Increase in cell mass during the division cycle of Escherichia coli B/rA.

Increase in the mean cell mass of undivided cells was determined during the division cycle of Escherichia coli B/rA. Cell buoyant densities during the division cycle were determined after cells from an exponentially growing culture were separated by size. The buoyant densities of these cells were essentially independent of cell age, with a mean value of 1.094 g ml-1. Mean cell volume and buoyant density were also determined during synchronous growth in two different media, which provided doubling times of 40 and 25 min. Cell volume and mass increased linearly at both growth rates, as buoyant density did not vary significantly. The results are consistent with only one of the three major models of cell growth, linear growth, which specifies that the rate of increase in cell mass is constant throughout the division cycle.     

Influence of sulfur accumulation and composition of sulfur globule on cell volume and buoyant density of Chromatium vinosum

Average cell volume and cell buoyant density of Chromatium vinosum DSM 185 growing in sulfide limited continuous cultures, were found to increase with increasing dilution rate. It was found that the increase in buoyant density was mainly a consequence of the accumulation of elemental sulfur. The contribution of other compounds such as protein, bacteriochlorophyll a and glycogen, was almost negligible. It was concluded that the sulfur globule is constituted by at least two fractions, sulfur and an unidentified moiety with a density lower than that of sulfur, probably water.A model was developed to explain the relation between the specific content of sulfur and cell buoyant density. The model also predicts the impact of elemental sulfur on the volume of the cell. It was found that in addition to the accumulation of sulfur the average cell volume also changes with the specific growth rate.In shift-up experiments (sulfur accumulation) the actual phenomena agreed with those predicted by the model, however, this was not so during shift-down (sulfur depletion). It is suggested that this difference is due to the fact that during the shift-down, elemental sulfur and the unidentified moiety are being depleted at different rates.Non-standard abbreviations BChl bacteriochlorophyll - PHB poly--hydroxybutyric acid - D dilution rate - specific growth rate - S _R reservoir concentration of limiting substrate     

Two-dimensional impedance studies of BSA buoyant density separated human erythrocytes

Combined DC (Coulter Volume) and radio frequency impedance studies were performed on human erythrocytes which had been separated by buoyant density in linear, neutral, isotonic bovine serum albumin gradients. The individual buoyant density fractions showed no reproducible shift in volume with buoyant density but did show a shift with opacity, radio frequency impedance divided by dc impedance. This new electronic parameter of opacity can be related to cell age, since both it and cell age are directly related to buoyant density. This increase in opacity with buoyant density is correlated with a change in shape.     

Evaluation of an electrochemical model of erythrocyte pH buffering using 31P nuclear magnetic resonance data

When erythrocytes are suspended in a solution of known composition the resultant values of such basic cell parameters as volume and pH are difficult to predict. To facilitate such predictions, we developed a mathematical model describing the passive transmembrane distribution of permeant species; three simultaneous equations were produced. Certain essential data required for the model were determined experimentally; these included the pH dependence of the charge on the hemoglobin molecule and the variation of the osmotic coefficient of hemoglobin with cell volume. Finally, cells were added to various solutions, and then titrated to produce a wide pH range (pH 6-8). We measured the resultant cell volume, cellular and extracellular pH using both conventional and 31P NMR methods. The expected equilibrium values of these electrochemical parameters were also calculated by solving (numerically) the three model equations. The accuracy of the model simulations was evaluated by direct comparison of calculated and experimentally determined values.     

Buoyant Densities and Dry-Matter Contents of Microorganisms: Conversion of a Measured Biovolume into Biomass

Several isolates of bacteria and fungi from soil, together with cells released directly from soil, were studied with respect to buoyant density and dry weight. The specific volume (cubic centimeters per gram) of wet cells as measured in density gradients of colloidal silica was correlated with the percent dry weight of the cells and found to be in general agreement with calculations based on the partial specific volume of major cell components. The buoyant density of pure bacterial cultures ranged from 1.035 to 1.093 g/cm³, and their dry-matter content ranged from 12 to 33% (wt/wt). Average values proposed for the conversion of bacterial biovolume into biomass dry weight are 1.09 g/cm³ and 30% dry matter. Fungal hyphae had buoyant densities ranging from 1.08 to 1.11 g/cm³, and their dry-matter content ranged from 18 to 25% (wt/wt). Average values proposed for the conversion of hyphal biovolume into biomass dry weight are 1.09 g/cm³ and 21% dry matter. Three of the bacterial isolates were found to have cell capsules. The calculated buoyant density and percent dry weight of these capsules varied from 1.029 g/cm³ and 7% dry weight to 1.084 g/cm³ and 44% dry weight. The majority of the fungi were found to produce large amounts of extracellular material when grown in liquid cultures. This material was not produced when the fungi were grown on either sterile spruce needles or membrane filters on an agar surface. Fungal hyphae in litter were shown to be free from extracellular materials.     

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

Summary 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.     

Buoyant density studies of several mecillinam-resistant and division mutants of Escherichia coli.

The buoyant density of wild-type Escherichia coli cells has previously been reported not to vary with growth rate and cell size or age. In the present report we confirm these findings, using Percoll gradients, and analyze the recently described lov mutant, which was selected for its resistance to mecillinam and has been suggested to be affected in the coordination between mass growth and envelope synthesis. The average buoyant density of lov mutant cells was significantly lower than that of wild-type cells. Similarly, the buoyant density of wild-type cells decreased in the presence of mecillinam. The density of the lov mutant, like that of the wild type, was invariant over a 2.8-fold range in growth rate. In this range, however, the average cell volume was also constant. Analysis of buoyant density as a function of cell volume in individual cultures revealed that smaller (newborn) lov mutant cells had higher density than larger (old) cells; however, the density of the small cells never approached that of the wild-type cells, whose density was independent of cell size (age). A pattern similar to that of lov mutant cells was observed in cells carrying the mecillinam-resistant mutations pbpA(Ts) and rodA(Ts) and the division mutation ftsI(Ts) at nonpermissive temperatures as well as in wild-type cells treated with mecillinam, but not in mecillinam-resistant crp or cya mutants.     

Biochemical properties of macrophage fractions and their relation to the mechanism of superoxide production

Guinea pig alveolar macrophages are separable by density gradient centrifugation into three subpopulations whose capacity for biological activity (e.g. O2- production and chemotaxis) varies directly with buoyant density [(1983) J. Reticuloendothel. Soc. 33, 157-164]. This study demonstrates that the activity per cell of various other enzymes remains constant among the subpopulations. When normalized for cell volume, enzyme activity diminishes with decreasing buoyant density. Intracellular calcium mobilization, linked to formyl peptide and concanavalin A-stimulated O2- production, similarly diminishes. Formyl peptide receptor distribution and affinity remain constant. Decreased responsiveness of lower density cells is probably due to lower concentration of enzyme(s) involved in the transduction of signal distal to ligand recognition (or binding).     

Macromolecular distribution near the limits of density-gradient columns. Some applications to the separation and fractionation of glycoproteins.

1. Expressions are derived for the distribution at density-gradient equilibrium of macromolecules whose densities are (a) close to the values characterizing the solution limits or (b) outside the span of the gradient. 2. Density-distribution predicted by the expressions agree with those obtained by rigorous methods. 3. The distribution equations are applied to hypothetical mixtures of proteins and glycoproteins in commonly used density-gradient media to simulate separation and fractionation conditions. 4. It is shown that CsBr, although less efficient than CsCl for fractionation, is nevertheless adequate for most purposes; in analytical experiments it may often have advantages over CsCl. Limitations on the use of LiBr are explored. 5. An expression is derived which allows the variance of the partial specific volume of the macromolecular component to be determined from the variance of the buoyant density. It is shown that the relative resolving powers of different salts is expressed by their values of the quantity (formula: see text). 6. The equations are applied to a well-characterized glycoprotein preparation at equilibrium in CsCl and in Cs2SO4:it is shown that the much wider distribution in CsCl than in Cs2SO4 is explicable in terms of the variance in buoyant density and the solvation properties of the salts. 7. Limitations of the expressions arise when dispersity in density is represented by a low apparent molecular weight; realistic simulations can then only be obtained when the component is fully banded.     

Electrochemical sterilization of bacteria adsorbed on granular activated carbon

Subcellular distribution of chitin synthetase has been studied in germ tubes of Candida albicans. Two fractions with synthetase activity were separated from cell homogenates: (i) a mixed membrane fraction where the enzyme, partly in an active form, is associated with the plasma membrane (isopycnic centrifugation of mixed membrane fraction on linear sucrose gradients resolved a unique peak of activity matching with [3H]ConA-labelled membranes at a buoyant density of 1.195 g/ml); and (ii) a cytoplasmic fraction containing fully zymogenic enzyme associated with particles whose buoyant density (determined by isopycnic centrifugation on linear sucrose gradients) depended on the cell breakage conditions. The actual cytoplasmic fraction-enzyme may correspond to particles with buoyant density 1.135 g/ml (chitosomes), whereas the enzyme particles with other densities (1.085 and 1.165 g/ml) probably originated during cell disruption, as has been reported previously to occur during the preparation of yeast cell homogenates.     

Chitin synthetase activity is bound to the plasma membrane and to a cytoplasmic particulate fraction in Candida albicans germ tube cells

Abstract Subcellular distribution of chitin synthetase has been studied in germ tubes of Candida albicans . Two fractions with synthetase activity were separated from cell homogenates: (i) a mixed membrane fraction where the enzyme, partly in an active form, is associated with the plasma membrane (isopycnic centrifugation of mixed membrane fraction on linear sucrose gradients resolved a unique peak of activity matching with [³H]ConA-labelled membranes at a buoyant density of 1.195 g/ml); and (ii) a cytoplasmic fraction containing fully zymogenic enzyme associated with particles whose buoyant density (determined by isopycnic centrifugation on linear sucrose gradients) depended on the cell breakage conditions. The actual cytoplasmic fraction-enzyme may correspond to particles with buoyant density 1.135 g/ml (chitosomes), whereas the enzyme particles with other densities (1.085 and 1.165 g/ml) probably originated during cell disruption, as has been reported previously to occur during the preparation of yeast cell homogenates.     

Sedimentation equilibrium of proteins in density gradients.

The technique of sedimentation equilibrium in density gradients in the analytical ultracentrifuge has been applied to the study of proteins. A variety of effects and procedures including the use of density marker beads, the effects of pressure on buoyant density and pH, and the calculation of compositional density gradient proportionality constants and density--refractive index relations have been developed. The buoyant densities of twenty-four proteins have been measured and hydration values computed. The buoyant titrations of six proteins have been measured. These data have been interpreted in terms of the buoyant titrations which have been obtained for six ionizable homopolypeptides, five copolypeptides, two non-ionizable homopolypeptides and three chemically modified proteins. Spectropolarimetry and potentiometric titrations were employed to further interpret these data. Approximate values for dissociation constants, numbers of ionizable residues, and the nature of ions bound or dissociated upon ionization have been obtained. The relation between potentiometric and buoyant titrations and the use of density gradient centrifugation as a probe for protein structure have been explored.     

Buoyant density variation during the cell cycle of Saccharomyces cerevisiae

Cell buoyant densities of the budding yeast Saccharomyces cerevisiae were determined for rapidly growing asynchronous and synchronous cultures by equilibrium sedimentation in Percoll gradients. The average cell density in exponentially growing cultures was 1.1126 g/ml, with a range of density variation of 0.010 g/ml. Densities were highest for cells with buds about one-fourth the diameter of their mother cells and lowest when bud diameters were about the same as their mother cells. In synchronous cultures inoculated from the least-dense cells, there was no observable perturbation of cell growth: cell numbers increased without lag, and the doubling time (66 min) was the same as that for the parent culture. Starting from a low value at the beginning of the cycle, cell buoyant density oscillated between a maximum density near midcycle (0.4 generations) and a minimum near the end of the cycle (0.9 generations). The pattern of cyclic variation of buoyant density was quantitatively determined from density measurements for five cell classes, which were categorized by bud diameter. The observed variation in buoyant density during the cell cycle of S. cerevisiae contrasts sharply with the constancy in buoyant density observed for cells of Escherichia coli, Chinese hamster cells, and three murine cell lines.     

Buoyant and potentiometric titrations of synthetic polpeptides. II. Five copolypeptides and two nonionizable homopolypeptides in CsCl solutions.

The buoyant density titrations of five ionizable copolypeptides in concentrated CsCl solutions have been determined. The results are used to formulate models for predicting the buoyant density titration behavior of copolypeptides and proteins using the previously reported homopolypeptide buoyant density titration curves. It was determined for these copolypeptides that the best predictive model must include not only the buoyant densities of the constituent amino acid residues and the relative composition, but also hydration and salt binding. Hydrations determined for the homopolypeptides are used in the copolypeptide predictive model. The hydrations of the neutral homopolypeptides were readily calculable since their buoyant densities are thermodynamically defined in terms of their partial specific volumes and hydrations. For the case of a charged macromolecule, an expression for the buoyant density as a function of the number and nature of the bound ions, its partial specific volume, and its relative hydration has also been available for some time. This heretofore intuitive relationship is now derived from thermodynamic principles and allows calculations of hydrations to charged macromolecules which bind either cations, anions, or both. The potentiometric titrations of three of the five copolypeptides in concentrated CsCl solutions were determined in order to study the effect of residue interaction and solvation effects on their ionization behavior. The potentiometric results are also combined directly with the buoyant density titration results to determine the correlation of the buoyant density with the degree of ionization. As in the cases of poly(Glu) and poly(His), the buoyant density of the copolypeptides changed linearily with the degree of ionization. The buoyant density titrations of two nonionizable homopolypeptides, poly(Gly) and poly(Ala), were determined in concentrated CsCl solutions. The buoyant density was found to increase with increasing pH, despite the fact that side chains do not contain ionizable groups. This is the first evidence from homopolypeptide or copolypeptide data that buoyant density changes can be observed from effects other than side-chain ionizations.     

Water and ion balance in rat thymocytes under apoptosis induced with dexamethasone or etoposide. Ion-osmotic model of cell volume decrease

Cell ion and water balance was studied with respect to analysis of the osmotic model of apoptotic volume decrease (AVD) in rat thymocytes under dexamethasone (1 microM, 4-6 h) or etoposide (50 microM, 5 h) treatment. Intracellular water content was determined by measurement of cell buoyant density in continuous Percoll gradient, while intracellular potassium and sodium contents were determined by flame emission analysis. Apoptosis was verified by an increase in cell buoyant density, fluorescence of cells stained with Acridine orange and Ethidium bromide (flow cytometry), by changes in the cell cycle and the appearance of sub-diploid peak in the DNA histogram (flow cytometry), and by a decrease in cell size examined with light microscope. A separate fraction of dense cells with reduced size was found to appear after dexamethasone or etoposide treatment. This fraction was considered as apoptotic. An increase in buoyant density of apoptotic cells corresponded to a decrease in cell water content. In apoptotic cells vs. cells with normal buoyant density, the intracellular potassium content was lower, but sodium content was higher. The sum of potassium and sodium contents was lower in apoptotic cells. Taken into account the loss of anions, associated with the loss of cations, the bulk decrease in ions content has been sufficient to be accounted for cell volume decrease on the basis of the ion-osmotic model.     

Morphometric analysis of fetal rat hepatocytes cultured in monolayer or following gyratory shaking

The stereological technique was used to quantify glycogen areas and endoplasmic reticulum in fetal rat hepatocytes cultured for 24 hr in monolayer (monolayer cells) or following shaking by gyratory rotation (shaken cells). The volume density and volume per cell of glycogen areas decreased in order of freshly isolated hepatocytes, monolayer cells, and shaken cells. The surface density and area per cell of smooth endoplasmic reticulum increased in order of freshly isolated cells, monolayer cells, and shaken cells. The results show that the decrease of glycogen areas and proliferation of the smooth endoplasmic reticulum are more prominent in shaken cells than in monolayer cells. Prominent proliferation of the smooth endoplasmic reticulum in shaken cells may be due to the consumption of glycogen for energy release as a result of gyratory rotation.     

Simple equations to estimate light interception by isolated trees from canopy structure features: assessment with three-dimensional digitized apple trees

* Simple models of light interception are useful to identify the key structural parameters involved in light capture. We developed such models for isolated trees and tested them with virtual experiments. Light interception was decomposed into the projection of the crown envelope and the crown porosity. The latter was related to tree structure parameters. * Virtual experiments were conducted with three-dimensional (3-D) digitized apple trees grown in Lebanon and Switzerland, with different cultivars and training. The digitized trees allowed actual values of canopy structure (total leaf area, crown volume, foliage inclination angle, variance of leaf area density) and light interception properties (projected leaf area, silhouette to total area ratio, porosity, dispersion parameters) to be computed, and relationships between structure and interception variables to be derived. * The projected envelope area was related to crown volume with a power function of exponent 2/3. Crown porosity was a negative exponential function of mean optical density, that is, the ratio between total leaf area and the projected envelope area. The leaf dispersion parameter was a negative linear function of the relative variance of leaf area density in the crown volume. * The resulting models were expressed as two single equations. After calibration, model outputs were very close to values computed from the 3-D digitized databases.     

Changes in buoyant density and cell size of Escherichia coli in response to osmotic shocks.

The buoyant density of Escherichia coli was shown to be related to the osmolarity of the growth medium. This was true whether the osmolarity was adjusted with either NaCl or sucrose. When cells were grown at one osmolarity and shocked to another osmolarity, their buoyant density adjusted to nearly suit the new osmolarity. When cells were subjected to hyperosmotic shock, they became denser than expected. When cells were subjected to hypoosmotic shock they occasionally undershot the new projected density, but the undershoot was not as dramatic as the overshoot seen with hyperosmotic shocks. Shrinkage and swelling of the cells in response to osmotic shocks could account for the change in their buoyant density. The changes in cell size after osmotic shocks were measured by two independent methods. The first method measured cell size with a Coulter Counter, and the second method measured cell size by stereologic analysis of Nomarski light micrographs. Both methods gave qualitatively similar results and showed the cells to be flexible. The maximum swelling recorded was 23% of the original cell volume, while the maximum shrinkage observed was 33%.     

Buoyancy regulation and aggregate formation in Amoebobacter purpureus from Mahoney Lake

Abstract The meromictic Mahoney Lake (British Columbia, Canada) contains an extremely dense layer of purple sulfur bacteria ( Amoebobacter purpureus ). The buoyant density of Amoebobacter cells grown in pure culture at saturating light intensity was significantly higher (1027–1034 kg m⁻³) than the density of lake water (1015 kg m⁻³). When stationary cultures were shifted to the dark, the gas-vesicle content increased by a factor of 9 and buoyant density decreased to 1002 kg m⁻³ within three days.
A novel mechanism of cell aggregation was detected for the Mahoney Lake strain. Dense cell aggregates were formed after depletion of sulfide. Formation of aggregates was correlated with an increase in cell surface hydrophobicity. Cell aggregates could be disintegrated within less than 1 s by addition of sulfide or various thiol compounds. Mercaptanes with a branched structure in the vicinity of the terminal thiol group, compounds with esterified thiol groups (methylmercaptanes), reducing compounds lacking thiol groups and detergents did not influence aggregate stability. Cell aggregates disintegrated upon addition of urea or of proteinase K. Addition of various sugars had no effect on aggregation; this points to the absence of lectins. The results indicate that cell-to-cell adhesion in A, purpureus ML1 is mainly caused by a hydrophobic effect and includes a specific mechanism possibly mediated by a surface protein.
Extrapolation of laboratory results to field conditions demonstrated that both regulation of buoyant density and formation of cell aggregates result in passive accumulation of cells at the chemocline and contribute to the narrow stratification of A. purpureus in Mahoney Lake.     

Bouyancy regulation and aggregate formation in Amoebobacter purpureus from Mahoney Lake

Abstract The meromictic Mahoney Lake (British Columbia, Canada) contains an extremely dense layer of purple sulfur bacteria ( Amoebobacter purpureus ). The buoyant density of Amoebobacter cells grown in pure culture at saturating light intensity was significantly higher (1027–1034 kg m⁻³) than the density of lake water (1015 kg m⁻³). When stationary cultures were shifted to the dark, the gas-vesicle content increased by a factor of 9 and buoyant density decreased to 1002 kg m⁻³ within three days.
A novel mechanism of cell aggregation was detected for the Mahoney Lake strain. Dense cell aggregates were formed after depletion of sulfide. Formation of aggregates was correlated with an increase in cell surface hydrophobicity. Cell aggregates could be disintegrated within less than 1 s by addition of sulfied or various thiol compounds. Mercaptanes with a branched structure in the vicinity of the terminal thiol group, compounds with esterified thiol groups (methyl-mercaptanes), reducing compounds lacking thiol groups and detergents did not influence aggregate stability. Cell aggregates disintegrated upon addition of urea or of proteinase K. Addition of various sugars had no effect on aggregation; this points to the absence of lectins. The results indicate that cell-to-cell adhesion in A. purpureus ML1 is mainly caused by a hydrophobic effect and includes a specific mechanism possibly mediated by a surface protein.
Extrapolation of laboratory results to field conditions demonstrated that both regulation of buoyant density and formation of cell aggregates result in passive accumulation of cells at the chemocline and contribute to the narrow stratification of A. purpureus in Mahoney Lake.