Cell-specific Parameters Research Articles

Theoretical analysis of calcium wave propagation based on inositol (1,4,5)-trisphosphate (InsP 3) receptor functional properties

In the presence of inositol (1,4,5)-trisphosphate (InsP 3) repetitive waves of elevated cytosolic free Ca 2+ (Ca waves) that travel through cellular cytoplasm are observed. Investigation of this phenomenon stimulated the view of cellular cytoplasm as ‘an excitable medium composed of Ca release processes (InsP 3R), coupled by a common stimulatory signal (Ca) through diffusion’ [Lechleiter JD. Clapham DE. (1992) Molecular mechanisms of intracellular calcium excitability in Xenopus laevis oocytes. Cell, 69, 283–294]. Using a kinetic model of InsP 3R gating, an analytical expression for the amplitude of Ca wave propagating through this excitable medium has been obtained. The ampiitude of the Ca wave is determined by the combination of cell-specific parameters and the functional properties of a single InsP 3R. An analytical expression for Ca wave propagation velocity has been also obtained using the Luther equation for diffusion-driven autocatalytic reaction. Both equations provided reasonable estimations for Ca wave amplitude (1.3 μM free Ca) and the velocity of the wave propagation (21 μm/s) for Ca waves in Xenopus oocytes when numerical values of parameters were used. The duration of refractory period has been shown to be determined mainly by the activity of CaATPase. Obtained results provide an insight into the mechanisms underlying the process of Ca wave propagation and define the interrelationship between different factors involved in this process. Some experimentally testable predictions can be done based on the analytical expressions obtained for Ca wave amplitude, the velocity of Ca wave propagation and the duration of refractory period.

A model of diffusion-limited ice growth inside biological cells during freezing

A theoretical model for predicting the kinetics of ice crystallization inside cells during cryopreservation was developed, and applied to mouse oocytes, by coupling separate models of (1) water transport across the cell membrane, (2) ice nucleation, and (3) crystal growth. The instantaneous cell volume and cytosol composition during continuous cooling in the presence of glycerol were predicted using the water transport model. Classical nucleation theory was used to predict ice nucleation rates, and a nonisothermal diffusion-limited crystal-growth model was used to compute the resulting crystallization kinetics. The model requires knowledge of the nucleation rate parameters Ω and κ, as well as the viscosity η of a water-NaCl-glycerol solution as a function of both the composition and temperature of the solution. These dependences were estimated from data available in the literature. Cell-specific biophysical parameters were obtained from previous studies on mouse oocytes. A sensitivity analysis showed that the model was most sensitive to the values of κ and η. The coupled model was used to study the effect of cooling rate and initial glycerol concentration on intracellular crystal growth. The extent of crystallization, as well as the crystal size distribution, were predicted as functions of time. For rapid cooling at low to intermediate glycerol concentrations, the cells crystallized completely, while at high concentrations of glycerol, partial or total vitrification was observed. As expected, the cooling rate necessary for vitrification dropped with increasing glycerol concentration; when cells initially contained ∼7.5 M glycerol, vitrification was achieved independent of cooling rate. For slow cooling protocols, water transport significantly affected the results. At glycerol concentrations greater than ∼3 M, the final intracellular ice content decreased with increasing glycerol concentration at a fixed cooling rate. In this regime, the cooling rate at which a critical amount of ice was formed increased as more glycerol was used. When less than ∼3 M glycerol was initially present in the cell, an increase in glycerol concentration was predicted to cause an increase in the final intracellular ice content at a given cooling rate. In this regime, the critical cooling rate decreased with increasing glycerol concentration. These predictions clarify previous empirical observations of slow freezing phenomena.

Growth, metabolic, and antibody production kinetics of hybridoma cell culture: 1. Analysis of data from controlled batch reactors.

A mouse-mouse hybridoma cell line (167.4G5.3) was cultivated in a 1.5-L stirred-tank bioreactor under constant pH and dissolved oxygen concentration. The transient kinetics of cell growth, metabolism, and antibody production were followed by biochemical and flow cytometric methods. The cell-specific kinetic parameters (growth and metabolic rates) as well as cell size were constant throughout the exponential phase. Intracellular protein and RNA content followed a similar trend. Cell growth stopped when the glutamine in the medium was depleted. Glucose could not substitute for glutamine, as glucose consumption ceased after glutamine depletion. Ammonia and lactate production followed closely glutamine and glucose consumption, respectively. Alanine, glutamate, serine, and glycine were produced but other amino acids were consumed. The cells are estimated to obtain about 45% of the total energy from glycolysis, with the balance of the metabolic energy provided by oxidative phosphorylation. The antibody was produced at a constant rate in both the exponential and decline phases of growth. The intracellular antibody content of the cells remained relatively constant during the exponential phase of growth and decreased slightly afterwards.

Kinetics of cell agglutination: A quantitative assay of Concanavalin A mediated agglutination of acanthamoeba castellanii

ConA-mediated cell agglutination is studied by measuring changes in light transmission of cell suspensions. The results of experiments are compared with those from a model of agglutination kinetics. The agglutination rate, k′ 1, is proved to be dependent on initial cell concentration, amount of agitation and temperature. In all cases it has a maximum at a given ConA concentration which is not influenced by the other agglutination conditions. From the light transmission data we found an average of 2.6 × 10 9 receptor sites/cell and a mean affinity constant for ConA of 0.5 × 10 6 M −1. The differences between model and experiment give information on cell specific parameters. Especially the contribution of the acanthapodia in the agglutination process is discussed. Reproducible results cannot be obtained unless temperature and amount of agitation are kept constant.