Activation of transcription function in lymphocytes after irradiation with He-Ne-laser

The influence of He-Ne-laser irradiation (lambda = 632.8 nm) in dose 56 J/m2 on the ultrastructure of the nucleolus from human peripheral lymphocytes was studied electronmicroscopically. After 1 h irradiation a well-expressed reaction of the nucleolus was observed in 70% of the lymphocytes under examination. Changes consist in the appearance of a wrong-shaped fibrillar center or in its fragmentation, the increase of RNP-containing fibrillar and granular components, and also in expansion of vacuoli. In a number of irradiated lymphocytes nucleoli with several fibrillar centres and with a strand-like organization of RNP part were observed. The size of these nucleoli increases. Following the accepted functional interpretations the observed changes can be connected with the intensification of RNA metabolism including the synthesis, processing of pre-rRNA and preribosome transport from the nucleolus. Similar rearrangements of the nucleoli were revealed in parallel experiments with phytohemagglutinin-treated lymphocytes. They were observed 1 h after the stimulation of lymphocytes. Taking into account the absence of mitogenic action of He-Ne-laser irradiation on lymphocytes, the ultrastructural changes of nucleoli under the action of irradiation are considered as functional activation of rRNA synthesis in the Go-period.     

A comparative study of chromatin from lymphocyte nuclei upon activation of transcription by irradiation from an He-Ne-laser or phytohemagglutinin]

The influence of He-Ne laser radiation (632.8 nm, 56 J/m2, t = 10 s) and phytohaemagglutinin (PHA, 2 micrograms/ml) on chromatin structure in human lymphocytes was studied by electron microscopy using ultrathin cell sections. Morphometric analysis of extranuclear condensed chromatin masses was performed 1 h after the irradiation or after the beginning of PHA treatment. In the irradiated cells the following insignificant changes were revealed: decrease in the relative area of the nucleoplasmic chromatin, increase in the relative area of decondensation zones as well as increase in the number of clumps of nucleoplasmic chromatin and relative length at their boundary with nucleoplasma. The tendency of these morphological changes may be interpreted as functional activation of extranucleolar RNA synthesis in response to irradiation by red laser light. Action of PHA results in significant changes of the surfaces of chromatin clumps, namely increase in relative length of nucleoplasmic chromatin boundary and decrease in relative length of perimembranous chromatin boundary with nucleoplasma as well as some less expressed delamination of the chromatin masses from the nuclear membrane. These essential changes may reflect chromatin activation by proliferative stimulus. Peculiarities of the ultrastructural reorganisation in the condensed chromatin after irradiation and PHA-treatment probably reflect the differences in the processes of gene activation caused by the two agents.     

Morphometric study of yeast cells of Torulopsis sphaerica after He-Ne-laser irradiation

A study was made of structural organization of Torulopsis sphaerica cells irradiated with He-Ne (lambda = 632.8 nm; dose--460 J/m2) and then cultured in the nutrient with 1% glucose and O2 for 6 h. The computer analysis of electron images of cell sections was carried out. Evidences of stimulation of cell proliferation were found, including decrease in the areas of cell and chondriome profiles, decrease in the number of mitochondria on sections, elongation of cells and mitochondria, and increased variability of cell parameters. In addition, cells of irradiated cultured were characterized by the increase in the number of mitochondria contacting the endoplasmic reticulum (in this case the outer mitochondrial membrane presumably associates with the ER membrane), which may suggest the activation of ATP synthesis. Thus, He-Ne laser irradiation activates cell metabolism even at the early stage of culture growth.     

Changes in chromatin structure during the mitotic cycle

Summary Optical density profiles of Feulgen-stained nuclei ofBryonia dioica at different stages of the mitotic cycle were determined. Nuclei in the G₂ phase have a greater fraction of dense chromatin than nuclei in G₁ phase. However, nuclei at the end of the S phase have dispersed chromatin of minimal density. Thus, chromatin density oscillates during the mitotic cycle of this species, consequently the progressive increase in density previously recorded throughout the intermitotic period of two other species (onion and mouse) cannot be a general rule.     

Changes in the human nuclear chromatin induced by ultra wideband pulse irradiation

The effects of ultra wideband pulse radiation on human cells were investigated. The density of the flow of energy on the surface of irradiated object varied from 10⁻⁶ to 10⁻² W/cm² with exposure of 10 s. It was shown that heterochromatin granule quantity in cell nuclei increased under the influence of radiation from 10⁻⁴ to 10⁻² W/cm². In some intervals the effect increased with irradiation dose. At irradiation intensity 10⁻³ W/cm² the process of heterochromatin granule formation was fully reversible after 2 h of recovery; at intensity 10⁻² W/cm² the reversion of irradiation effects was not full. The data obtained indicated the strong biological activity of ultra wideband ultra short pulse radiation.     

Activation of the synthesis of RNA in lymphocytes following irradiation by a He-Ne-laser]

The rate of 14C-uridine incorporation during the first 12 hours after exposure of human peripheral blood lymphocytes to He-Ne laser radiation (lambda = 632.8 nm, D = 56 J/m2) has been determined. The stimulation of RNA synthesis is maximum 2 and 4 h following irradiation. The same regularity is noted after the addition of phytohemagglutinin (PHA). In 7 h the rate of RNA synthesis in irradiated cells is at the control level whereas in PHA stimulated cells the rate of 14C-uridine considerably increases.     

Alterations of neuronal nuclear matrix and chromatin structure after irradiation under aerobic and anoxic conditions

This study was undertaken to determine if structural alterations of the bulk chromatin and the amount of protein associated with the nuclear matrix in cerebellar neurons depend on radiation dose and a cell's state of oxygenation. After irradiation with 2.5 to 25.0 Gy under both aerobic and anoxic conditions, the sensitivity of the neuronal chromatin to m. nuclease digestion increase linearly with dose up to about 5 Gy, beyond which there was no further increase. The same increase in accessibility of chromatin to micrococcal nuclease digestion was observed when neuronal nuclei were irradiated at 4 degrees C. Neuronal nuclei were stained with propidium iodide (PI) for DNA and with fluorescein isothiocyanate (FITC) for protein, both before and after complete digestion with DNase I, and analyzed by flow cytometry. There was no change in either the PI (P greater than 0.4) or the FITC (P greater than 0.9) fluorescence of undigested nuclei after irradiation. For the DNase I digested nuclei, the PI fluorescence was unchanged after irradiation (P greater than 0.4), but the FITC fluorescence increased significantly (P less than 0.02). This increase in the FITC fluorescence was linear with dose up to about 5 Gy, beyond which there was no further increase. The flow cytometry results from DNase I digested nuclei were identical for neurons irradiated under aerobic or anoxic conditions, indicating that this phenomenon is oxygen independent. This increase in FITC fluorescence after irradiation was inhibited at ice-cold temperatures and probably reflects an increase in protein content at the nuclear matrix that requires metabolism. This may explain our previously observed resistance of nuclear matrix-associated DNA to digestion by DNase I. This protein increase at the nuclear matrix appears to follow "saturation" kinetics identical to that previously reported for repair of DNA strand breaks in cerebellar neurons. However, the exact molecular nature of this process and its role in DNA repair or cell survival remains to be determined.     

Changes in osteoclasts after irradiation with carbon ion particles

We examined the effects of radiation on decreases in osteoclast numbers after regional irradiation of rats with carbon ions and gamma rays. Male Wistar rats were subjected to hind-leg irradiation with carbon ions (290 MeV/u) or gamma rays at doses of 15, 22.5, or 30 Gy. The effects of carbon ions and gamma rays on osteoclasts were studied using histologic and morphometric methods. At doses of 15 Gy and 22.5 Gy, osteoclast numbers increased transiently until day 5 after irradiation and then decreased rapidly in both the carbon ion and gamma ray irradiation groups. The carbon ion group showed reduced osteoclast size compared with the gamma ray group. Carbon ion irradiation had a more marked effect on osteoclast activity, and suppressed maturation to a greater extent than gamma irradiation. These observations suggest that carbon ion irradiation induces differential modulation of osteoclast growth factor expression.     

Changes in bone volume after irradiation with carbon ions

The effects of carbon ions on bone volume were studied by histological and morphometrical methods. Anesthetized rats irradiated with three different single doses (15, 22.5, or 30 Gy) of either carbon ions or gamma rays were sacrificed (5 rats/group) at 10 time points together with non-irradiated controls. Carbon ion-induced bone responses were qualitatively and quantitatively different from those induced by gamma irradiation at the same dose level. Irradiation with carbon ions resulted in a dose-dependent increase in bone volume, while gamma irradiation induced loss of bone volume, which was found to be independent of dose in the range studied over the same time course. After irradiation with carbon ions, bones showed thickening of the trabeculae, and the bone marrow became fibrous with fewer vessels. Carbon ion irradiation showed a greater effect on the bone-absorbing capability of osteoclasts than gamma irradiation. These observations suggest that irradiation with carbon ions may produce differential modulation of radiation-induced growth factor expression.     

Changes in chromatin structure during aging of human skin fibroblasts

Human skin fibroblasts from embryo, 16-, 30- and 60-year-old adults were cultivated and passaged in vitro. Their chromatin structures were examined by the sensitivity to micrococcal nuclease and by electron microscopy. When the mode of DNA degradation by the nuclease was analysed during in vitro aging of the embryo skin fibroblasts, the discrete ladder of nucleosomal DNA became obscure in old cells. Analogous change of chromatin structure was also observed even in young cells as their donor ages increased. From the observation with electron microscopy, it became clear that chromatin of fibroblasts from 30-year-old adults does not have regularly spaced nucleosomes, compared with chromatin from embryo. These results suggest that the length of the linker DNA which connects core particles becomes to be heterogeneous by aging, both in vivo and in vitro in human skin fibroblasts.     

Changes in the circular dichroic spectrum of calf thymus solubilized chromatin caused by ultraviolet irradiation

Summary UV irradiation of the chromatin caused an increase of the positive circular dichroic band in the vicinity of 275 nm (corresponding to DNA) and a deepening of the negative band of proteins at about 225 nm. These changes in the circular dichroic spectrum are monotonous in the range of doses studied (< 6 × 10⁴ J.m^–2). The increase of the positive circular dichroic band probably reflects the occurrence of local conformational changes in DNA, which include changes in base position (tilting, distance from helix axis) in the close neighbourhood of photoproducts. The presence of photoproducts in chromatin reduces changes in its circular dichroic spectra with temperature.     

Changes in chromatin structure associated with germination of maize and their relation with desiccation tolerance

Morphological and physicochemical measurements of chromatin condensation were made on germinating maize (Zea mays L.) radicles to determine whether the loss of genetic activities that occurs during the loss of desiccation tolerance is linked to irreversible changes in chromatin condensation. Chromatin samples were compared at different stages of germination (0, 24 and 72 h after imbibition), before (control) and after 24 h of desiccation. Morphological changes in chromatin structure and condensation were characterized by a qualitative and quantitative electron microscope study of chromatin which was allowed to spread in 0.2 mol m^?3 EDTA and then laid on coated microscope grids. The experiments showed similar levels of chromatin condensation in quiescent embryos and 24-h-old radicles (desiccation-tolerant material). After 72 h of imbibition, when radicle emergence and desiccation intolerance had ceased, the chromatin underwent a major decondensation towards various lower order folded structures. Regardless of the desiccation tolerance stage, an in vivo drying treatment of 24- and 72-h-old radicles before chromatin extraction did not induce significant changes in the extent of condensation compared to their respective controls. Similar conclusions were drawn from measurements of several spectroscopy properties (absorbance ratios: A₂₆₀/A₂₄₀, A₂₆₀/A4₀₀; thermal denaturation, and linear electric dichroism) of chromatin fragments that were obtained after nuclease digestion and then dissolved in 0-2 mol m^?3 EDTA. In quiescent and 24-h-old material, chromatin fragments were poorly soluble but highly stable during thermal denaturation. Chromatin fragments were 3-5-fold more soluble and less thermally stable in 72-h-old material than in 24-h-old material. In vivo desiccation had no significant effects on these properties compared to the respective controls. Collectively these data suggest that desiccation did not induce irreversible changes in the condensation properties of chromatin. The likelihood that the decondensation process occurring during germination is linked to the loss of desiccation tolerance is discussed.     

Changes in the blood system after its extracorporeal irradiation

One-stage extracorporeal beta-radiation of dog blood in a dose of 6-12 Gy is not followed by the development of radiation after-effects. Favourable changes in the peripheral blood content are marked. Erythrocyte carbohydrates undergo adaptation reconstruction with an increase in the percentage of nucleic acids. Over 7 to 30 days of observation the activity of nonspecific immunity factors such as lysozyme, lytic activity of beta-lysines, and phagocytosis also increases. At the same time the antimicrobial action of penicillin is potentiated. The data obtained can be used for correction of upset immune responsiveness.     

Changes in non-histone chromatin proteins during the storage of chromatin

We have described here the changes in stored chicken reticulocyte chromatin which take place among non-histone protein fractions based on SDS-polyacylamide gel electrophoresis and hybridization of globin cDNA with RNAs transcribed on native and reconstitited chromatin templates.     

Gamma irradiation induced apoptotic changes in the chromatin structure of human erythroleukemia K562 cells

Exponentially growing human erythroleukemia K562 cells were synchronized by centrifugal elutriation prior to and after Co⁶⁰ γ-irradiation (4 Gy). Forward scatter flow cytometry used for size analysis revealed the increase of an early apoptotic cell population ranging from lower (0.05 C-value) to higher DNA content (∼1 C) as the cells progressed through the S phase. The increase in cellular DNA content expressed in C-values correlated with apoptotic chromatin changes manifested as many small apoptotic bodies in early S phase and larger but less numerous disintegrated apoptotic bodies in late S phase. Most significant changes after exposure to γ-irradiation took place in early S phase resulting in an increase of nuclear size by more than 50%. Cell fractions containing irradiated cells showed enhanced growth arrest at 2.4 C-value, which was accompanied by apoptosis. Apoptotic cell cycle arrest near to the G₁/G₀ checkpoint and apoptotic changes indicate that the radiation resistance of K562 cells is related to the bypass of the early stage of the p53 apoptotic pathway. Apoptotic changes in chromatin structure induced by γ-irradiation indicate that these injury-specific changes can be identified and distinguished from chromatin changes induced by UV radiation or heavy metals.