The nuclear desoxyribonuclease (DNAse) spectrum of normal rat thymus cells and after whole-body x-ray irradiation

Spectra of thymocyte nuclear DNAases of control and irradiated (4 Gy) rats have been investigated. Using the method of SDS-electrophoresis of nuclear proteins in DNA-polyacrylamide gel (PAAG) the authors managed to discover a number of polypeptides of 35, 32, 17.7, 17.2, and 16.4 kDA molecular mass possessing a DNAase activity. The enzyme of 35 kDA is only active in the presence of Ca2+ and Mg2+ ions. Nucleases of 32, 17.7, 17.2, and 16.4 kDA are active in the presence of Ca2+ ions and inactive in the presence of Mg2+ ions or in the absence of divalent cations. A simultaneous addition of Ca2+ and Mg2+ ions to the incubation medium causes a synergistic effect with respect to the manifestation of these DNAase activities. Nucleases of 32, 17.7, 17.2, and 16.4 kDa only emerge after the preliminary removal of histones by ion exchange chromatography on a column with CM-sephadex C-50. The enzymic activity of 32 kDA protein increases 60 min after irradiation and drops to the control value in 4 h. At the same time, the postirradiation increase in DNAase activity of a low-molecular weight enzyme group remains invariable throughout the entire period of observation (1-4 h). The preinjection of cycloheximide (CHI) prevents the postirradiation degradation of chromatin and, simultaneously, makes the enzymic activity, corresponding to 35 kDA protein, disappear at the electrophoregrams. The experiments with CHI permit to identify the given enzymic fraction as Ca/Mg-dependent endonuclease. This indicates the participation of normally pre-existing Ca/Mg-dependent endonuclease in implementing the process of chromatin enzymic degradation in the irradiated thymocytes.     

Effect of infrared impulse laser irradiation on the development of albino rat embryos]

Development of the white rat embryos has been studied under effect of infrared laser beams (wave length--0.89 mcm, impulse frequency--300 Hz) during various periods of pregnancy. Exposure of the female rats for 256 and 128 sec produces an increased preimplantational death of embryos and certain disturbances in formation of the osseous skeleton in some embryos.     

Disorders of vaginal development in the progeny of white rats exposed to x-ray irradiation

One hundred and twenty white rat embryos 13-22-day-old have been irradiated with x-rays (the dose 250 R) on the 12th-14th day of embryogenesis. The embryos have been divided into series of sagittal, frontal, transversal sections and stained by means of general histological methods. The irradiation performed on the days mentioned does not affect formation of the paramesonephric ducts. In all the experimental animals the caudal end of the paramesonephric duct is separated from the mesonephric duct as a solid cellular cord in which the lumen appears later. In the experimental females the disturbances developed after irradiation are manifested first of all in retardation of the main stages of the organ's formation; the retardation is observed: in fusion of the paramesonephric ducts, in resorption of the medial septum between the fused ducts, in separating the sinuous part of the vagina from the urogenital sinus, in recanalization of the vaginal epithelial cord. More severe lesions are presented as agenesia of the vaginal sinuous part and as its atresia represented by a transversal septum of the organ. The disorders in the vagina development are depended on massive primary necrobiotic radial lesions of the mesenchymal cells around the epithelial anlages of the small pelvis.     

Studies on the development of the eye cup and optic nerve in normal mice and in mutants with congenital optic nerve aplasia.

With the use of quantitative histological techniques, we have described, in normal mice, the formation of a system of intercellular channels within the embryonic retina and continuing without interruption into the optic stalk. The channels develop in advance of the morphological differentiation of the retinal ganglion cells and their neurites. Moreover, they appear at predictable times during gestation and are localized along the potential route to be taken by the earliest developing fibers of the optic nerve. A functional relationship may exist between the development of the channels and the subsequent outgrowth of the optic nerve from the eye. We have also examined a series of mouse embryos homozygous for the mutant gene ocular retardation (or^J), which causes optic nerve aplasia. In the or^J mutant, there is a reduction in area of these extracellular spaces and the optic nerve fails to exit from the eye. The lack of intercellular space within the mutant retina is associated with an increased number of cells which, in turn, may result from a continuing absence of normal cell death during earlier stages.     

The fine structure of the limulus optic nerve

Two complete composite photographs of the optic nerve of Limulus, made by electron microscopy, reveal the presence of neurosecretory granules in the large axons of the rudimentary eye neurons. The number of intermediate sized, (3–7 μ), of eccentric cells corresponds with the number of ommatidia as expected, but only their sheath of Schwann cells show an intimate interfolding. Based on the number of fine axons within the nerve each ommatidium has an average of 12–13 retinular cells. The diameter of their fibers is between 0.2 and 3 μ although the majority are between 1 and 1.5 μ. They are aggregated into bundles of six to seven fibers by the sheath cells although some bundles contain only two, others as many as 181 fibers. There is no indication in these studies that retinular cell axons within a bundle are associated with the same, adjacent, or other pattern of ommatidia. The photographs suggest that physiological activity in retinular cell axons might be detected most easily in the smallest bundles because they contain the fewest, but the larger retinular cell axons.     

The differentiation of oligodendrocytes in the rat optic nerve

The time of appearance and the rate of accumulation of specific myelin lipids and proteins were measured in the rat optic nerve during the period from birth to 18 days. The appearance of the activities of several enzymes involved in the synthesis of these lipids was also monitored. Correlation of these biochemical data with previously known morphological findings indicated that the “active” oligodendrocytes (detected between 5 and 15 days after birth) displayed maximal levels of synthesis of the components of myelin, and that these cells appeared to be responsible for the initial synthesis of myelin. Both young and mature oligodendrocytes showed limited capacity to synthesize these compounds. Furthermore, induction of the enzymes involved in the synthesis of myelin components appeared to take place in a simultaneous, rather than a sequential manner.     

Dynamics of rat hepatocyte histone glycoxidation after general x-ray irradiation

Dynamics of structural parameters of hepatocyte histone glucoxidative modification 3, 9 and 24 h after general X-ray irradiation of rats at dose 5 Gy was studied. Dynamics of these parameters (content of carbonyl groups, bityrosyl cross-linkings, pentosidines, advanced glycation end products) was compared with alterations in DNA structure (according to agarose gel electrophoresis) and lipid peroxidation extent (by malondialdehyde content). Oxidative stress induced by hepatocyte irradiation results in structural damage of DNA and histones accompanied by an increase of histone bityrosyl cross-linking and carbonyl content. The content of advanced glycation end products in histones corresponds to the extent to DNA damage and malondialdehyde content. The described postradiation modifications of histones may be important for regulation of chromatin function.     

Action of x-ray irradiation on the nuclear envelope of rat liver cells]

X-irradiation of isolated nuclear envelopes (NE) has revealed their high radiosensitivity, while irradiation of isolated intact nuclei in vitro, in the doses up to 5000 r 18--20 hours after partial hepatectomy, produced no morphological changes in NE. The damaging effect of irradiation on both nuclei and mitochondria (Mt) was revealed only with a decrease in cytochrome-c-oxidase (CO) activity in parallel with an increase in the radiation dose. One hour after the whole body irradiation of rats in the beginning of S-period, the damaging effect was recorded in both NE and Mt at the doses of 50 and 150 t, and was enhanced with the increase of irradiation dose. Morphological changes were observed mostly in the outer nuclear membrane, which lost its distinct outline and disappeared from some nuclear regions. Lethal radiation doses produced a decrease in the number of pore complexes (PC) with their evident segregation from the membranes. After irradiation in a dose of 1200 r, only the residue or "ghosts" of the PCs remained. After irradiation in doses up to 400 r, the CO-activity recovered during the first hour in Mt and during first two hours in the nuclei.     

Development of the notochord in normal and malformed human embryos and fetuses.

In view of its possible involvement in early embryogenesis and teratogenesis, the developmental characteristics of the human notochord were studied by light and electron microscopy and immunohistochemistry on 20 human conceptuses (5th-22nd week). At the earliest embryonic stages examined, the notochord is closely related to both the pharyngeal endoderm and the neuroectoderm of the posterior (tail) end of the neural tube. In both regions the interspace is bridged by slender cytoplasmic processes, lined with basal lamina and filled with amorphous extracellular material containing collagen types III and IV and laminin. The notochordal cells express cytokeratin brightly and vimentin weakly. As embryonic age progresses, the notochord gradually separates from the epithelia, becomes the axis of developing spinal column and undergoes progressive cell degeneration and rearrangement within the vertebral bodies. This is associated with extensive production of extracellular material and the first appearance of fibronectin. Intracellularly, the expression of vimentin gradually increases, while that of cytokeratin slightly weakens. Changes in the notochord parallel other developmental events in axial organs. In anencephalic fetuses the course of the notochord is irregular and partly interrupted with segments outside the basichondrocranium in specimens with craniorachischisis.     

DNA synthesis in the mono- and binuclear cells of regenerating rat liver after x-ray irradiation

The effect of X-irradiation on the dynamics of DNA synthesis during the S-period in bi- and mononucleated of regenerating rat liver was studied autoradiographically and microphotometrically. Rats were treated with X-rays at doses 3.84 X 10(-2), 15.48 X 10(-2), and 30.96 X 10(-2) Kl/kg 23 hours after a partial hepatectomy, and were sacrificed one hour after irradiation. In the control liver the rate of DNA synthesis was the lowest at the beginning of the S-period and the highest at the last quarter of this period in both mono- and binucleated cells. The irradiation results in the inhibition of DNA synthesis mainly at the end of the S-period depending on doses employed. This inhibition was the same in bi- and mononucleated cells. In addition, the increase of correlation of the 3H-thymidine incorporation rate and DNA content was found between nuclei of binucleated cells after irradiation.     

Nuclear pore density in rat liver cells during regeneration and x-ray irradiation of the animals]

Cytoplasmic as well as nucleoplasmic surfaces of the pore complexes (PC) could be observed using freeze-etching method. The density of PCs per 1 micron2 of nuclear envelope (NE) surface in regenerating liver (9.9) is twice as that in resting liver (5.3). 1 hour after 1200 R X-ray irradiation the pore density in regenerating liver decreases 5.8-fold, consisting only of 1.7 PCs per 1 micron2 of the NE. The structure of the PC after irradiation undergoes degradation and normal PCs practically disappear; only their "ghosts" remain. Peripheral and possibly central granules of the PC appear to consist of some subunits with their diameter of 4--5 nm. The central granule forms a channel through which RNA containing material may be transported from the nucleus to the cytoplasm. The non-uniform state of the PC, observed on platinum-carbon replicas of cleaved nuclei, and the non-altered PC associate with the dense lamina of the NE, after detergent treatment of isolated nuclei indicate that the PC could be formed inside the nuclei and to be "inserted" into the NE membranes in the course of their processing.     

Kinetic characteristics of Ca2+-phospholipid-dependent protein kinase from the rat liver in the early stages after x-ray irradiation]

The changes of kinetic characteristics (apparent Km and Vmax) of the Ca2+ phospholipid-dependent protein kinase (protein kinase C) from the rat liver for substrates ATP and histone Hl 2 and 24 hours after total X-ray irradiation have been established. The obtained results evidence for the important role of these changes in early radiosensitivity of protein kinase C.