The link between yeast cell wall porosity and plasma membrane permeability after PEF treatment.

An investigation of the yeast cell resealing process was performed by studying the absorption of the tetraphenylphosphonium (TPP+) ion by the yeast Saccharomyces cerevisiae. It was shown that the main barrier for the uptake of such TPP+ ions is the cell wall. An increased rate of TPP+ absorption after treatment of such cells with a pulsed electric field (PEF) was observed only in intact cells, but not in spheroplasts. The investigation of the uptake of TPP+ in PEF treated cells exposed to TPP+ for different time intervals also showed the dependence of the absorption rate on the PEF strength. The modelling of the TPP+ uptake recovery has also shown that the characteristic decay time of the non-equilibrium (PEF induced) pores was approximately a few tens of seconds and this did not depend on the PEF strength. A further investigation of such cell membrane recovery process using a florescent SYTOX Green nucleic acid stain dye also showed that such membrane resealing takes place over a time that is like that occurring in the cell wall. It was thus concluded that the similar characteristic lifetimes of the non-equilibrium pores in the cell wall and membrane after exposure  to  PEF indicate a strong coupling between these parts of the cell.

Contribution of mitochondria to injury of hepatocytes and liver tissue by hyperthermia.

OBJECTIVE: The aim of this study was to investigate functional changes of liver mitochondria within the experimentally modeled transition zone of radiofrequency ablation and to estimate possible contribution of these changes to the energy status of liver cells and the whole tissue. MATERIALS AND METHODS: Experiments were carried out on mitochondria isolated from the perfused liver and isolated hepatocytes of male Wistar rats. Hyperthermia was induced by changing the temperature of perfusion medium in the range characteristic for the transition zone (38-52°C). After 15-min perfusion, mitochondria were isolated to investigate changes in the respiration rates and the membrane potential. Adenine nucleotides extracted from isolated hepatocytes and perfused liver subjected to hyperthermic treatment were analyzed by HPLC. RESULTS: Hyperthermic liver perfusion at 42-52°C progressively impaired oxidative phosphorylation in isolated mitochondria. Significant inhibition of the respiratory chain components was observed after perfusion at 42°C, irreversible uncoupling became evident after liver perfusion at higher temperatures (46°C and above). After perfusion at 50-52°C energy supplying function of mitochondria was entirely compromised, and mitochondria turned to energy consumers. Hyperthermia-induced changes in mitochondrial function correlated well with changes in the energy status and viability of isolated hepatocytes, but not with the changes in the energy status of the whole liver tissue. CONCLUSIONS: In this study the pattern of the adverse changes in mitochondrial functions that are progressing with increase in liver perfusion temperature was established. Results of experiments on isolated mitochondria and isolated hepatocytes indicate that hyperthermic treatment significantly and irreversibly inhibits energy-supplying function of mitochondria under conditions similar to those existing in the radiofrequency ablation transition zone and these changes can lead to death of hepatocytes. However, it was not possible to estimate contribution of mitochondrial injury to liver tissue energy status by estimating only hyperthermia-induced changes in adenine nucleotide amounts on the whole tissue level.

Electric field-induced effects on yeast cell wall permeabilization.

The permeability of the yeast cells (Saccharomyces cerevisiae) to lipophilic tetraphenylphosphonium cations (TPP(+) ) after their treatment with single square-shaped strong electric field pulses was analyzed. Pulsed electric fields (PEF) with durations from 5 to 150 µs and strengths from 0 to 10 kV/cm were applied to a standard electroporation cuvette filled with the appropriate buffer. The TPP(+) absorption process was analyzed using an ion selective microelectrode (ISE) and the plasma membrane permeability was determined by measurements obtained using a calcein blue dye release assay. The viability of the yeast and the inactivation of the cells were determined using the optical absorbance method. The experimental data taken after yeasts were treated with PEF and incubated for 3 min showed an increased uptake of TPP(+) by the yeast. This process can be controlled by setting the amplitude and pulse duration of the applied PEF. The kinetics of the TPP(+) absorption process is described using the second order absolute rate equation. It was concluded that the changes of the charge on the yeast cell wall, which is the main barrier for TPP(+) , is due to the poration of the plasma membrane. The applicability of the TPP(+) absorption measurements for the analysis of yeast cells electroporation process is also discussed.