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.     

Interrelationships between water and cellular metabolism in Artemia cysts VII. Free amino acids

The concentration of total ninhydrin-positive material (NPM) soluble in 5% trichloroacetic acid was measured in cysts of the brine shrimp, Artemia salina, as a function of hydration level. No net change in NPM was observed until the cysts had achieved a water content of about 0.65 g H₂O/g of initially dry cysts. Above this hydration threshold the NPM content increased markedly. Examination of the free amino acid composition of cysts incubated at selected hydration levels revealed that almost all of the amino acids underwent net change above the hydration threshold. However, just below this threshold, the free amino acid composition was essentially the same as in fully dried cysts. The activity generating net changes in the concentration of free amino acids above the hydration threshold was shown to be metabolic in nature and restricted to the cellular component of the cyst.     

Interrelationships between water and cellular metabolism inArtemia cysts

Cysts of the crustaceanArtemia 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 (ρ_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 (ρ_c) were determined over the entire range of water content from 0 to 0.63 weight fraction of water (W _f), and temperature dependence was measured for 0.61W _f over 2–41°C. The following refer to 25°C. No marked change was detected in ρ_c until the water content exceeded 0.15W _f, at which ρ_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 ofW _f. 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.     

Interrelationships between water and cellular metabolism in Artemia cysts. IV. Adenosine 5'-triphosphate and cyst hydration.

The concentration of ATP in cysts of the brine shrimp, Artemia salina, has been studied as a function of cyst hydration. Cysts dried over CaSO4 contain 0.02 gH2O/g cysts and 0.54 +/- 0.05 (S.D.) mumoles of ATP/g dried cysts. Addition of water up to 0.05 g/g cysts produced no net change in the level of ATP during incubation. Hydration levels between 0.05 and 0.62 g/g cysts resulted in a net loss of ATP, whereas above 0.65 g/g cysts a net increase was observed with incubation time. No net change in the amount of ATP, compared with dried cysts, was detected between the latter two hydrations. These results, when integrated with those from previous work, indicate that conventional aerobic energy metabolism does not begin until cyst hydrations of about 0.65 g/g are achieved. The fate of ATP in cysts hydrated to levels lower than 0.65 g/g was discussed.     

Interrelationships between Water and cellular metabolism in Artemia cysts. VIII Sorption isotherms and derived thermodynamic quantities

An analysis of water vapor sorption by cysts of the brine shrimp, Artemia salina, has shown that at environmental water activities (aw) of 0.95 or less, the cysts equilibrate with the aw of their environment. Above this aw the metabolic activity of the cysts participates directly in their water content, and equilibration does not occur. In contrast, dried cysts killed by heat treatment or exposure to ammonia fumes equilibrated with all values of aw examined. Analysis of the temperature dependence of sorption isotherms revealed that below cyst hydrations of about 0.3 g H2O/g dried weight the temperature coefficient for water sorption was negative, but became positive at hydrations appreciably in excess of this value. Estimates for the differential and integral net enthalpy and entropy changes accompanying the sorption of water have been calculated from isotherms. These results have been interpreted and integrated with those from previous work on the hydration-dependence of metabolic activity. All of the examined hydration properties of the cysts have been shown to be due chiefly to the cellular component, and not the acellular shell. Analysis of the data by the Bradley equation has shown that the hydration behavior of the shell obeys this relationship, whereas that of the cellular component does not.     

Interrelationships between Water and Cellular Metabolism in Artemia cysts. V. 14CO2 incorporation

The ability of cysts of the brine shrimp, Artemia salina, to incorporate ¹⁴CO₂ into organic compounds soluble in cold-trichloroacetic acid was examined over a broad range of cellular water concentrations. Carbon dioxide was not incorporated by cysts containing less than about 0.3 g H₂O/g dried cysts, the “critical hydration” for CO₂-fixation. This relationship held whether the cysts were hydrated from the liquid or the vapor phase. The incorporation of radioactivity was shown to be due exclusively to metabolic activity in the cellular component of the cyst. Above the critical hydration, the amount of ¹⁴CO₂ incorporated was a function of cyst water content, but the kinds of metabolites labelled with this precursor, and their relative proportions, were found to be similar in cysts of greatly different hydration. Almost all of the radioactivity was associated with amino acids, Krebs cycle intermediates and related acids, and pyrimidine nucleotides. The fact that the pathway involved with CO₂-fixation, and subsequent metabolism of the fixation products are all initiated in cysts containing as little as 0.3 g H₂O/g is particularly noteworthy since this hydration level is well within the range of the amounts of “bound water” described in the literature for a wide array of cells and tissues.     

Interrelationships between water and cell metabolism in Artemia cysts X. Microwave dielectric studies

Artemia cysts are composed of an inner mass of about 4000 cells surrounded by an acellular shell. This system can undergo cycles of hydration-dehydration without viability loss, and is a useful model for the study of intracellular water. We have measured the relative permittivity (ε′) of these cysts as a function of water content over the frequency range 0.8–70 GHz. Detailed analysis of the data for cysts containing close to 1 g H₂O/g dry weight indicates that a significant fraction of the total water in this system exhibits dielectric behavior different from that of pure water: the distribution parameter (α) for the dispersion analyzed by the Cole-Cole equation deviates from zero, and the permittivity of cyst water appears to be significantly lower than that of pure liquid.     

Early cellular responses to diverse growth stimuli independent of protein and RNA synthesis.

Serum, elevated pH, excess Zn++, 9,10 dimethyl-1,2 dibenzanthracene (DMBA) and insulin accelerate the progress of growth-inhibited chick embryo cells into the S-period of DNA synthesis. A comparative study was made of their capacity to elicit other cellular responses within two hours after their application. All the agents studied stimulated the uptake of the glucose analogue 2-deoxy-D-glucose (2-dGlc). Elevated pH elicited a more striking increase than the other agents in the uptake of the amino acid analogue alpha-amino isobutyric acid (AIB). The application of subtoxic concentrations of Zn++ or DMBA did not stimulate the uptake of uridine by cells nor its incorporation into RNA when tested at 2 hours. However, it was found that the stimulation of uridine utilization did occur but was delayed several hours. Similarly, the accelerated onset of DNA synthesis was also delayed for several hours by these agents. Insulin acted like serum in stimulating the utilization of 2-dGlc, AIB and uridine. Serum and DMBA were particularly effective in stimulating the utilization of choline. It was concluded that the utilization of 2-dGlc, uridine and thymidine are affected similarly by all the agents, but that there may be differential effects in the utilization of AIB and choline. The inhibition of RNA synthesis by actinomycin D did not prevent the relative stimulation of 2-dGlc, AIB and choline utilization by serum and pH treatment. The inhibition of protein synthesis by cycloheximide did not prevent the relative stimulation of 2-dGlc and choline utilization by serum and pH treatment. It partially blocked the increased uptake of AIB and had erratic effects on the utilization of uridine. It was concluded that neither RNA nor protein synthesis is required for some, if not all, the early responses to growth stimuli measured here. The inhibited cell appears to be a poised system which carries out a programmed array of reactions characteristic of the cell type following perturbation by a variety of unrelated agents. In vivo specificity is provided by the physiological reagents available (i.e., hormones) and their capacity to interact with different cell types.     

Interrelationships in experiments in vitro between the migration activity of leukocytes and RNA and DNA synthesis]

The following correlations were revealed in the parallel study of leukocyte migration in vitro in the presence of a specific antigen and of spontaneous RNA and DNA synthesis in the cultured lymphocytes: 1) a direct correlation between the RNA and DNA synthesis in lymphocytes; 2) a close correlation between the antigen-induced migration and the levels of RNA and DNA synthesis. The effect of the antigen was evidenced by the inhibition or stimulation of leukocyte migration. A high ratio of RNA synthesis to DNA synthesis corresponded to the migration inhibition and a low one--to the migration stimulation. The ratio value varied mainly on account of the changes in the level of DNA synthesis. Participation of T and B cells in the regulation of the antigen-induced leukocyte mobility is discussed.     

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.     

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.