Population screening of the Arctic variant, which has arisen due to the G > A mutation at locus rs80356779 in the CPT1A gene, has been performed for the first time among indigenous peoples of Siberia (Chukchi, Koryaks, Evens, Evenks, Yakuts, Buryats and Altaians) and East Asia (Koreans). It is assumed that CPT1A Arctic variant originated from Eskimo ancestors, probably as a result of adaptation to a high-fat diet and/or to the extremely cold environment. It is also known that the homozygous Arctic variant is associated with hypoketotic hypoglycemia attributable to CPT1A deficiency and high infant mortality and occurs at high frequency in American Eskimo. On the other hand, the association of CPT1A Arctic variant with increased levels of HDL-cholesterol and apolipoprotein A1 in blood plasma suggests that this mutation might have a cardioprotective role. In the present study, a high frequency of CPT1A Arctic variant has been found in coastal populations of Northeast Asia – in Koryaks (66 %), Chukchi (56 %) and Evens (30 %), and singularly (at a frequency of 1 %) in Evenks of Central Siberia. Five polymorphic loci relevant to the haplotypic structure of CPT1A gene (rs2278908, rs2278907, rs2924699, rs7112615 and rs2229738) were revealed by high-throughput DNA sequencing in addition to locus rs80356779 studied here. It was found that the Arctic variant haplotype has arisen only once on the basis of the haplotype, which is widespread in modern populations of Eurasia. We assume that the expansion of Eskimo culture of the sea mammal hunting as well as Eskimo assimilation by Chukchi and Koryaks have contributed to the spread of the CPT1A Arctic variant across the populations of indigenous peoples of Northeast Asia.

Cattle breeding, including sheep farming, is an important sector of agriculture. Increasing productivity and improving meat quality are considered today as the priorities in the industry. Significant advances have been achieved in sheep breeding through the use of genetics. The commonplace of all selection programs is using manufacturers selected on the basis of the quality of the offspring, relatives or ancestors. At the same time, using the achievements of molecular genetics can lead breeding to a new methodological level. The problem of finding reliable communication between productivity features and genetic markers has not yet been solved, because productivity is a set of features (unlike, for example, monogenic diseases) and its expression depends on the balance between various physiological functions. By contrast, imbalance may cause reduced productivity as a whole even if there is a positive role of prevailing element. Selection on the basis of genetic markers of productivity aims to work with animals with high genetic potential for weight gain and meat quality. This review considers promising genes – potential markers of productivity in sheep farming, such as growth hormone gene, callipyge, calpain and calpastatin, which have promise as genetic markers for sheep selection. However, it should be stated that in spite of numerous reports about potential genetic markers of productivity there is still no data about the influence of molecular genetic methods on improving the economic performance of sheep selection.

The present work is phenomenological and purely descriptive. It shows polymorphism of colors in a breeding group of mini-pig in the Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences (ICG SB RAS) and makes some assumptions about the nature of this genetic diversity. In the ICG SB RAS mini-pig herd, wild-type coloration (agouti), black-spots, black and white color are present. All these options have variants, including previously undescribed, with genetically determined differences. In addition, the ICG SB RAS mini-pigs have variants inherent only to heterozygous individuals and archaic gray color described only in primitive domestic pig. Comparison of the observed color phenotypes in the breeding with literature data showed that EDNRB, KIT, KITLG and the MC1R are polymorphic loci, while ASIP, OCA2 and TYRP1 are monomorphic loci. Thus, the loci that control the most common coat colors in domestic pigs are polymorphic, while those controlling rare or relatively rare colors and monomorphic. The research allowed us to put forward some assumptions concerning the genetic determination of the phenotypic characteristics of color seen in the study group selection. Among these assumptions is one about the genetic control of juvenile colors in domestic pigs. Observations made in the ICG SB RAS mini-pig breeding group allowed us to examine and describe the features of age-related changes of an embodiment of black-motley suit, when newborn piglets originally have black-and-motley fur color on the brick-red background and at an age of one year, a gray roan background formed by a mixture of white and black hairs. In addition, it was shown that with the help of technical means black spots can also be detected on a black background.

The genetic diversity of potato varieties from the VIR collection was analyzed based on microsatellite analysis. These varieties have been bred in Russia and near-abroad countries since the 1931. Application of 14 highly polymorphic nuclear microsatellites (SSR) enabled the complete discrimination of all 113 varieties. Additionally, we have studied these varieties for the distribution of 8 DNA markers associated with three R-genes involved in the control of resistance to two quarantine objects: the potato wart Synchytrium endobioticum and golden potato cyst nematode Globodera rostochiensis, which occur locally in some regions of the Russian Federation. All the analyzed varieties with resistance to S. endobioticum pathotype 1 revealed the diagnostic marker Nl-251400 of the Sen1 gene and a few susceptible cultivars lost this diagnostic fragment. The tested markers of the H1 and Gro1-4 genes, which confer resistance to G. rostochiensis pathotype Ro1 revealed different predictiveness. In the molecular screening of potato varieties, it is better to use several markers of these genes. Results of molecular screening using six markers of the H1 and Gro1-4 genes allowed us to detect 6 haplotypes in the tested subset. Five haplotypes include varieties with different combinations of the markers tested, the majority (87.9 %) of these varieties were highly resistant or moderately resistant to G. rostochiensis. The most numerous haplotype H1/0 included 76 varieties, which did not possess any marker; 96.1 % of these varieties were susceptible to G. rostochiensis. Predictive associations between haplotype content, wart and nematode resistance, pedigree and ‘variety age’ are discussed.

The results of years of studying new and promising varieties of Apple trees (Malus domestica Borkh) grown in Southern Russia. Evaluation of Apple cultivars was carried out for resistance to the major fungal pathogens scab (Venturia inaequalis (Cooke) G. Winter) and powdery mildew (Podosphaera leucotricha Salm.), the quality and marketability of fruits, the content of biologically active substances underlying the antioxidant  properties of fruits. An advanced method of polyploidy based on inclusion of parental forms with immunity to scab was applied in Apple selection. Integrated assessment of key biological traits of varieties and forms of Apple with diverse ploidy in unstable environments resulted in a selection of donors and sources of significant characteristics for Apple breeding such as resistance and immunity to major fungal pathogens, large fruit, bright red color of the fruit, improved chemical composition. High-quality varieties of Apple breeding in SKZNIISiV together with VNIISPK combining immunity to scab (the Vf gene) with high resistance to powdery mildew were selected: Fortune, Amulet, Carmen, Nika, Margo, Orfei, Souyz and others. Apple-tree grades, immune to scab, with a high viability of pollen (83–98%) were revealed: Azimuth, Vasilisa, Carmen, Lubava, Margo, Fortune, Liberty, Prima, Florina, Freedom. The promising parental forms passing to most of their hybrid progeny good tastes of fruits are Golden Delicious tetraploid, Wealthy tetraploid, Belle de Boskoop, Balsgard 0247 E, Papirovka tetraploid, and diploidy grades to Alkmene, Prima, Redfree and Kuban. Apple cultivars were revealed with high chemical indicators including the content of sugars: Orfei (10.8 %), Champion (10.5 %); vitamin C: Vasilisa and Souyz (more than 9.0 mg/100 g), hybrid form 44-24-49-uy (more than 11.4 mg/100 g); and P-active substances – Vasilisa, Lubava, Margo (more than 110.0 mg/100 g), whose use in breeding programs encourages the rapid creation of new genotypes with improved biochemical composition of fruits. 

Scald (Rhynchosporium secalis (Oudem.) J.J. Davis pathogenic agent) is one of the most harmful barley diseases. A high variability of this fungi explains the occurrence of new aggressive pathotypes and, accordingly, loss of cultivar resistance. It was found that many recently selected varieties of barley and previously identified sources of resistance to R. secalis are now susceptible to the pathogen. There is only one gene, Rrs9, that maintains efficiency against pathogen populations in Russia. The aim of this study is to find donors of genes for effective resistance to scald with the ability to easily transfer this trait for hybridization. The inheritance of scald resistance in 33 barley landraces was studied. Analysis of the interaction between the pathogen test clones and the host plant revealed a difference between the alleles determining fungal resistance in 32 barley forms, the previously effective genes Rrs4, rrs6, rrs7, and currently effective Rrs9. It was found that 30 accessions are protected by new unidentified genes for scald resistance. Accessions k-18398 and k-16231 from China are likely to possess allelic genes for scald resistance, while the gene (or genes) of accession k-31075 from Nepal is allelic to the Rrs9 gene. It was demonstrated by hybridological analysis that accessions k-3307, k-15868, k-18989 and k-3481 are protected by effective genes for scald resistance, which differ from each other and which are not allelic to the Rrs4, rrs6, rrs7 or Rrs9 genes. Accessions k-15868 and k-3481 possess two complementary recessive genes for scald resistance, k-18989 has two recessive genes, and k-3307 carries one recessive gene for pathogen resistance. The resistance genes of these forms are manifested during all the stages of plant ontogenesis.

Results of a study of economically valuable parameters of barley regenerant lines obtained by cell selection on selective media with aluminum, hydrogen ions and polyethylene glycol are presented. Genotypes superior to the initial variety and standard variety have been identified under laboratory conditions: root length index (regenerants: 0.9–1.2 %; standard: 0.8 %) and drought resistance (regenerants: 17.8–45.2 %; standard: 8.5 %); productive parameters under growth chamber conditions (12 % increase in germination ability, 21 % increase in productive plant stand, 2.3- fold increase in grain number per ear, 1.5-fold increase in weight of grains per plant). The parameter ”weight of grains per plant” correlates with the level of alkalization of the rhizosphere zone of the investigated plants under stressful conditions in growth chamber experiments (r = 0.908). Regenerant pants are typically much less affected by phytopathogenic fungi. The genotypes screened in selective systems in vitro had advantages over the initial varieties and standard varieties in productivity, especially under provocative conditions of acid soils and moisture deficit in 2010. As a result of field tests, the genotypes of regenerant origin were identified that had consistently shown a 10.0–43.2 % higher productivity than the standard variety over several years. The proportion of varieties regenerated at the stage of competitive variety trials increased from 8.3 % (2006) to 32.4 % (2014). The varieties regenerated with high combining ability are used as the parental forms in crosses. New barley varieties Forward and Bionik have been developed on the basis of regenerant lines 917-01 and 496-07. Under edaphic stress (pH 3.8–4.5; Аl3+ 0.5–9.6 mg/100 g soil), Forward has productivity up to 5.5 t/ha and Bionik, 6.6 t/ha; which is 113–128 % higher than the standard.

The search for and creation of the initial material steady against adverse environmental factors and adapted to constantly changing weather conditions have always been some of the most promising directions of selection of agricultural plants. Results of studying the resistance of spring soft wheat (F4, F5) hybrids to the main leaf phytopathogens (Erysiphe graminis DC. f. sp. tritici Em. Marchal, Alternaria spp., Helminthosporium spp. and Puccinia recondita Rob. ex. Desm f. sp. tritici Eriks) under different soil and climatic conditions are presented. Tests were carried out within two vegetative periods (2013–2014) in three geographic locations: Tyumen region (Russia), Baden-Württemberg (Germany) and Lower Saxony (Germany). These areas significantly differed in climatic conditions during the study years. The assessment of resistance to leaf diseases in the hybrids was carried out in the settings of natural infection. The influence of a complex of abiotic factors on the prevalence of phytopathogenic fungi at the study sites has been demonstrated. Hybrid combinations that were less susceptible to powdery mildew, brown rust and spot blotch than other hybrids have been identified, and so have been the hybrids that have resistance to all the phytopathogens identified. It was noted that the hybrid forms under the ecological test had different indicators of biological resistance of plants across the geographic study sites. The ♀Cara × ♂Lutescents 70 hybrid at both German sites had rather a high index of biological resistance of plants and possessed, according to field data, complex resistance to the main phytopathogenic fungi at all geographic sites.  Ecological testing of new plant genotypes under environmental conditions strongly differing in biotic and abiotic factors is one of effective methods to reveal resistance to phytopathogens and to identify highly adapted cultured plants.

Seed metabolomic profiles have been investigated in wild and cultivated forms (cultivars) of oat (Avena L.). Seed accessions from the VIR oat collection were used for the research. Metabolomic analysis employed gas liquid chromatography-mass spectrometry (GLC- MS) using an Agilent 6850 chromatographer (USA). The analysis covered the composition and content of organic and fatty acids, amino acids, polyatomic spirits and sugars. The content fluctuation range for the studied groups of compounds was found to be narrower (significantly in some cases) in cultivars than in the wild species. Along with a sharp increase in oleic acid content, cultivars demonstrated a decrease in that of linoleic acid. The general conclusions from the comparison of seed metabolomic profiles in wild species and cultivars are presented below. A number of wild species can be recommended as a potential source of biochemical quality traits for breeding purposes. A series of metabolites (compounds), the content of which changes during domestication or which differentiate wild oat species from cultivars has been identified was found. Along with such well-known healthy food chemical factors as oleic acid, glucose and fructose, etc., differences concerning monoacylglycerol compounds (MAG 16 : 0 and MAG-2 18 : 2, etc.) have been found. The latter have been proposed to be related to the formation of adaptive traits, in particular, resistance to diseases and pests, and to environmental abiotic stresses.

The use of monosomic lines significantly increases the effectiveness of molecular-genetic analysis and the development of superior quality breeding lines via substitutions by alien chromosomes. A complete set of aneuploid series for each cotton chromosome was required. Simple sequence repeats (SSR) have been applied as useful markers for understanding cotton genetics. Several DNA markers have already been assigned to the individual chromosomes of Gossypium hirsutum. The primary objective of this paper is to report the creation of new monosomic lines and the use of chromosome specific simple sequence repeat (SSR) markers and translocation lines for chromosome identification to confirm chromosome specificity of monosomic lines in the Uzbek collection. Here, we summarize data on the development of a monosomic stock collection of cotton (G. hirsutum L.) from Uzbekistan, including the origin of 95 primary monosomics, their cytogenetic characteristics, morphological features and their identification by translocation tests and chromosome SSR-markers. Our results indicated several different monosomic lines identified by chromosomes 2, 4, 6 and telosome 11 of the At subgenome and chromosomes 18 and 22 of the Dt subgenome, 22 monosomes were identified as duplicates of three monosomes (chromosomes 2, 4 and 6). Chromosome 4 of the At subgenome was recovered more frequently (18 times) than chromosome 2 of the At subgenome (4 times) and chromosome 6 of the At subgenome (3 times) during pollen irradiation at different doses and in the progeny of the desynaptic plants. These lines will be useful for molecular mapping and enhancement of Upland cotton.

This paper presents the results of studying the inheritance of protein markers in different generations of interspecific hybrids of cotton Gossypium hirsutum L. × G. barbadense L. and G. barbadense L. × G. hirsutum L. While these hybrids were in development, the species G. hirsutum was represented by dwarf plants and the species G. barbadense, by variety C-6037 plants. G. hirsutum plants had protein marker H-0.13 and G. barbadense plants had the homologous marker, protein B-0.18. Previous studies demonstrated that in interspecific crosses these markers behave as alleles of the same locus. This allowed these markers to be used for analysis of the ratios of phenotypic classes in the succession of generations of interspecific hybrids, including F2, F9 and F10. In these studies, the plants with only one type of protein (H-0.13 or B-0.18) were regarded as homozygous for the corresponding gene, while plants with both proteins (H-0.13 and B-0.18), as heterozygous. Some polymorphic progeny showed a significant shift in the ratios of phenotypic classes. These changes were observed as an increased or a decreased share of a particular phenotypic class, or as a significant increase or decrease in the proportion of the heterozygotes. The electrophoretic protein composition of cotton seed samples remained unchanged until the tenth generation of hybrids, but in the tenth generation, some heterozygous plants gave offspring, the seeds of which lacked protein with an electrophoretic mobility of 0.70. Plants in which this protein was absent belonged to the homozygous phenotypic class with marker H-0.13.

The garden chrysanthemum (Chrysanthemum morifolium Ramat.) is a highly demanded autumn flowering culture for parks and gardens and as cut flowers and has long, abundant and colorful flowering. The lack of winter-hardy and season-adequate flowering cultivars and poor resistance to diseases limit its distribution in most regions of Russia. The development of new plant-breeding material by artificial interspecific hybridization able to combine valuable traits of different Chrysanthemum species in a single organism is therefore of importance. Reciprocal crossing has been conducted between the species of the Manchurian (C. chanetii H. Lév., C. coreanum (H. Lév. et Vaniot) Nakai, C. maximoviczii Kom., C. naktongense Nakai, C. zawadskii subsp. acutilobum (DC.) Kitag., C. tenuisectum Kitag., C. zawadskii subsp. latilobum (Maxim.) Kitag., C. leiophyllum Nakai), Mongolian-Siberian (C. mongolicum Y. Ling, C. zawadskii Herbich) flora and interspecific hybrid forms with the involvement of subtropical Chrysanthemum species. Crossing and backcrossing of species with identical numbers of chromosomes (hexaploid × hexaploid, 2n = 54 and tetraploid × tetraploid, 2n = 36) were easy. Tetraploids × hexaploids showed good compatibility. Diploids (2n = 18) are not always compatible between themselves and are partly compatible with hexaploids. Interspecific hybrids in the first generation (F1) were characterized by phenotypic uniformity with intermediate inheritance or predominance of traits of the highploidy parent and differed in hybrid power, intensity of emergent rhizomes and complex of adaptive qualities. Multicomponent interspecific hybrids have been obtained from combinations of crossing, consisting of three different components. The selected highly decorative interspecific and multicomponent forms have promise for planting of greenery and serve as complex sources of adaptiveness for the development of garden chrysanthemum native to Russia.

Biologically active compounds on the basis of highly efficient strains of bacteria of the genus Bacillus are currently being sought for to increase the yield of grain crops in the North. There is a number of biological products available, including those based on bacteria in the genus Bacillus (Phytosporin-M, Bactofit, Gamair, Integral, and other). However, the effectiveness of these drugs in the northern regions may be reduced, because the activity of introduced microorganisms depends on their adaptability in the rhizosphere and rhizoplane. This article describes the effect of bacteria of the genus Bacillus in permafrost on the morphological and physiological indicators of seedlings of soft spring wheat variety Irgina. These indicators are germination energy, laboratory seed germination, length and wet weight of roots and shoots of seedlings, as well as the length of coleoptile and number of chlorophylls a, b and carotenoid in the pigment extract from the plant material. It has been demonstrated that germination and morphometric parameters were significantly higher following exposure to Bacillus strains in permafrost than following treatment with the current strain of Phytosporin-M. It is likely that in the process of swelling of seeds (during their preplant treatment) Bacillus bacteria reach the caryopsis with water and begin to cleave spare nutrients actively, making them easier accessible for assimilation by seedlings. It is proposed that the increase in morphophysiological indicators of spring wheat variety Irgina is also a consequence of stimulation of the photosynthesis system and, consequently, an increased efficiency of absorption of light energy. Considering how differently different strains of bacteria isolated from permafrost influence the morphological and physiological and biochemical parameters of the plant, it appears that these strains or their combinations can be used for the development of biologics ensuring a comprehensive effect.

Apple chlorotic leaf spot virus (ACLSV), apple stem pitting virus (ASPV), and apple stem grooving virus (ASGV) are common in all apple cultivating countries including Belarus. The aim of this investigation was to study the genetic diversity of the apple-tree viruses in Belarus. Virus-infected apple trees were identified by RT- PCR in modern horticultural plantations and were not found among old trees aged more than 50 years. The genome fragments of the ACLSV, ASPV, and ASGV viruses detected were cloned and sequenced. The analysis of their nucleotide sequences gives evidence of high molecular variability generated mostly by single nucleotide substitutions and rarely by single nucleotide deletions and insertions. Recombination processes could also make some contribution to the existing genetic diversity of the virus populations studied. Estimates of selective pressure on the genome fragments of ACLSV, ASPV and ASGV obtained in this study as well as homologous sequences from the GenBank database indicate negative selection, except for one codon in the sequence of the ACLSV coat protein, which is under positive selection. The study of phylogenetic relationships between viruses isolated in different countries did not reveal any clear-cut relationship between their geographical origin and the degree of similarity of virus genome fragments.

Heterosis as the increased performance of hybrid progeny compared to their inbred parents is one of the most intriguing phenomena in genetics. The first attempts to find out about underlying mechanisms were based on theoretical models, which were useful, but could not characterize this unique phenomenon as a whole. With the advent of molecular markers great efforts were made to identify genomic regions causing heterotic response and clarify prospects of using information about molecular divergence of parental forms as a criterion for the prediction of F1 performance. Despite some achievements, the effec-tiveness of both molecular divergence and prospective heterotic QTL for practical goals was limited, confirming that genetic heterogeneity is necessary, but not sufficient to produce perfect phenotype. Current methodological tools of functional genomics and related disciplines have provided new opportunities for searching for mechanisms of heterosis at different levels in the context of relative importance of dominance, overdominance and epistasis. To date, differences in genome organization, gene expression and epigenetic status have been found between hybrids and their parents. At the genomic level, some QTLs associated with heterosis were identified and the impact of DNA divergence on F1 performance was evaluated. At the level of transcriptome, it was shown that heterosis in hybrids occurs along with changes in gene expression regulation under the influence of circadian clock genes. Several studies have been conducted to clarify the role of epigenetic DNA modification and genomic imprinting in the manifestation of heterosis. Taken together, data indicates that heterosis cannot been explained by a single common mechanism, because this complex phenomenon involves many components, a cumulative effect of which leads to the formation of an outstanding phenotype.

A study of phylogenetic relationships in closely related groups of epidemically important species is an important task of parasitology and evolutionary theory. One of these species groups is the maculipennis complex, with its members widespread in Eurasia, North Africa and North America. We studied phylogenetic schemes of the Palaearctic part of this Complex, which are based on crossability of species, polytene chromosomes, isoenzymes, cuticular hydrocarbons and ITS2 of rRNA genes. The lack of high compliance between them prompted us to create a consensus scheme of phylogenetic relationships in the Palaearctic part of this Complex. We constructed phylogenetic schemes on the sequences of a COI gene fragment, the D2 variable region of 28S rDNA and ITS2 of rRNA genes using various algorithms (Neighborjoining, Minimum Evolution, Maximum parsimony and Maximum likelihood). Two consensus schemes of the Palaearctic branch of the maculipennis complex were constructed, validated and discussed. The first scheme was based mostly on molecular genetics data. The second scheme relies on polytene chromosome, geographical spread and ecological data. Phylogenetic relationships of An. beklemishevi are contradictory. This species is similar to other Palaearctic species by molecular markers but it is far from them by fixed chromosome inversions. It was shown that this species is distant from Neoarctic species of the An. quadrimaculatus branch. The proposed schemes show two possible scenarios of the maculipennis complex evolution. Differences between them are explained by facts and associated speculations as well as by assumptions made under different phylogenetic reconstruction methods. A hypothesis about a significant role of the disparity between the astronomical and biological times in the interpretation of evolutionary processes was proposed. To address the phylogeny problem of the Palaearctic branch of the maculipennis complex at a more detailed level, it is required to increase our knowledge about the underlying species.

The immune response to immunogenic stimuli depends on various factors like cytokine context, way of entry, and immune status of the organism. In mice, female chemosignal entry into the male organism via the respiratory system causes activation of the mucosal immune response, which leads to the development of enhanced resistance to infections and is of adaptive value. However, the activation of mucosal immunity depends on the genetic predispositions of the immune response. BALB/c and C57BL/6 are prototypically Th2- and Th1-type mouse strains, respectively, therefore, they can serve as perfect model organisms for studying mechanism of lung mucosal immune activation in response to female chemosignals. Respiratory tract mucosal immune response to intranasal application of LPS, urea solution, saline and female urine used as a chemosignal was investigated in BALB/c and C57BL/6 male mice. Application of both female urine and LPS increased total white blood cell count and protein concentration in bronchoalveolar lavage fluid in BALB/c, but not in C57BL/6 male mice, suggesting an important role of Th2 pathway in lung mucosal immune response. At the same time, urine application provoked a significantly lower plasma corticosterone elevation than LPS. Thus, sexual signals associated with infection risks provide genotype-dependent mobilization of innate immunity without significant activation of physiological stress mechanisms.

Various mutationally impaired genes are often found in malignant tumors of animals and humans. At the same time, a large number of carcinogens demonstrate positive activity in different in vitro tests for mutagenicity. These findings are indicative of a geno- toxic mechanism of carcinogen action. It is considered that chemically active carcinogens induce mutations (and tumors) directly interacting with DNA, while inactive substances are mutagenically activated in the processes of cellular metabolism in target tissues. The aminoazo dyes was found to be activated by N-hydroxilation and subsequent conjugation with sulfuric acid catalyzed by the enzyme sulfotransferase. Previously we found that it is activated metabolites of ortho-aminoazotoluene that are responsible for its inhibitory effect on hormonal induction of tyrosinaminotransferase activity in the liver of sensitive mice. Inhibition of sulfoconjugation of 4-aminoazobenzene, another hepatocarcinogen for mice, by pentaclorophenol was reported to reduce its both mutagenic and carcinogenic activity. In this paper, we confirmed this observation. But we found that, when used ortho-aminoazotoluene, pentaclorophenol inhibited its mutagenic activity, but significantly stimulated the hepatocarcinogenic potency. It seems that carcinogenic action is provoked by unmetabolysed ortho-aminoazotoluene per se or some of its nonsulfated derivatives. The results of our comparative study with ortho-aminoazotoluene and 3.4-benzopyrene are in contradiction with the genotoxic theory of carcinogenesis: both are similarly activated by mouse liver enzymes, but induce tumors in different tissues: the former, hepatocellular carcinomas and the latter, splenic lymphoma. The conclusion was made that the accepted notion about the mechanism of carcinogenesis has to be revised.

Previously, we reported on the development of a therapeutic regimen allowing eradication of primary murine Krebs-2 ascites transplants. This protocol involved multiple injections of dsDNA preparations administered during the NER and HR phases of repair of interstrand DNA cross-links induced by prior cyclophosphamide treatments. Mice treated under this protocol frequently developed secondary ascites, which indicated that some tumor-inducing cancer stem cells could survive the treatment and caused relapse. Further, we observed that animals receiving multiple dsDNA injections developed pronounced systemic inflammatory response. This prompted us to develop a more straightforward treatment regimen based on the synergistic activity of cyclophosphamide and dsDNA preparations, which would allow complete eradication of established primary Krebs-2 ascites and also be less toxic for the treated animals. This protocol relies on a precisely timed single injection of dsDNA during the NER/HR transition period of each repair cycle. Under this protocol, 8-day remission of Krebs-2 engrafted mice was achieved, which was similar to the results of the multiple-injection treatment schedule. We observed an increase in the average life span of Krebs-2- transplanted mice on a single-injection regimen, which was consistent with reduced toxicity of such treatment.

Cumulative evidence obtained in this series of studies has guided the logic behind the development of a novel composite dsDNA-based preparation whose therapeutic application according to the specific regimen completely cures the mice engrafted with otherwise lethal Krebs-2 ascites. The likely mechanism involves elimination of TAMRA+ tumor-inducing stem cells (TISCs) from Krebs-2 tumors. We performed quantitative analysis of TISC dynamics in Krebs-2  ascites following treatment with the cytostatic drug cyclophosphamide (CP) and untreated control cells. In intact ascites, TISC percentage oscillates around a certain value. Following CP treatment and massive apoptosis of committed cancer cell subpopulation, we observed relative increase in TISC percentage, which is consistent with reduced susceptibility of TISCs to CP. Nonetheless, this treatment apparently synchronizes TISCs in a cell cycle phase when they become sensitive to further drug treatments. We describe the regimen of synergistic DNA + CP activity against Krebs-2 ascites. This protocol results in a complete cure of 50 % of Krebs-2 engrafted mice and involves three metronomic injections of CP exactly at the timepoints when repair cycles are about to finish combined with dsDNA injections 18 hours following each CP injection. The “final shot” uses CP + DNA treatment, which targets the surviving yet highly synchronized and therefore treatmentsensitive cells. The first three CP/DNA injections appear to arrest Krebs-2 cells in late S-G2-M phase and result in their simultaneous progression into G1-S phase. The timing of the “final shot” is crucial for the successful treatment, which eradicates tumorigenic cell subpopulation from Krebs-2 ascites. Additionally, we quantified the changes in several biochemical, cellular and morphopathological parameters in mice throughout different treatment stages.