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J S Clegg

Publications and source records attributed to J S Clegg.

At least 37 records · Page 2Linked to original sources
PubMed1990

Glucose metabolism and the channeling of glycolytic intermediates in permeabilized L-929 cells.

L-929 cells (mouse fibroblasts) permeabilized with dextran sulfate (DSP cells) carry out vigorous and linear rates of glycolysis when supplied with a suitable incubation medium. Glycolysis in DSP cells is pH dependent, being strongly inhibited at pH 6.5. Compared to their nonpermeabilized counterparts, DSP cells exhibit faster glycolytic rates, but tend to convert a smaller proportion of the glucose utilized to lactate. [14C]Glucose is converted to lactate by DSP cells without dilution from endogenous substrates. When exogenous 12C-labeled glycolytic intermediates (12C-I) are added to glycolyzing DSP cells the [14C]lactate produced from [14C]glucose is diluted to varying extents, depending on the intermediate. However, the extent of that dilution (reduced specific activity) is not that expected from the complete mixing of exogenous 12C-I with their corresponding 14C-labeled intermediates coming from [14C]-glucose. DSP cells also respire and convert glucose to CO2. The amount of 14CO2 produced from [14C]glucose is also reduced by addition of most 12C-I, an interesting exception being pyruvate, which had no measurable effect on 14CO2 production and caused only a modest stimulation of respiration in glycolyzing DSP cells. These results suggest that channeling, or some other form of coupling, takes place between the glycolytic production of pyruvate and its further oxidation. These observations confirm previously published data and add further support to the proposition that channeling of glycolytic intermediates occurs in DSP cells but is of the "leaky" type. Although abundant evidence in the literature indicates that various glycolytic enzymes associate with F-actin, as well as other elements of the cytomatrix, we observed no effect of cytochalasin D on lactate production even at very high concentrations of this compound. Our results are compared with those from other laboratories and discussed in the context of metabolic organization.

Animals↗
PubMed1990

Glycolysis compared in intact, permeabilized and sonicated L-929 cells.

The effects of incubation time and cell density on glycolytic rate were examined in suspensions of intact, permeabilized and sonicated L-929 cells. Sonicates exhibited strong dependence on cell density and a distinct lag in glycolytic rate, while intact cells showed no cell density dependence and linear glycolytic rates. Permeabilized cells exhibited linear glycolytic rates, but sometimes showed dependence on cell density. Rates of lactate production (nmol at 30 min/10(6) cells) were highest in sonicates and lowest in intact cells. These results are interpreted as support for the previously proposed hypothesis that enzymes of the glycolytic pathway are highly organized in intact L-929 cells.

Animals↗
PubMed1990

Effects of reduced cell volume and water content on glycolysis in L-929 cells.

Mouse L-929 cells were subjected to increasing concentrations of sorbitol, which remove cell water and reduce volume osmotically. The rate of lactate production from glucose was significantly higher in osmotically perturbed cells than in controls, both in monolayers and in suspensions. L cells can apparently use sorbitol as a glycolytic substrate; however, studies using other solutes (trehalose and sucrose) and permeabilized cells showed that the major effect of sorbitol on glycolysis in intact cells is mediated through a reduction in cell water content and volume. It is possible to explain some of these results by an increase in the chemical potentials of dissolved components of the glycolytic pathway caused by water loss; however, the relationship between water loss and glycolytic rate increase in not a simple linear one, suggesting that the situation is more complex than would result merely from increased concentrations of pathway components. Whatever the complete explanation might be, these studies show that glycolysis continues in an orderly fashion in cells that have lost about 85% of their original water content, suggesting that the operation of this pathway is not unduly sensitive to events taking place in the bulk aqueous phase.

Animals↗
PubMed1989

Evidence for intermediate channelling in the glycolytic pathway of permeabilized L-929 cells.

L-929 cells permeabilized by dextran sulfate (DSP cells) carry out vigorous and linear rates of glycolysis when supplied with a suitable incubation medium. Unlabeled 3-phosphoglycerate (PGA) added to DSP cells reduces the specific activity of lactate coming from [14C]glucose but the extent of this reduction can not be accounted for on the basis of free diffusion of PGA coming from [14C]glucose. Studies on other glycolytic intermediates, although preliminary, yield similar results. PGA also inhibits the production of lactate from glucose; however, this effect, like that of the reduction of lactate specific activity, becomes apparent only at concentrations of PGA well in excess of those considered to be physiological. We conclude that channelling of PGA, and probably other intermediates, occurs but is of the "leaky" type.

Animals↗
PubMed1989

Dielectric properties of Artemia cysts at low water contents. Evidence for a percolative transition.

Cellular cysts of the crustacean Artemia provide a useful model for studies on water-dependent mechanisms in cellular function because they can undergo reversible cycles of dehydration-rehydration. We explored their dielectric behavior over the frequency range of 10 kHz to 1 MHz, at water contents between near zero and 0.5 g H2O/g dry weight (g/g). The dc conductivity and static dielectric permittivity were evaluated from electrostatic analysis of data obtained with a three-layered capacitor. Below cyst hydrations of 0.05 g/g, negligible dielectric response was observed at all frequencies. Between 0.05 and 0.25 g/g the permittivity increased sharply then reached a near plateau up to cyst hydrations close to 0.35 g/g, above which a second abrupt increase occurred. Values for the dielectric loss (tan delta) exhibited frequency-dependent peaks over the hydration range of 0.05-0.3 g/g, followed by an abrupt increase near 0.35 g/g, an hydration at which metabolism is first initiated in this system. These hydration-dependent dielectric changes are compared with previous studies on the biology and physics of this system, and evaluated by a model involving percolative ionic (likely protonic) conduction. Percolative behavior is characterized by a sharp increase in conductivity at a critical threshold of hydration (hc) according to a power law in which the exponent, t, equals 1.65 for a three-dimensional infinite lattice. For the Artemia cyst, t = 1.64 above hc = 0.35 g/g, which is in excellent agreement with theory. These results are compared to similar studies on lysozyme which also exhibits percolative behavior connected with the onset of biological function.

Animals↗
PubMed1988

Glycolysis in permeabilized L-929 cells.

Mouse L-929 cells permeabilized with dextran sulphate (DSP cells) carry out glycolysis when supplemented with glucose, ATP and NAD+ in a suitable incubation buffer. Glycolytic rates were linear and generally independent of cell density over the range examined (1 x 10(6)-10 x 10(6) cells/ml). Electron microscopy revealed characteristic changes in DSP cell ultrastructure, notably for nuclei and mitochondria. Some cells lacked plasma membranes, while others appeared intact. In the latter case, estimates of the lesion size in plasma membranes were obtained from volume of distribution studies using 14C-labelled proteins, and infiltration of fluorescein isothiocyanate dextran. The results indicated the presence of lesions large enough to allow globular proteins of about 400 kDa to cross the cell surface. In spite of that, only about 10% of total cell protein exited from DSP cells during a 30 min incubation period. We propose that none of the glycolytic enzymes in DSP cells can exist completely in solution in the 'cytosol', suggesting extensive enzyme organization. The results are interpreted within the broader picture of metabolic organization in animal cells and the nature of the 'cytosol'.

Adenosine Triphosphate↗
PubMed1988

L-929 cells under hyperosmotic conditions. Water, Na+, and K+.

Changes in cell water content resulting from sorbitol addition to the environment of L-929 cells were evaluated gravimetrically using 14C-labeled polyethylene glycol as a probe of extracellular space. Reductions in cell water were proportional to sorbitol supplements up to 0.6 molal, above which no further measurable decrease occurred. No volume regulation occurred for at least 1 h but the percentage of cell water lost was quickly regained when physiological conditions were restored. The amount of cell water lost because of a given hyperosmotic exposure was found to exceed the loss of cell volume. That discrepancy could be the result of an overestimation of extracellular space and/or an underestimation of cell volume reduction as a result of in-folding of the cell surface. Na+ and K+ were also measured in cells of variable water content and volume: no significant change occurred in the amounts of these ions per cell, but large increases in total cell concentration resulted from hyperosmotic exposure. The sum of Na+ and K+ concentrations exceeds the total osmotic pressure of the medium indicating that an appreciable fraction of Na+ and K+ must be bound to fixed charges within the cells. The results are evaluated in the context of intracellular organization.

Animals↗
PubMed1988

Contrasting views on the organization of cytoplasm.

The widely held conception of cytoplasm as a concentrated solution of dissolved proteins and other macromolecules, metabolites and inorganic ions, within which the various organelles and other formed elements are suspended appears to be a gross over-simplification. Evidence to be reviewed briefly here, from a wide variety of experiments and cell types, indicates that a much more extensive organization exists, in animal cell cytoplasm at least. Results from experiments on dextran sulfate permeabilized L-929 cells will be presented which support that organized paradigm. It seems likely that this intracellular architecture also exerts a strong influence on the water in the regions adjacent to it, resulting in the generation of altered aqueous phase physical properties compared to those of ordinary aqueous solutions. This analysis suggests consequences of far reaching importance to our understanding of cell structure and function.

Animals↗
PubMed1986

L-929 cells under hyperosmotic conditions: volume changes.

Volume changes resulting from the addition of sorbitol to the environment of mouse L-929 cells were evaluated from cell diameter measurements. Over periods of 1 hour or less, this solute was effectively excluded from intracellular water. The reduction in cell volume was inversely related to sorbitol concentration up to levels of about 0.6 molal, above which no further significant reduction occurred. Reduced cell volumes were maintained for at least 1 hour without measurable volume regulation. The percentage of volume lost was independent of the initial cell volume and was quickly regained when physiological conditions were restored. However, cell volume was influenced strongly by cell density or by some variable related to it. L-cells store surface area when dehydrated, apparently by means of plasma membrane convolutions and microvilli, based on the rapid kinetics of reversible volume changes and on observations from scanning electron microscopy. These results are related to current views on the nature of intracellular organization.

Animals↗
PubMed1986

Some structural, biochemical and biophysical characteristics of L-929 cells growing in the presence of hyperosmotic sorbitol concentrations.

Mouse L-929 cells (a fibroblast-like line) were transferred from normal growth medium to one supplemented with 0.3 M sorbitol, doubling the normal external osmotic pressure. After a short lag phase and minimal cell death, the cells began to grow, and the growth rate reached that of controls after about one week. These chronically grown cells (S) have been compared to those of control cultures (C) with regard to general morphology, ability to reverse when returned to normal condition, water content, volume and selected metabolic parameters. S-cell cultures exhibited considerable heterogeneity but most contained vesicle-like cytoplasmic structures, sometimes in abundance. These structures do not appear to be completely bounded by membranes, but that is uncertain. S cells become larger and contain more water than C cells; however, the ratio of total water to total dry mass is indistinguishable from controls suggesting regulation at that level. S and C cells were found to be remarkably similar, on a per cell basis, with regard to their rate of respiration and the incorporation of glucose into metabolites and macromolecules. These results are interpreted in terms of current views on the composition and organization of the aqueous compartments of animal cells.

Animals↗
PubMed1985

Respiratory metabolism of L-929 cells at different water contents and volumes.

Oxygen consumption was measured in mouse L-929 cells whose volumes and water contents were reduced by adding sorbitol to the medium. The volume of water lost due to a given sorbitol supplement exceeded the loss in apparent cell volume. An explanation is given for this discrepancy. The rate of oxygen uptake in the absence of exogenous respiratory substrate was essentially the same in cells whose total volume was reduced by 45%, amounting to a loss of about 70% of the total cell water, compared to controls at 'physiological' volume and water content. Cells under these same conditions responded to added substrates (pyruvate, glucose, and glutamine) and inhibitors (iodoacetate and 2-deoxyglucose) in nearly the same way as control cells. These observations are in accord with and add to previous work showing that very large fluctuations in cell volume and water content have only modest effects on the rates and directions of a variety of metabolic processes. The results are interpreted in terms of current views on the composition and organization of the aqueous compartments of eucaryotic cells.

Animals↗
PubMed1984

Interrelationships between water and cellular metabolism in Artemia cysts. XI. Density measurements.

Cysts of the crustacean Artemia are a useful model for studies on intracellular water because they are capable of essentially complete and reversible desiccation. We have used a variety of techniques on this system, the present work being an attempt to estimate the density of intracellular water (rho w). The density of individual cysts was evaluated from sedimentation velocity. Heptane displacement methods were used to determine the volume of a known mass of cysts, from which the density was calculated. The two methods produce comparable results. It was shown that the densities and water contents of large masses of cysts accurately reflect those of individual cysts. Cyst densities (rho c) were determined over the entire range of water content from 0 to 0.63 weight fraction of water (Wf), and temperature dependence was measured for 0.61 Wf over 2-41 degrees C. The following refer to 25 degrees C. No marked change was detected in rho c until the water content exceeded 0.15 Wf, at which rho c decreased as a linear function of Wf to maximum water content. However, the cyst does not behave ideally in the sense that the densities of the nonaqueous components and added water are not additive as a function of Wf. The partial specific volume of water in cysts at maximum hydration was estimated to be 3% larger than that of pure water. These observations are compared with density measurements on other systems, and with previous findings on the physical properties of water in this system.

Animals↗
PubMed1984

Diffusive properties of water in Artemia cysts as determined from quasi-elastic neutron scattering spectra.

Results have been obtained on the quasi-elastic spectra of neutrons scattered from pure water, a 20% agarose gel (hydration four grams H2O per gram of dry solid) and cysts of the brine shrimp Artemia for hydrations between 0.10 and 1.2 grams H2O per gram of dry solids. The spectra were interpreted using a two-component model that included contributions from the covalently bonded protons and the hydration water, and a mobile water fraction. The mobile fraction was described by a jump-diffusion correlation function for the translation motion and a simple diffusive orientational correlation function. The results for the line widths gamma (Q2) for pure water were in good agreement with previous measurements. The agarose results were consistent with NMR measurements that show a slightly reduced translational diffusion for the mobile water fraction. The Artemia results show that the translational diffusion coefficient of the mobile water fraction was greatly reduced from that of pure water. The line width was determined mainly by the rotational motion, which was also substantially reduced from the pure water value as determined from dielectric relaxation studies. The translational and rotational diffusion parameters were consistent with the NMR measurements of diffusion and relaxation. Values for the hydration fraction and the mean square thermal displacement [u2] as determined from the Q-dependence of the line areas were also obtained.

Animals↗
PubMed1984

Intracellular water and the cytomatrix: some methods of study and current views.

The extent to which the properties of water in cells are like those of water in dilute aqueous solutions is a question of broad significance to cell biology. A detailed answer is not available at present, although evidence is accumulating that the properties of at least a large fraction of intracellular water are altered by interactions with cell ultrastructure, notably the cytomatrix. That and related evidence also suggests that the properties, composition, and activities of the "aqueous cytoplasm" of intact cells bear little resemblance to those of the "cytosol" obtained by cell fractionation. This paper will consider some of the evidence for these possibilities and some of their potential consequences with regard to cellular structure and function.

Animals↗
PubMed1984

Microwave dielectric measurements (0.8-70 GHz) on Artemia cysts at variable water content.

Dielectric permittivity measurements are reported for cysts of Artemia, a crustacean known as the brine shrimp. Using coaxial and waveguide techniques we examined the frequency range from 0.8 to 70 GHz. Taking advantage of the ability of this system to reversibly lose essentially all intracellular water, we determined the permittivity over the entire range of cyst water contents. Although experimental errors prevent a rigorous treatment of the data, we advance the general conclusion that little of the water in this system behaves dielectrically like pure water, regardless of water content. This conclusion is supported by, and is consistent with, the results of previously published studies that probe the motional properties of water in this system using nuclear magnetic resonance spectroscopy and quasi-elastic neutron scattering.

Animals↗
PubMed1984

Properties and metabolism of the aqueous cytoplasm and its boundaries.

The nucleoplasm, the interiors of cytoplasmic membrane-bound organelles, and the aqueous cytoplasm make up the aqueous compartments of animal cells. The extent to which these compartments are concentrated solutions of macromolecules, metabolites, ions, and other solutes is a matter of some importance to current thinking about cell structure and function. This paper will focus on the aqueous cytoplasm. It will show that the composition and metabolic activities of the cytosol, obtained by methods of cell disruption and fractionation, bear almost no resemblance to those of the aqueous cytoplasm in intact cells. The consequences of this to contemporary views on cell structure and function are considered. A closely related topic concerns the physical properties of the dominant component of these compartments, water: Are these properties the same as those of water in aqueous solutions, or are they altered as a result of interaction with cell architecture? Available evidence strongly suggests that at least a large fraction of the total cell water exhibits properties that markedly differ from those of pure water. Selected examples of these studies will be reviewed, and the roles of cell water will be discussed, notably as they relate to metabolism and cell ultrastructure. Although dimly perceived at present, it appears that living cells exhibit an organization far greater than the current teachings of cell biology reveal.

Animals↗
PubMed1983

Cellular and molecular consequences of reduced cell water content.

Cell volumes, viability, ultrastructure, and metabolism have been studied in mouse L cells at reduced water contents. Dehydration was achieved by addition of sorbitol to the incubation medium at concentrations of 0.15, 0.3, and 0.6 m. In 0.3 m sorbitol, cell volume was reduced to about 35% of the initial control level and cell ultrastructure was markedly altered. Nevertheless, such cells retained viability and their metabolism was much the same as that of control cells, evaluated by [U-14C]glucose. Except for a lesser volume reduction, cells in 0.15 m sorbitol behaved similarly. However, when placed in 0.6 m sorbitol the cells exhibited quantitative and qualitative differences in their metabolism compared with controls, and grew very poorly or not at all. Their ultrastructure was not obviously different from cells in 0.3 m sorbitol although cell volume was slightly reduced. These results are considered in the context of different conceptions of the properties and metabolic activities of the aqueous intracellular compartments (cytosol, nucleoplasm, and interiors of cytoplasmic membrane-bound-organelles). We interpret the data as additional evidence for the existence of extensive spatial organization of enzyme and the pathways they catalyze in the aqueous compartments. On this basis it is also suggested that at least part of the damage that occurs in severely dehydrated mammalian cells could result from the irreversible disruption of this organization and loss of metabolic control. We speculate about the evolutionary involvement of intracellular water and how it might have participated in the development of structure and function in contemporary cells.

Animals↗