Cell volume changes during rapid temperature shifts.

The effect of a rapid temperature increase on the volume of different types of cells was investigated. Experiments were carried out using continuous microscopic image analysis. Volume variation of yeast cells, yeast spheroplasts and human leukaemia cells was measured during the transient phase after a thermal shift. The thermal shift was found to induce rapid increase in cell volume for cells lacking a cell wall (yeast spheroplasts and human leukaemia cells). This increase in cell volume is assumed to be a main cause of the heat shock-induced cell death. A theoretical mechanistic model that explains the behaviour of these cells is finally proposed.

Effects of 131I-labeled TNT-1 radioimmunotherapy on HT-29 human colon adenocarcinoma spheroids.

Radiolabeled murine monoclonal antibody TNT-1, directed against the nuclear histones of degenerating cells, was used to treat human colon adenocarcinoma HT-29 spheroids in vitro. The therapeutic effects of 131I-TNT-1 were investigated as a function of the radioactive dose, treatment time, and number of treatments. Efficacy of treatment was assessed by TNT-1 antibody uptake, spheroid growth delay, and morphological examination using light microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). From these studies, it was determined that the therapeutic effect increased with the number of doses and the duration of treatment. Spheroids treated for 24 h showed approximately two to four times more cell death than those with a 2-h treatment. As previously shown in animal models, additional treatment with radiolabeled TNT-1 produced an expanding number of TNT-1 targets, and subsequent treatments were more effective as shown by antibody uptake studies. Microscopic examinations demonstrated that morphological changes consistent with spheroid destruction correlated well with antibody uptake data and increased gradually with dose, treatment time, and frequency of treatments. At the ultrastructural level, destruction of cells in the treated spheroids included the formation of porous cell membranes, crater-like holes (SEM), blebbing, and dissolution of cytoplasmic organelles (TEM). With continued culture, the injured spheroids were found to disaggregate after intensive 131I-TNT-1 therapy (e.g. 50 microCi/ml or 100 microCi/ml with two or three 24-h treatments). These findings suggest that tumor spheroids can be used as an in vitro model to evaluate monoclonal antibody therapy using TNT-1 and other candidate mAbs directed against intracellular antigens exposed in degenerating cells of tumors.

[Fusion of Erwinia chrysanthemi spheroplasts in an electric fields]. / Sliianie sferoplastov Erwinia chrysanthemi v élektricheskom pole.

A new approach has been elaborated for electrofusion of Erwinia chrysanthemi spheroplasts. The new approach consists of superimposition of high voltage impulses on the pellet of tightly contacting cells in the course of centrifugation. The mixture of spheroplasts of two genetically marked strains was placed into the special centrifuge chambers and spinned for 15 min at 2500 g to get a compressed pellet between chamber electrodes. Three successive pulses of 6.6 kv/cm amplitude and 30 microseconds duration were applied to spheroplast pellet during centrifugation. Fusion products were viable and after plating on the surface of hypertonic medium regenerated to the rod forms. As a result, the hybrid clones carrying the markers of both parents were isolated.

Effects of microwave irradiation on some membrane-related processes in bacteria.

In a series of experiments performed on intact cells or spheroplasts of E. coli and Bac. subtilis a possibility of non-thermal effects induced by continuous microwave irradiation of a low power density (at wave length range from 0.0 to 7.8 mm) was studied. Thymidine and thymine uptake, leakage of potassium and hydrogen ions as well as the uptake of the transforming DNA by the component cells of Bac. subtilis were shown to be affected in a way typical of that due to heating of a sample. No specific dependence of the effects observed on wavelength was found.

Characterization of postreplication repair in Saccharomyces cerevisiae and effects of rad6, rad18, rev3 and rad52 mutations.

Postreplication repair of nuclear DNA was examined in an excision defective haploid strain of yeast lacking mitochondrial DNA (rad1 rho 0). The size of the DNA synthesized in cells exposed to various fluences of ultraviolet light (UV) corresponds approximately to the average interdimer distance in the parental DNA. Upon further incubation of cells following exposure to 2.5 J/m2, the DNA increases in size; by 4 h, it corresponds to DNA from uniformly labeled cells. The alkaline sucrose sedimentation pattern of DNA pulse labeled at various times after UV irradiation, for up to 4 h, does not change substantially, indicating that dimers continue to block DNA replication. A significant amount of postreplication repair requires de novo protein synthesis, as determined by its inhibition by cycloheximide. The rad6 mutant does not carry out postreplication repair, the rad18 and rad52 mutants show great inhibition while the rev3 mutation does not affect postreplication repair. Both recombinational and nonrecombinational repair mechanisms may function in postreplication repair and most of postreplication repair is error free.

Measurement of pyrimidine dimers in spheroplasts of Bacillus subtilis.

A method is described for making spheroplasts of Bacillus subtilis which are permeable to exogenous enzymes. Conditions are described for measuring small numbers of pyrimidine dimers in the DNA of UV-irradiated cells by use of a partially purified Micrococcus luteus extract containing an enzyme specific for pyrimidine dimers. The system will detect as few as 10-12 pyrimidine dimers per genome.

Degradation of cellular deoxyribonucleic acid after gamma irradiation of spheroplasted Escherichia coli CR thy- cells.

Gamma-ray-irradiated Escherichia coli CR thy(-) cells and spheroplasts, prelabeled with (14)C-thymine, were assayed for acid-insoluble activity as a function of incubation time after irradiation. Under similar irradiation and incubation conditions, degradation profiles of cells and spheroplasts were virtually identical. Similar results were found for cells and protoplasts irradiated in the presence of rifampin (20 mug/ml). These results suggest that postirradiation deoxyribonucleic acid degradation enzymes are probably not loosely localized in the periplasm, unlike endonuclease I.