Ultrastructure of erythroblast mitochondria in mice treated with mixtures of radioprotectors.

Unirradiated mice were given i.p. a mixture of mexamine (20 mg/kg body weight) and AET (150 mg/kg body weight) or mexamine (20 mg/kg body weight) and cystamine (150 mg/kg body weight). Acute effects of these mixtures of radioprotective agents on the ultrastructure of basophilic and polychromatic bone marrow erythroblast mitochondria were studied. One hour after injecting mice with above-mentioned mixtures, statistically significant differences in the size of mitochondria were found between control mice and mice given a mixture of mexamine and cystamine. The differences in the number of dilated cristae per micron 2 of the mitochondrion area between control mice and both experimental groups given mixtures of radioprotective agents were also statistically significant. Thus it was proved that also mixtures of radioprotectors induced acute ultrastructural changes of mitochondria which were so far described only after treating mice with only one radioprotective agent.

A morphological study of continuously irradiated lymphosarcoma cells.

Mouse lymphosarcoma LS/BL cells, cultivated as a free suspension in the peritoneal cavity of C57Bl mice, were continuously irradiated by 60Co unit at an exposure rate of 35.8 x 10(-5) mC kg-1 s-1 (5 R/h). After 250 and 350 weeks of irradiation these cells were examined in light and electron microscope. Both the cytoplasm and the nucleus underwent morphological alterations under continuous exposure. Areas of advanced cytoplasmic degradation were often observed and many nuclei showed wide indentations and prolongation of their shape. The number of mitochondria and vacuoles in the cytoplasm was increased markedly in the irradiated cells. The authors consider some of the described alterations caused by continuous irradiation to act as compensatory mechanisms of the impaired cell.

Light and electron microscopy of mouse lymphosarcoma LS/BL cells during the first hour after irradiation with x-rays.

Mouse lymphosarcoma LS/BL cells growing in tissue culture were irradiated with 100, 316 and 1000 R respectively and fixed 5, 15 or 60 minutes later. Semithin sections were studied under a light and ultrathin under an electron microscopes. Under the light microscope, differences between the morphology of individual nuclei were found in all groups, including controls; the rate of their occurrence was without any relation to the irradiation. They may reflect the differences in the state of LS/BL cells which were not completely adapted to cultivation in small glass tubes. On the other hand, the frequency of some ultrastructural changes seen under the electron microscope showed a relation to the irradiation. They were: undulation of the outer nuclear membrane and dilatation of the space between the outer and inner nuclear membranes, slots in karyosomes adjacent to the surface of the nuclei, enlargement of cisternae and vesicles of endoplasmic reticulum and Golgi apparatus. The importance of a careful examination of control groups in such experiments is stressed.

Study on incorporation of 3H-DNA in the L-cells by means of electron microscopic autoradiography.

Uptake of isologous DNA (labeled with 3H-thymidine) by L-cells was investigated by means of electron microscopic autoradiography. It was found that exogenous 3H-DNA occurred not only in the cytoplasm, but, already from the 1st hr of incubation, permeated as far as in the host cell nuclei. During continuous cultivation, radioactivity established in the nuclei and cytoplasm increases with maximum value after 12 hr incubation. Beginning at the 3rd hr of incubation, in cytoplasma, grain clusters over loci of higher radioactivity were found. After 24 hr incubation, similar grain clusters in L-cell nuclei were observed. In the cytoplasma of the cells, the grains were often determined over or in the vicinity of lysosomes; in the nuclei they were localized predominantly over heterochromatine. The manner of 3H-DNA and 3H-TdR labeling was also compared.

Low survival of mice following lethal gamma-irradiation after administration of inhibitors of prostaglandin synthesis.

An impairment of the survival of mice subjected to whole-body gamma-irradiation with a lethal dose of 10 Gy and treated with a repeated postirradiation administration of prostaglandin synthesis inhibitors (PGSIs), indomethacin or diclofenac, was observed. Morphological examination of the gastrointestinal tract and the estimation of blood loss into its lumen in animals treated with diclofenac did not show serious damage such as haemorrhages or perforation, but revealed structural injury to the intestinal mucosa indicating inflammatory processes. The lesions found are supposed to be connected with increased intestinal permeability which leads to endotoxin escape from the gut and a subsequent increased mortality rate of irradiated animals. It may be concluded that PGSIs are not suitable for the management of radiation sickness after an exposure to lethal doses of ionizing radiation.

Delivery of liposome content into plant protoplasts.

The interaction of various types of liposomes with protoplasts of Nicotiana tabacum and Capsicum annuum was studied by means of fluorescence microscopy and autoradiography. The liposomes with a positive charge displayed the strongest affinity to the protoplast membrane, whereas the maximum intracellular delivery of encapsulated protein and DNA molecules into protoplasts was exhibited by the negatively charged liposomes. The results showed that the negatively charged unilamellar liposomes prepared by the reverse-phase evaporation method represent a very promising universal vehicle system for introducing genetic information into plants.

The kinetics of endogenous erythroid haemopoietic spleen colonies in mice between 4 and 10 days after irradiation with doses of 5, 7, or 9 Gy.

The kinetics of post-radiation haemopoietic tissue recovery was studied by evaluating quantitatively the results from histologic studies of the kinetics of endogenous erythroid spleen colony counts in subserial sections of spleens from mice irradiated with 5, 7, or 9 Gy of 60Co gamma-rays at intervals of 4, 6, 8, and 10 days following irradiation. Emphasis was put on individual types of colony-forming cells and on time intervals at which these cells enter into action. Large numbers of rapidly maturing microscopic erythroid colonies appeared between 8 and 10 days after irradiation. From this finding we conclude that in addition to colony-forming cells giving rise to macroscopic colonies, large numbers of already differentiated progenitor cells--the products of the first regeneration wave with a peak approximately 6 days following irradiation--participate in the second wave of regeneration which is typically manifested from day 10 after irradiation.

Haemopoiesis in murine bone marrow and spleen after fractionated irradiation and repeated bone marrow transplantation. I. Erythropoiesis.

Erythropoiesis was studied in mice repeatedly subjected to individual doses of 3 Gy of 60Co gamma-rays at 4-day intervals up to a total dose of 24 Gy on the basis of total bone marrow and spleen cellularity follow-up and analysis of myelograms and splenograms. Half of the mice received 10(6) nuclear cells of syngeneic bone marrow after each fractional radiation dose. It was mainly the spleen which was involved in the adaptation and regeneration of erythropoiesis, its contribution to total erythropoiesis in bone marrow recipients having been as much as 73.9% (day 20 of experiment, total dose 15 Gy). In mice only irradiated, the number of nuclear cells of erythroid lineage decreased to zero values sooner in the spleen (day 16 of experiment, total dose 12 Gy) when compared to the bone marrow (day 24 of experiment, total dose 18 Gy). Analysis of the results of collections made on day 9 after the last irradiation, however, revealed that the haemopoietic microenvironment of the spleen and haemopoietic cells capable of differentiation in the erythroid direction are so resistant to irradiation in only irradiated mice that erythropoiesis in their spleens exhibits signs of regeneration even after the highest total dose of 24 Gy.

Haemopoiesis in murine bone marrow and spleen after fractionated irradiation and repeated bone marrow transplantation. II. Granulopoiesis.

Granulopoiesis was studied in mice repeatedly subjected to individual doses of 3 Gy of 60Co gamma-rays at 4-day intervals up to a total dose of 24 Gy on the basis of total bone marrow cellularity follow-up and analysis of myelograms and splenograms. Half of the mice received 10(6) nuclear cells of syngeneic bone marrow after each fractional radiation dose. After an initial steep decrease, the number of granuloid cells in the spleen increased about 30-fold between days 12 and 16 of the experiment (total dose 9 and 12 Gy, respectively). This increase was temporary and between days 20 and 24 (total dose 15 and 18 Gy, respectively) a steep decrease again occurred. At a low level (below 10% of the control value) remained the granuloid cells in the spleens of bone marrow recipients until the end of the experiment (day 37, total dose 24 Gy). The behaviour of the granuloid compartment of haemopoiesis thus contrasts with findings in the erythroid compartment (see Hofer et al., 1989b) when high numbers of erythroid nuclear cells remained in the spleens of bone marrow recipients until the end of the experiment. On the whole, the influence of repeated bone marrow transplantation on granulopoiesis in the bone marrow and spleen is positive. Of the 22 comparisons made between bone marrow recipients and mice only irradiated, 14 differences are statistically significant, always in favour of bone marrow recipients.

Effect of indomethacin on the proliferative and differentiation activity in the haemopoietic system of lethally irradiated mice after syngeneic bone marrow transplantation.

The effects of indomethacin treatment on the proliferation and differentiation of haemopoietic stem cells of bone marrow grafts in lethally irradiated mice were investigated. Indomethacin was given subcutaneously, on days 3 to 5 after irradiation, in 6 doses of 0.05 mg per mouse. On day 6 after irradiation, an increased self-renewal of the settled colony-forming cells and an increased amount of differentiated cells were observed in the spleens of the mice treated. Simultaneously, a decreased maturation of the erythroid cells was demonstrated. The effects observed may be explained by the indomethacin-induced inhibition of prostaglandin biosynthesis.

Interaction of protein- and DNA-loaded liposomes with sperm cells.

Liposomes were loaded with fluorescence-labelled serum albumin and their interaction with mouse spermatozoa was examined in a fluorescence microscope. In the presence of polyethylene glycol 1500, fluorescence became unevenly distributed along the entire body of mature spermatozoa, mostly in the form of aggregates, indicating adsorption of liposomes to the surface of the cells. The fluorescence could be largely removed by washing the cells. In the case of immature sperm cells, fluorescence was evenly distributed on the cells and was not removed by extensive washing, suggesting an internalization of the labelled albumin. A more direct evidence for the entrance of liposome-entrapped macromolecules into the immature sperm cells was provided by autoradiography: radioactivity was found in discrete spots in spermatocytes upon their incubation with liposomes containing isotopically labelled DNA.

A contribution to the morphological study of acute changes in the blood vessels of the central nervous system of rats following irradiation with high-energy (4 GeV/nucleon) helium ions.

Rats were irradiated with helium ions (4 GeV/nucleon; 2 or 4 Gy). After 4-9 hours or three days, a perfusion was performed and sections of the cerebral cortex were investigated under light and electron microscopes. Changes observed in the vessels of the telencephalic cortex are described. Most of the vessels in the sections analysed showed no signs of damage. In some cases there was a dilated perivascular space; a comparison of its occurrence in irradiated and control animals showed a statistically significant increase in frequency of the phenomenon at a shorter interval after irradiation with a larger dose. Electron microscopical analysis showed that the main locality of damage was the border of the perivascular foot of the astrocytes, which exhibited various degrees of edema. The discussion stresses the peculiarities of the interaction of helium ions with living tissues, particularly the heterogeneity of the distribution of absorbed energy. The consequence is in accordance with the nature of the changes observed.

Electron-microscopic study of a Mycoplasmatales virus, strain MV-Lg-pS2-L 172.

Morphology and adsorption of a globular virus, lysing Acholeplasma laidlawii were studied in ultrathin sections of plaques in a lawn of the host strain. The virus was globular, about 50 to 90 nm in diameter, with a clearly defined membrane, 6.5 to 8 nm thick. A protuberance about 25 to 35 nm long and 12 to 20 nm thick was observed on numerous virus particles. The evenly granulated, electron-optically dense content of the cells became clearer in cells affected by the viruses. Fibrillar structures of different thickness and small dense areas appeared in cells assumed to be in the preliminary stages of lysis. The interactions in the virus-host system and possible development stages of the virsu are discussed.

Properties of the cured oncogenic strain 37400 of Agrobacterium tumefaciens.

The properties of the 37400 oncogenic strain of Agrobacterium tumefaciens are described. This strain was derived from the VI lysogenic strain originally isolated by Hamilton from a Zinnia elegans tumour. Strain 37400 has a number of properties which render it suitable for quantitative and genetic studies. It is cured of prophages and can serve as a universal sensitive indicator for a number of phages isolated from various lysogenic strains of Agrobacterium tumefaciens. Its good growth properties in synthetic media and at elevated temperatues enable the isolation of auxotrophic mutants and temperature sensitive phage mutants. Preliminary experiments show that strain 37400 will serve as suitable starting material for conjugation experiments under defined conditions.

Size and shape of mycoplasma virus MV-Lg-L 172 DNA.

Sedimentation of DNA of mycoplasma virus MV-Lg-L 172 in neutral and alkaline density gradients showed that the chromosome of this virus is most probably formed by a double-stranded circular DNA molecule with a molecular weight of approx. 10(7) daltons. These results were confirmed by direct electron microscopy of the viral DNA. Gamma-irradiation of the virus caused in its DNA single breaks on the average per each 66 eV of absorbed energy. Virus survival depended exponentially on the irradiation dose, D0 being between 60,000 and 70,000 rads. This dose induced on the average one single break in the DNA.