration of Na and K in single red blood cells from electron probe microanalysis of a cell's Na and K content. To this end, red blood cells were separated into subpopulations according to their buoyant density by means of bovine serum density gradient centrifugation. Cell water and Na + K contents were then determined in each fraction by conventional analytic methods with cell volume estimated from measurements of hematocrits and cell number. It was found that an inverse relationship obtains between the mean cell volume and buoyant cell density since cells increased in size as density decreased. Although the amount of hemoglobin per cell was found to slightly increase as cell density decreased, hemoglobin concentration showed the inverse relationship, indicating that buoyant cell density differences are primarily the result of differences in hemoglobin concentration. In confirmation of Funder and Wieth (Funder, J., Wieth, J.O. 1966. Scand. J. Lab. Invest. 18:167-180) cell water and cell volume was found to vary directly with the summed content of Na + K. Finally, by means of electron probe microanalysis of single cells, the cellular concentration of hemoglobin was found to vary inversely with the Na + K content, providing a quantitative basis for directly estimating cell volume, and thus ionic concentration, with this technique.

Independence of buoyant cell density and growth rate in Escherichia coli

The relationship between growth rate and buoyant density was determined for cells from exponential-phase cultures of Escherichia coli B/r NC32 by equilibrium centrifugation in Percoll gradients at growth rates ranging from 0.15 to 2.3 doublings per h. The mean buoyant density did not change significantly with growth rate in any of three sets of experiments in which different gradient conditions were used. In addition, when cultures were allowed to enter the stationary phase of growth, mean cell volumes and buoyant densities usually remained unchanged for extended periods. These and earlier results support the existence of a highly regulated, discrete state of buoyant density during steady-state growth of E. coli and other cells that divide by equatorial fission.

Isolation and Freezing Tolerances of Mesophyll Cells from Cold-hardened and Nonhardened Winter Rye

A method is described for the release of large quantities of mesophyll cells from leaves of both cold-hardened and nonhardened winter rye seedlings by a combination of enzymic and physical maceration. Such preparations usually contain a certain percentage of nonviable cells and are thus not suitable for quantitative biochemical studies. A method is also described whereby pure preparations of viable cells could be obtained by centrifugation on Percoll, using the observation that upon replacement of the isolation medium by water the viable and dead cells exhibited very different buoyant densities. The buoyant density of cells isolated from cold-hardened seedlings differed significantly from their nonhardened counter-parts. Survival of the isolated cold-hardened and nonhardened cells following extracellular freezing in water and following plasmolysis in balanced salt solutions was found to be in very close agreement with that of the plants and tissues from which they were isolated.

Prediction of survival in head and neck cancer based on leukocyte sedimentation in ficoll‐hypaque gradients

A battery of immunologic tests were performed on 42 patients with recurrent cancer of the head and neck whose median survival time was 6.8 months. Two tests correlated with survival: total white blood cell (WBC) count and the percentage of lymphoid cells (%LG) in the Ficoll-Hypaque gradient separated leukocyte fraction of blood. Patients with abnormally low values of %LB had a poor prognosis, as did patients who had greater than 8000 WBC count. Of the variation in %LG, 43% was due to the differences in differential WBC counts among the patients, with the remaining variation attributed to changes in the buoyant densities of the leukocytes. These data suggest that further study of the factors effecting buoyant density of cells may be clinically useful.

Cellular heterogeneity in the chick wing mesoblast: I. Analysis by equilibrium density gradient centrifugation

An analytic and preparative cell separation technique, equilibrium density gradient centrifugation, has been applied to the heterogeneous cell population of the developing chick wing mesoblast in an effort to isolate separately and characterize cell populations differing in buoyant density. When dissociated cell suspensions are distributed throughout a continuous bovine plasma albumin-balanced salt solution gradient and centrifuged to equilibrium, reproducible patterns characteristic of the tissue source or developmental stage of origin are obtained. Such patterns differ both qualitatively and quantitatively. More than 90% of the centrifuged cells remain viable as judged by dye exclusion, and nearly 90% are recovered from the gradients. Cellular subpopulations prepared on these gradients can be cloned yielding cartilage, muscle, or fibroblastic colonies. At stage 25 there appears a population of cells with a mean buoyant cell density of 1.0437 g-cm −3 which exhibits a markedly enhanced ability to form cartilage-making clones when compared to cells of differing density or developmental age.