[Loosening of condensed chromatin in human blood lymphocytes exposed to irradiation with a low-energy He-Ne laser].

It was shown that, 1 h after irradiation of human blood lymphocytes with a He-Ne laser at 56 J/m2 (5.6 W/m2, 10 s), the relative optical density of condensed chromatin masses observed in ultrathin sections was decreased (p < 0.01); i.e., the condensed chromatin became less compact. Such transition of condensed chromatin to a more "open" state may improve its availability for regulatory proteins and transcriptional factors. The irradiation also results in dispersion of condensed chromatin clumps in the nucleoplasm and enhancement of their angularity, i.e., in extension of the clump surface. These shifts, correlating with the activation of transcription, may be due to decompaction of the chromatin fibers not only on the periphery of chromatin clusters in the center of the nucleus, but also within the masses of condensed chromatin.

[Structure of mitochondria and activity of their respiratory chain in subsequent generations of yeast cells exposed to He-Ne laser light].

The data on the effect of He-Ne laser light (lambda = 632.8 nm) on mitochondria of yeasts in late log phase were reviewed. The qualitative analysis of the ultrathin sections of cells demonstrated a nonuniform thickness of the giant branched mitochondria typical for budding yeasts. Exposure to a dose of 460 J/m@2 accelerated cell proliferation, activated respiratory chain enzymes (cytochrome c oxidase and NADH dehydrogenase), and also changed the microstructure of the giant mitochondria--much of the narrow regions of the mitochondrial tube with sections < or = 0.06 microm2 were dilated (while no signs of organelle damage were observed). Relative surface area of the cristae increased in such mitochondria, which can be due to the activation of their respiration and ATP synthesis. The number of associations between mitochondria and endoplasmic reticulum increased in cells in early log phase, which reflects high capacity of mitochondria to absorb Ca2+. Altered giant mitochondria configuration can increase the efficiency of both energy transfer and Ca2+ distribution in the cytoplasm.

[Increase in the number of contacts of endoplasmic reticulum with mitochondria and plasma membrane in yeast cells stimulated to division with He-Ne laser light]. / Uvelichenie kolichestva assotsiatsii éndoplazmaticheskogo retikuluma s mitokhondriiami i plazmaticheskoi membranoi v drozhzhevykh kletkakh, stimulirovannykh k deleniiu svetom He-Ne-lazera.

We studied the ultrastructure of Torulopsis sphaerica yeast cells irradiated with He-Ne laser (lambda = 632.8 nm, dose--460 J/m2) and then cultured for 6 h in the nutrient with 1% glucose by aeration. The length of membranes of endoplasmic reticulum (ER) and the number of its associations with mitochondria (M) and plasma membrane (PM) were measured on ultrathin sections. A distance of less than 50 nm between heterogeneous membranes was considered as an "association". The cells from irradiated cultures are characterized by the following features: 1) the length of cortical ER membranes in relation to cellular perimeter, and the length of perinuclear ER membranes in relation to nuclear perimeter increase, resp., by 21 and 79%; 2) the number of ER-PM associations per cellular section, and that per unit of PM length increase, resp., by 26 and 41%; 3) the number of ER-M association in relation to the total mitochondrial perimeter, and to perimitochondrial ER increase by 80 and 87%, resp. The latter may be associated with Ca2+ uptake by mitochondria associated with ER, which results in activation of respiration and ATP production.

[Morphometric study of yeast cells of Torulopsis sphaerica after He-Ne-laser irradiation]. / Mopfometricheskoe issledovanie drozhzhevykh kletok Torulopsis sphaerica posle oblucheniia svetom He-Ne-lazera.

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.

[Formation of gigantic mitochondria in human blood lymphocytes under the effect of an He-Ne laser]. / Poiavlenie gigantskikh mitokhondrii v limfotsitakh krovi cheloveka pod vliianiem izlucheniia He-Ne lazera.

A phenomenon of formation of giant mitochondria in lymphocytes after He-Ne-laser irradiation (56 J/m2, 5.6 W/m2) was discovered. Reconstruction of mitochondria from ultrathin sections through the whole lymphocyte showed that 1 h after the irradiation the number of mitochondria was reduced to 9-12 compared to 40-45 in the control cells. In the irradiated lymphocytes 2-4 branching giant mitochondria were revealed instead of small discrete mitochondria in the control cells. Other 6-7 mitochondria were found to retain their shape and volume. It is suggested that the giant mitochondria are formed by fusion of small mitochondria. Formation of giant mitochondria may reflect the increase in the energy exchange level.

[A comparative study of chromatin from lymphocyte nuclei upon activation of transcription by irradiation from an He-Ne-laser or phytohemagglutinin]. / Sravnitel'noe issledovanie khromatina iader limfotsitov pri aktivatsii transkriptsii deistviem izlucheniia He-Ne-lazera ili fitogemaggliutinina.

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.

[The effect of irradiation by a He-Ne laser and phytohemagglutinin on lymphocyte mitochondria]. / Reaktsiia mitokhondrial'nogo appparata limfotsitov na obluchenie He-Ne-lazerom i na mitogen fitohemaggliutinin.

Electron-microscopic morphometry has been applied to study mitochondria on ultrathin sections of lymphocytes from human peripheral blood. It has been shown that the stimulation of lymphocytes by the mitogen phytohemagglutinin (PHA) 1 h causes increases in the quantity of mitochondria per cellular section (17%) as well as in the total area of mitochondria per cell section (35%), i.e. an increase in mitochondrial mass. Taking into account known facts about growth and division of mitochondria in late phases of cellular cycle, one can suppose that described above changes in mitochondria during G0----G1 transition under action of PHA belong to an early phase of biogenesis of mitochondria. In the contrary, irradiation of lymphocytes with He-Ne-laser (lambda = 632.8 nm) in dose 56 J/m2 which does not cause the G0----C1 transition, results in the increase in the number of mitochondria per cellular section (20%) but not increase in the total area of mitochondria per cell section. The last finding indicates to some modification of space configuration of the mitochondria without any changes in their mass. The increase in the quantity of mitochondria per cellular section after the irradiation could be related with the increase in electrochemical proton gradient and in phosphorylating activity of mitochondria. He-Ne-laser radiation as well as mitogen PHA cause some deaggregation of mitochondria (this is more pronounced in case of PHA) which may be related to their functional activation.

[Activation of transcription function in lymphocytes after irradiation with He-Ne-laser]. / Aktivatsiia transkriptsionnoi funktsii v limfotsitakh pod vliianiem oblucheniia He-Ne-lazerom.

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.

[The ultrastructure and possible role of the vacuolar component of hepatocyte nucleoli]. / Ul'trastruktura i vozmozhnaia rol' vakuoliarnogo komponenta iadryshek gepatotsitov.

A study was made of the nucleolar vacuoles in guinea pig hepatocytes that are poorly investigated for animal cells. A comparison of ultrathin sections, contrasted by heavy metals, with those treated according to Bernhard allowed to reveal the following intravacuolar structures: 1) fibrils 10-15 nm and 20-30 nm thick similar to perinucleolar chromatin fibrils; 2) RNP-granules 15-20 nm in diameter resembling the granular component of the nucleolus; 3) RNP-fibrils 15-20 nm thick with high electron density. The latter were visualized for the first time, their function still remains obscure. Upon stimulation of hepatocytes with partial hepatectomy, the vacuolar component changed. In 2.5 and 5 hours after the operation the vacuoles became smaller, the number of RNP structures of two types increased. Further, in 9 hours, the enlarging of vacuoles was accompanied by a sharp decrease in the number of these RNP-structures. The results obtained allow to suppose that the vacuoles of nucleolonemic nucleoli may be functioning elements, linking intra- and perinucleolar chromatin fibrils. They are depots for the RNP synthesized in the nucleolus; the rRNP is transported through the vacuolar system.

[Electron microscope study of chromatin in nuclei of rat hepatocytes during age-related changes in the genome]. / Elektronno-mikroskopicheskoe issledovanie khromatina v iadrakh gepatotsitov krys pri vozrastnykh izmeneniiakh genoma.

On ultrathin liver sections, condensed chromatin of rat hepatocyte nuclei was studied. The animals were 2 days, 6 and 28 months old. It was established that neither maturation nor senescence were accompanied by the change of the relative square of total condensed chromatin. Relative square of perimembrane, nucleoplasmic and perinucleolar condensed chromatin were non changed either. Intensively proliferating hepatocytes of nascent animals were characteristic of maximal values of the following parameters (i) the relative length of the perimembrane condensed chromatin boundary with nucleoplasma. (ii) amount of chromatin clumps, (iii) the relative length of the nuclear membrane without condensed chromatin. For mature animals all these parameters are significantly decreased. For old rats as compared with mature ones the following parameters are significantly diminished: (i) the relative length of the perimembrane chromatin boundary with nucleoplasma, (ii) the relative length of the nuclear membrane without condensed chromatin, (iii) the mean square of the nucleolus. So, the known diminishing of the RNA synthesis at senescence is expressed morphologically in margination of condensed chromatin, in smoothing of the condensed chromatin surface responsible for the hnRNA synthesis and also in diminishing of the nucleolus responsible for the rRNA synthesis.

[Rearrangement of nucleoli in hepatocytes stimulated for proliferation and enhancement of their transcription function]. / Perestroiki iadryshek v stimulirovannykh k proliferatsii gepatotsitakh i usilenie ikh transkriptsionnoi funktsii.

The nucleoli of normally functioning guinea-pig hepatocytes that have a nucleolonemal (strand-like) organization differ from identical nucleoli of other cells. Their nucleolonema consists as a rule of a fibrillar component with 45S RNA and is poor in granulas that contain pre-rNA molecules of an intermediate size and 28S rRNA, a dense fibrillar component with nascent rRNPs in its composition was not revealed. In hepatocytes stimulated by a 2/3 liver resection rearrangements in nucleoli were found. This brought to a conclusion that rRNA metabolism undergoes some changes. In 2.5 and 5 hours after the resection the hepatocytes' nucleoli were characteristic of a greater thickness of strands and a smaller size of vacuoles, appearance of distinct zones of the dense fibrillar component and an increased amount of RNP-granules. All these observations taken together point out at an increased synthesis and processing of rRNA at early stages of the prereplicative period. In 9 hours the character of changes in nucleoli was different: the vacuoles were considerably widened, whereas the thickness of strands that consisted of a well-expressed dense fibrillar, fibrillar and granular components was lesser. Such rearrangement points out at an increased transport of preribosomes from the nucleolus, a high level of synthesis and processing of nascent RNP-product being maintained. The changes of nucleolar RNP-component were followed by appearance of greater blocks of perinucleolar condensed chromatin, which may be connected with "cutting-off" some tissue-specific genes and initiation of functioning of the mitotic operon genes.

[Changes in the ultrastructure of inactive circular nucleoli from mature rat lymphocytes at early stages of blast transformation]. / Izmenenie tonkoi organizatsii neaktivnykh kol'tsevidnykh iadryshek zrelykh limfotsitov krys na rannikh stadiiakh blasttransformatsii.

We studied the ultrastructure of lymphocyte nucleoli from rat peripheral blood within the first 6 hrs of their cultivation in the presence of phytohaemagglutinin (PHA). In the mixed population of intact lymphocytes there were observed several types of nucleoli, the localization and the ratio of individual nucleolar components being different. This seems to reflect functional peculiarities of these nucleoli. Some nucleoli are characteristic of the presence of several sites of intranucleolar condensed chromatin. However, all these nucleoli could be related to the so-called ring-shaped nucleoli with a fibrillar center surrounded by fibrillar and granular RNP-components, which points out to their low synthetic activity. The major changes in the nucleoli of stimulated lymphocytes were observed after the action of PHA within 1 hr. These changes were expressed in the elevation of electron density of the fibrillar centre and the loss by it of clear borders and correct shape, in the appearance of large zones of the dense fibrillar component with acquired an increased electron density, and also in the disappearance of condensed chromatin sites. By that time, there was revealed a peculiar shape of nucleoli with a "fragmented" fibrillar centre and the presence of vacuoles and strands consisting of fibrillar and granular RNP-components. The different types of changed nucleoli in the population of stimulated lymphocytes seem to be the consequence of a gradual transition of ring-shaped nucleoli to active nucleolonema-like ones. So, it is obvious that changes in the ultrastructure of nucleoli occur already by 1 hr after the PHA action. This points out to the intensification of pre-rRNA transcription that takes place by that time.

[Electron microscope study of chromatin in hepatocyte nuclei during the first hours after partial hepatectomy. V. Changes in the relative area of condensed chromatin and the density of chromatin fibril packing in the ultrathin sections]. / Elektronno-mikroskopicheskoe izuchenie khromatina v iadrakh gepatotsitov v pervye chasy posle chastichnoi gepatéktomii. V. Izmenenie otnositel'noi ploshchadi kondensirovannogo khromatina i plotnosti upakovki fibrill na ul'tratonkikh srezakh.

The degree of chromatin condensation was studied on ultrathin cell sections of guinea pig hepatocytes during the prereplicative period after partial hepatectomy. Three time points were chosen for analysis namely 2,5, 5 and 9 hrs after operation since they show marked increasing (2.5 hrs), decreasing (5 hrs) and repeated increasing (9 hrs) of the amount of ethidium bromide binding to chromatin. The degree of chromatin condensation was determined by measuring the area occupied by condensed chromatin and also by measuring the number of chromatin fibrils per a certain length. The condensed chromatin with varying localization in the nucleus were studied separately. The changes of nucleoplasmic chromatin were most pronounced: at 2.5 and 9 hrs after operation the decrease of the relative area and of the density of chromatin fibrils package was observed; these parameters were near to control at 5 hrs after operation. In general the changes in nucleoplasmic chromatin were correlated with the changes of the activity of the chromatin in the whole nucleus. The decondensation of the perimembranous chromatin was manifested in the decrease of its area and was expressed only at 9 hrs after operation. The perinucleolar chromatin was found to show the gradual decondensation which was manifested mainly by the decrease of its relative area. Thus the condensed chromatin seems to be a labile structure which undergoes essential changes in the process of the exit of the hepatocytes from G0-stage of the cell cycle, during the prereplicative period.

[Electron microscopic morphometric study of activated chromatin of lymphocytes. I. Variation of chromatin condensations during early action of phytohemagglutinin]. / Elektronno-mikroskopicheskoe morfometricheskoe izuchenie aktivirovannogo khromatina limfotsitov. I. Izmenenie stepeni kondensatsii khromatina v rannii period deistviia fitogemaggliutinina.

We made a quantitative electron microscopic investigation of rat blood lymphocyte chromatin on cell sections in the early period of action of phytohaemoagglutinin (PHA). According to the dynamics of binding of acridine orange to chromatin, there were chosen time lapse after the beginning of stimulation of cells. In one hour of PHA action when the binding of acridine orange is intensified two-fold the total area of profiles of condensed chromatin was not changed, however, the area of membrane-free chromatin profiles was simultaneously diminished by 36%. At that period, the packing of DNP-fibers of condensed chromatin loosened. After 1.5 hr, parallel to a noticeable decrease of binding of acridine orange, there were observed tendencies for restoration of area of chromatin profiles and partial diminishing of relative distances between DNP-fibers. In 6 hrs, the binding of acridine orange was sharply intensified, which was accompanied by a decrease by 26% of the total area of chromatin profiles at the expense of diminishing of perimembranous chromatin. At the same time, the low-electron--density regions of condensed chromatin were enlarged, and the packing of DNP-fibers was strongly loosened.

[Electron microscopic study of the chromatin in hepatocyte nuclei in the 1st hours after a partial hepatectomy. IV. Increased sensitivity of active chromatin to EDTA exposure]. / Elektronno-mikroskopicheskoe izuchenie khromatina iader gepatotsitov v pervye chasy posle chastichnoi gepatéktomii. IV. Povyshennaia chuvstvitel'nost' aktivnogo khromatina k vozdeistviiu EDTA.

In the period of increasing the guinea-pig hepatocyte chromatin template activity, 2.5 hours after partial hepatectomy, an increased susceptibility of condensed chromatin to the bleaching action of EDTA in the Bernard reaction has been found. The condensed chromatin of the activated by partial hepatectomy guinea-pig hepatocytes, studied on ultrathin sections, is bleached under the action of EDTA more intensely compared to the chromatin of the control (non-activated) cells. Five hours after partial hepatectomy, when hepatocyte chromatin, according to its physico-chemical properties and functional activity, is the same as that of the control (non-operated) animals, its capacity of being bleached by EDTA also returns to the control level. In one nucleus studied on ultrathin sections the perinucleolar chromatin was found to be more sensitive to EDTA than the chromatin of other parts of the nucleus. It is suggested that the treatment with EDTA under given conditions can be used for revealing the functional state of chromatin on ultrathin sections.

Electron microscopic studies of guinea pig hepatocyte chromatin.

Chromatin from guinea pig hepatocytes has been studied by electron microscopy using the spreading technique. Among DNP fibers with nucleosomal organization numerous structures with different organization have been observed. The structures consist of the main axial fiber from which the lateral fibrils branch off normal to the axis. The distances between the bases of lateral fibrils vary to a considerable extent. The lateral fibrils are composed of regular granules with an average diameter of 257 A, and the axial fiber of irregularly and rarely arranged granules. The structures described are thought to be nonribosomal transcription complexes, and the granules of lateral fibrils--nuclear informosomes. The longest lateral fibril may have up to 80 granules (informosomes), which corresponds to a pre-mRNA chain of about 15-17 x 10(6) daltons.

[Electron microscopic examination of hepatocyte nuclei within first hours after partial hepatectomy. III. Changes of DNP fiber diameters of condensed chromatin]. / Elektronno-mikroskopicheskoe izuchenie khromatina v iadrakh gepatotsitov v perbye chasy posle chastichnoi gepatektomii. III. Izmenenie tolshchiny DNP-fibrill v sostave kondensirovannogo khromatina.

The DNP fibers have been measured on electron microscope photographs of sections of guinea-pig hepatocytes. The condensed chromatin was found to have fibers from 4 to 27 nm in diameter. Within the first hours after partial hepatectomy the distribution of fibers by their diameters is changed. This is correlated with the ethidium bromide binding to chromatin DNP, which reflects the level of the chromatin template activity. Maximal binding to ethidium bromide was observed at 2.5 hours after the operation and the distribution of fiber diameters was shifted towards a smaller diameter, simultaneously, the amount of thinner fibers from 4 to 14 nm in diameter is larger. Five hours after the operation the content of thicker fibers from 14 to 24 nm is reduced, and the intensity of the ethidium bromide binding is decreased. Nine hours later, the ethidium bromide binding was intensified, and the distribution of fiber diameters again shifted to a smaller width. Our data prove that the thickness of DNP fibers of condensed chromatin, which has been thought inactive, depends on the intensity of the template synthesis.

[Electron microscope examination of chromatin in hepatocyte nuclei within the first hourse after partial hepatectomy, II. The degree of chromatin condensation and the organization of fibrillar RNP components]. / Elektronno-mikroskopicheskoe izuchenie khromatina v iadrakh gepatotsitov v pervye chasy posle chastichnoi gepatektomii. II. Stepen' kondensatsii khromatina i organizatsiia fibrilliarnykh RNP-komponentov.

The state of hepatocyte chromatin (the area occupied by the regions of condensed chromatin on ultrathin sections and the quantity of perichromatin RNP fibrils which was estimated by the area of the fibrillar zone and the concentration of fibrils within the same zone) were studied within the first hours after partial hepatectomy of guinea pigs. The area occupied by the regions of condensed chromatin on preparations with differentially revealed DNP and RNP components decreased by 12% in 2.5 hours since the operation had been performed, became normal in 5 hours, and again decreased by 30% in 9 hours. Decondensation of chromatin was accompanied with the increase of the number of perichromatin RNP fibrils, products of template activity of chromatin, and the rise of ethidium bromide binding. The binding of ethidium bromide by the chromatin of hepatocytes increased by 39% in 2.5 hours, returned to the control level in 5 hours and again increased by 22% in 9 hours.