V. Yu. Polyakov

Levels of granular organization of chromatin fibres.

The molecular organization of chromatin is a much discussed problem. One of the questions is the size of fibrils in isolated deoxyribonucleoprotein (DNP) and its relation to the size of fibrils in intact nuclei. Recently a number of authors have reported the detection of spherical chromatin untis 80-100 A in diameter connected to each other by a DNA strand [1 -4 ] . However, according to many morphological descriptions chromatin fibrils have an average diameter of 200-250 A [5 -7 ] . It is also known that the diameter of chromatin fibrils in nuclei and DNP preparations may vary from 100 to 200 A, depending on the isolation procedure used [8]. The aim of this study was to elucidate the factors determining the levels of structural organization of chromatin fibrils and to understand the way in which globular components participate in the formation of the 200 A chromatin fibril. We have shown that the basic structural unit of chromatin is a fibril consisting of 200 A globules which converts into a fibril with 100 A globules when part of the magnesium is removed.

Fusion, fragmentation, and fission of mitochondria.

Individual mitochondria which form the chondriom of eucaryotic cells are highly dynamic systems capable of fusion and fragmentation. These two processes do not exclude one another and can occur concurrently. However, fragmentation and fusion of mitochondria regularly alternate in the cell cycle of some unicellular and multicellular organisms. Mitochondrial shapes are also described which are interpreted as intermediates of their “equational” division, or fission. Unlike the fragmentation, the division of mitochondria, especially synchronous division, is also accompanied by segregation of mitochondrial genomes and production of specific “dumbbell-shaped” intermediates. This review considers molecular components and possible mechanisms of fusion, fragmentation, and fission of mitochondria, and the biological significance of these processes is discussed.

ORGANIZATION OF HIGHER-LEVEL CHROMATIN STRUCTURES (CHROMOMERE, CHROMONEMA AND CHROMATIN BLOCK) EXAMINED USING VISIBLE LIGHT-INDUCED CHROMATIN PHOTO-STABILIZATION

The method of chromatin photo‐stabilization by the action of visible light in the presence of ethidium bromide was used for investigation of higher‐level chromatin structures in isolated nuclei. As a model we used rat hepatocyte nuclei isolated in buffers which stabilized or destabilized nuclear matrix. Several higher‐level chromatin structures were visualized: 100nm globules—chromomeres, chains of chromomeres—chromonemata, aggregates of chromomeres—blocks of condensed chromatin. All these structures were completely destroyed by 2M NaCl extraction independent of the matrix state, and DNA was extruded from the residual nuclei (nuclear matrices) into a halo. These results show that nuclear matrix proteins do not play the main role in the maintenance of higher‐level chromatin structures. Preliminary irradiation led to the reduction of the halo width in the dose‐dependent manner. In regions of condensed chromatin of irradiated nucleoids there were discrete complexes consisting of DNA fibers radiating from an electron‐dense core and resembling the decondensed chromomeres or the rosette‐like structures. As shown by the analysis of proteins bound to irradiated nuclei upon high‐salt extraction, irradiation presumably stabilized the non‐histone proteins. These results suggest that in interphase nuclei loop domains are folded into discrete higher‐level chromatin complexes (chromomeres). These complexes are possibly maintained by putative non‐histone proteins, which are extracted with high‐salt buffers from non‐irradiated nuclei.

Structural-functional model of the mitotic chromosome

In the present review the structural role of noncoding DNA, mechanisms of differential staining of mitotic chromosomes, and structural organization of different levels of DNA compactization are discussed. A structural-functional model of the mitotic chromosome is proposed based on the principle of discreteness of structural levels of DNA compactization.

Chromosome scaffold and structural integrity of mitotic chromosomes

Chromosome scaffold represents a continuous protein substructure revealed in isolated metaphase chromosomes after harsh extraction. According to postulates of the widespread radial loop model the scaffold plays an important role in the formation and maintenance of structural integrity of the mitotic chromosomes. Here, the data concerning the structure and major components of the chromosome scaffold are presented. The experiments suggesting that the scaffold represents a system of discrete linker proteins and the data about high mobility of scaffolding proteins are discussed. Furthermore, the data about higher-level chromatin structures (elementary chromonema and 200–250 nm fibers) and behavior of scaffolding proteins are compared. The results presented agree with the idea that at the present stage it is possible to discriminate chromatin complexes, whose structural integrity is not maintained by the chromosome scaffold.

Antipodal complex development in the embryo sac of wheat

Dynamics of an antipodal complex formation in wheat (Tritiñum aestivum L.) has been observed in detail using a reconstruction of serial semifine sections. Three consecutive crucial stages have been identified in the development of the antipodal complex: (1) proliferation of initial cells, (2) growth and functional differentiation of antipodal cells, and (3) cell apoptosis. Specific features of the mitotic division of antipodal cells have been characterized. It has been shown that the structure of interphase nuclei and mitotic chromosomes of proliferating antipodal cells is similar to that of nucellar cells surrounding the embryo sac. According to the reconstruction of appropriately oriented serial sections, the division of antipodal cells is asynchronous. DNA content in differentiated antipodal cells has been determined by a cytophotometric analysis; in the case of a mature embryo sac, the ploidy of antipodal cells varied from 8 to 32C. Proliferation and DNA endoreduplication processes in the antipodal complex proceed at different time; the second process starts only after the termination of the first one. DNA endoreduplication is accompanied by total chromatin remodeling; as a result, giant chromosomes are formed in the nuclei of antipodal cells. The final stage of the antipodal complex development is programmed cell death or apoptosis. A model for the structural organization of an antipodal complex has been proposed based on the layer arrangement of cells. The secretory activity of antipodal cells directed towards the endosperm syncytium has been detected for the first time. The analysis of “truncated” ovules with an undeveloped endosperm has shown that developing endosperm can be a possible inductor, which stimulates the functional activity of antipodal cells and triggers their terminal differentiation. The obtained results evidence the functional role of antipodal cells in the development of the endosperm and embryo.

Structural organization of chloroplast of tomato plants Solanum lycopersicum transformed by Fe-containing superoxide dismutase

In this work we analyzed the results of the transformation of tomato plants Solanum lycopersicum by gene FeSOD1 from Arabidopsis thaliana, equipped with the signal sequence for targeting into chloroplasts. PCR analysis showed that the gene was integrated into the genome of several tomato plants that underwent transformation followed by selection in the kanamycin-containing medium. Two lines, provisionally denoted as nos. 6 and 8, were selected from the independent transformants. Line 6 was characterized by a reduced growth rate and altered leaves and line 8, by normal growth and leaves typical for control plants. Both lines showed a significant increase in SOD activity. In line 8 the increase in SOD activity was accompanied by an increase of ascorbate peroxidase activity, and in line 6 this effect was not present. Electron microscopic analysis of parenchymal and guard cells of both lines was performed, with an emphasis on the ultrastructural organization of chloroplasts. It is shown that the chloroplasts of the two transgenic lines differ in the number and size of starch grains and deposited plastoglobules as well as in the organization of lamellae and grana. Taken together, the results indicate that the expression of the introduced gene FeSOD1 has a significant effect on metabolic processes in the plastids. The findings are discussed in relation to the hypothesis about the importance of low concentrations of ROS for the integration of structure and function of chloroplasts.

Chromatin folding in human spermatozoa. I. Dynamics of chromatin remodelling in differentiating human spermatids

Changes in chromatin structure at different stages of differentiation of human spermatids were studied. It was shown that, in nuclei of early spermatids, chromatin is loosely packed and its structural element is an 8-nm fiber. This “elementary” fiber is predominant at the initial stages of differentiation; in the course of maturation, it is replaced by globular elements approximately 60 nm in diameter. In intermediate spermatids, these globules start to condense into fibrillar aggregates and reduce their diameter to 30–40 nm. At all stages of spermatid maturation, except the final stages, these globules are convergence centers for elementary fibers. This remodelling process is vectored and directed from the apical (acrosomal) to the basal pole of the nucleus. In mature spermatids, the elementary 8-nm fibers are almost absent and the major components are 40-nm fibrillar aggregates. The nuclei of mature spermatids are structurally identical with the nuclei of spermatozoa with the so-called “immature chromatin,” which are commonly found in a low proportion in sperm samples from healthy donors and may prevail over the normal cells in spermiogenetic disorders. The cause of this differentiation blockade remains unknown. Possibly, the formation of intermolecular bonds between protamines, which are required for the final stages of chromatin condensation, is blocked in a part of spermatids. The results of this study are discussed in comparison with the known models of nucleoprotamine chromatin organization in human spermatozoa.

Role of DNA methylation and histone modifications in structural maintenance of heterochromatin domains (chromocenters)

Effects of DNA methylation inhibitor; 5-azacytidine (5-aza-C); and histone acetylation inhibitor, trichostatine A (TSA), on the structure of pericentric heterochromatin of L929 mouse cells have been studied. 5-aza-C treatment for 48 h resulted in the transformation of ovoid chromocenters into elongated structures in 85% of cells. Hypotonic treatment of these cells reveals tandemly arranged DAPI-positive globules that are well distinguishable by light microscopy. Similar globular units can be observed in hypotonic-treated control cells. TSA treatment for 48 h causes dramatic decrease in HP1α content in cells. In 25% of treated cells chromocenters became highly decondensed and can not be reliably detected by light and electron microscopy. 85% cells demonstrate globular chromocenters with low HP1α content. Hypotonic treatment induces transformation of compact chromocenters into ring-like structures that can be either single or clustered. Rings are formed by uniform fiber in which no globular subunits are detected. The data obtained are discussed concerning several mechanisms of heterochromatin structure maintenance and the role of epigenetic factors.

A drug based on water-ethanol extract of oyster mushroom mycelium displays hepatoprotective properties in experimental model of steatohepatitis

Nonalcoholic steatohepatitis in rats induced by the high-fat diet was used as an experimental model for testing hepatoprotective properties of drug based on water-ethanol extract from oyster mushroom mycelium. Progression of pathology development was monitored histologically and by biochemical assay of blood samples. Subcellular response was analyzed using electron microscopy. It was shown that administration of the high-fat diet for 14 days leads to a development of adipose degeneration of liver (steatosis). Histological evidences were confirmed biochemically. Alkaline phosphatase, bilirubin, free cholesterol and lowdensity lipoprotein cholesterol levels were increased in blood of experimental animals in comparison to control. Administration of the drug was performed in parallel with high-fat food. It was shown that pathological alterations of liver in this case were reduced at both organ and cellular levels. Cholesterol and triglyceride levels were close to those in control animals. The data obtained confirm that the drug assayed can be used in clinical practice for prevention and treatment of nonalcoholic steatohepatitis.