Stimulation of mouse hematopoietic stem cells by angiogenin and DNA preparations.

Immature hematopoietic progenitors are a constant source for renewal of hemocyte populations and the basic component of the tissue and cell repair apparatus. A unique property of these cells of internalizing extracellular double-stranded DNA has been previously shown. The leukostimulatory effect demonstrated in our pioneering studies was considered to be due to the feature of this cell. In the present research, we have analyzed the effects of DNA genome reconstructor preparation (DNAgr), DNAmix, and human recombinant angiogenin on both hematopoietic stem cells and multipotent progenitors. Treatment with bone marrow cells of experimental mice with these preparations stimulates colony formation by hematopoietic stem cells and proliferation of multipotent descendants. The main lineage responsible for this is the granulocyte-macrophage hematopoietic lineage. Using fluorescent microscopy as well as FACS assay, co-localization of primitive c-Kit- and Sca-1-positive progenitors and the TAMRA-labeled double-stranded DNA has been shown. Human recombinant angiogenin was used as a reference agent. Cells with specific markers were quantified in intact bone marrow and colonies grown in the presence of inducers. Quantitative analysis revealed that a total of 14,000 fragment copies of 500 bp, which is 0.2% of the haploid genome, can be delivered into early progenitors. Extracellular double-stranded DNA fragments stimulated the colony formation in early hematopoietic progenitors from the bone marrow, which assumed their effect on cells in G0. The observed number of Sca1+/c-Kit+ cells in colonies testifies to the possibility of both symmetrical and asymmetrical division of the initial hematopoietic stem cell and its progeny.

Stimulation of mouse hematopoietic stem cells by angiogenin and DNA preparations

Immature hematopoietic progenitors are a constant source for renewal of hemocyte populations and the basic component of the tissue and cell repair apparatus. A unique property of these cells of internalizing extracellular double-stranded DNA has been previously shown. The leukostimulatory effect demonstrated in our pioneering studies was considered to be due to the feature of this cell. In the present research, we have analyzed the effects of DNA genome reconstructor preparation (DNAgr), DNAmix, and human recombinant angiogenin on both hematopoietic stem cells and multipotent progenitors. Treatment with bone marrow cells of experimental mice with these preparations stimulates colony formation by hematopoietic stem cells and proliferation of multipotent descendants. The main lineage responsible for this is the granulocyte-macrophage hematopoietic lineage. Using fluorescent microscopy as well as FACS assay, co-localization of primitive c-Kit- and Sca-1-positive progenitors and the TAMRA-labeled double-stranded DNA has been shown. Human recombinant angiogenin was used as a reference agent. Cells with specific markers were quantified in intact bone marrow and colonies grown in the presence of inducers. Quantitative analysis revealed that a total of 14,000 fragment copies of 500 bp, which is 0.2% of the haploid genome, can be delivered into early progenitors. Extracellular double-stranded DNA fragments stimulated the colony formation in early hematopoietic progenitors from the bone marrow, which assumed their effect on cells in G0. The observed number of Sca1+/c-Kit+ cells in colonies testifies to the possibility of both symmetrical and asymmetrical division of the initial hematopoietic stem cell and its progeny.

[Characteristic of the active substance of the Saccharomyces cerevisiae preparation having radioprotective properties]. / Характеристика активной субстанции препарата дрожжей Saccharomyces сerevisiae, обладающей радиопротекторными свойствами.

The paper describes some biological features of the radioprotective effect of double-stranded RNA preparation. It was found that yeast RNA preparation has a prolonged radioprotective effect after irradiation by a lethal dose of 9.4 Gy. 100 % of animals survive on the 70th day of observation when irradiated 1 hour or 4 days after 7 mg RNA preparation injection, 60 % animals survive when irradiated on day 8 or 12. Time parameters of repair of double-stranded breaks induced by gamma rays were estimated. It was found that the injection of the RNA preparation at the time of maximum number of double-stranded breaks, 1 hour after irradiation, reduces the efficacy of radioprotective action compared with the injection 1 hour before irradiation and 4 hours after irradiation. A comparison of the radioprotective effect of the standard radioprotector B-190 and the RNA preparation was made in one experiment. It has been established that the total RNA preparation is more efficacious than B-190. Survival on the 40th day after irradiation was 78 % for the group of mice treated with the RNA preparation and 67 % for those treated with B-190. In the course of analytical studies of the total yeast RNA preparation, it was found that the preparation is a mixture of single-stranded and double-stranded RNA. It was shown that only double-stranded RNA has radioprotective properties. Injection of 160 µg double-stranded RNA protects 100 % of the experimental animals from an absolutely lethal dose of gamma radiation, 9.4 Gy. It was established that the radioprotective effect of double-stranded RNA does not depend on sequence, but depends on its double-stranded form and the presence of "open" ends of the molecule. It is supposed that the radioprotective effect of double-stranded RNA is associated with the participation of RNA molecules in the correct repair of radiation-damaged chromatin in blood stem cells. The hematopoietic pluripotent cells that have survived migrate to the periphery, reach the spleen and actively proliferate. The newly formed cell population restores the hematopoietic and immune systems, which determines the survival of lethally irradiated animals.

[Effect of Triton X-100 on genetic segregation and associated monocotyledonous and dicotyledonous traits in sugarbeet (Beta vulgaris L.)].

The effect of Triton X-100 (TX-100) on the ratio of phenotypic classes and the expression of morphological traits in the progeny of sugar beet hybrids (N12 and N2) was investigated. It was shown that the TX-100 exposition on the unopened flower buds of sugar beets has different effects on hybrid progenies. In agamospermic progeny of hybrid plant No 12km-4, a significant decrease in the heteroallelic (heterozygous) phenotypic classes of alcohol dehydrogenase (ADH1) fraction was determined in the nonagamospermic progeny of hybrid plant No 2km-2 appearance of sugar beet seedlings with one cotyledon leaf was detected. The obtained results indicate the high efficiency of the epimutagenic effect of TX-100 on the early stages of plant ontogenesis.

[Role of polyteny and chromosome-membrane interactions in plant genetic processes].

Existing data in the literature on polyteny in plants and in eukaryotes in general, data on the structure of interphase chromosomes and their association with the nuclear membrane are analyzed in the paper. In light of these data, the results of our studies on the ratios of phenotypic classes of marker enzymes in agamospermy (apomictic) progeny of sugar beet are considered. The given data allow to state that chromosome polyteny and their association with nuclear membrane provide not only hereditary information, but also determine the ratio of genotypes and phenotypes in progeny.

[Autosegregation of enzyme loci in agamospermous progenies of triploid plants of sugar beet (Beta vulgaris L.)].

The ratios of the phenotypic classes of glucosephosphate isomerase (GP12) and malate dehydrogenase (MDH1 and MDH2) were studied in agamospermous progenies of triploid sugar beet plants. The ratio of the phenotypic classes of these enzymes corresponds to the calculations based on the assumption of polyteny of chromosomes carrying alleles of the enzyme loci accompanied by the loss of extra copies of the alleles in the first division of a cell entering embryogenesis. An increase in the gene dosage due to polyteny leads to the appearance in the progeny with a definite frequency of alleles that were absent in the original parental plant. The notions of meiotic autosegregation and mitotic autosegregation characteristic of meiotic and mitotic agamospermy are introduced, as well as the term locus polygenotype characterizing not only the allelic composition and the number of chromosomes, but also the number of chromatids carrying alleles of the marker locus in the cell before its entry into embryogenesis.

[Effect of colchicine and Triton X-100 on expression of the enzyme-encoding genes in nongerminating seeds of sugarbeet (Beta vulgaris L.)].

The expression of the enzyme-coding genes, controlling glucose-phosphate isomerase (GPI), malate dehydrogenase (MDH), and alcohol dehydrogenase (ADH), was examined in nongerminating seeds of sugarbeet after Triton X-100 (TX-100) and colchicine treatment. Two types of changes revealed included modification of the enzymatic loci expression (change of the isozyme electrophoretic mobility) and inactivation of standard profiles. In the MDH and GPI systems, these processes were found to be associated. Complete isozyme modification was accompanied with the disappearance of standard profiles. In the ADH system, the treatment with TX-100 and colchicine gave rise to two independent processes, including silencing of the Adh1 locus and the appearance of the ADH isozymes with abnormal electrophoretic mobility, which were probably the products of the Adh2 locus. It was suggested that the effect of TX-100 and colchicine on the expression of the enzyme-encoding genes examined depended on the intracellular localization of the encoded enzymes.

[Polymorphism of PCR profiles and expression of alleles at the locus Adh1 in agamospermous progeny of beet root Beta vulgaris L].

A modification of the ISSR amplification method based on using a combination of microsatellite and specific unique primer is proposed and tested. This modification simplifies the detected PCR profiles and allows the examination of DNA regions containing definite genes. Combinations of microsatellite primer Mic2 (5'-gacag-acaga-cagac-a-3') and one of the primers specific to the Adh1 locus, which controls alcohol dehydrogenase (ADH1) in sugar beet, were employed in this work. The microsatellite primer was used in combination with the following specific primers: Adh1f (5'-agagt-gttgg-agagg-gtgtg-ac-3') containing the binding site at the fourth exon of gene Adh1, or Adh1r (5'-act(ct)a-cagca-ag(ct)cc-(ct)ac(ct)g-ctcc-3') that binds to the fifth exon of the same gene. In the agamospermous progeny of individual heterozygous diploid plants of sugar beet with the Adh1-F/Adh1-S genotype, polymorphism of PCR profiles obtained in plants of each of three phenotypic classes (FF, FS, and SS) was detected. Among plants of the progeny from an individual plant that represents the heterozygous phenotypic class FS, differences were revealed not only between the PCR profiles but also in the relative activity of allele isozymes of ADH1.

[Analysis of natural samples for studying the variation in sugar beat (Beta vulgaris L.) agamospermous seed progeny].

The existence of natural genetic and natural mixed samples of seeds was theoretically grounded and experimentally demonstrated. A natural genetic sample is detected when analyzing the seed agamospermous progeny in the sequence coinciding with the sequence of seed setting, i.e., in the order they are located on a branch. The method of natural genetic samples has demonstrated the presence of individual regions with nonrandom distribution of phenotypic classes of agamospermous seeds on the branches of sugar beet plants. This phenotype distribution reflects the state of somatic cells on extended branch regions and the manifestation of this state in seed-bud cells. In turn, the genotype of each seed reflects the state of the seed-bud cell (apozygote) that gave rise to embryo development. We also demonstrated heterogeneity in the ratios of enzyme phenotypes of the seeds set on individual branches, regarding it as a result of the changes arising in somatic tissues at the moment of branching. Analysis of the ratios of seed phenotypes on individual branches of the same plant was named the method of natural mixed samples. The combination of natural genetic and natural mixed samples provides for identifying and analyzing various modes of seed setting (gamospermy and agamospermy) and studying the factors that influence the variation in these processes. We postulated that the nonrandom distribution and ratio of the seed phenotypes on plant branches resulted from a nonrandom endoreduplication of the homologous regions in homologous chromosomes carrying the marker locus alleles.