The nuclear delivery of nucleic acid derivatives is an essential prerequisite for successful antisense therapy. Using laser confocal and electron microscopy, we have studied the uptake of fluorescently labeled oligonucleotides in the form of nanocomposites with polylysine and TiO 2 nanoparticles into Caco2, MDCK, and HeLa cells. In all three cell lines, bright fluorescence has been detected after 30 min in the nuclei (excluding the nucleoli) of the interphase cells; no substantial increase in the intensity of the signal was observed for next 24 hours. In all cells undergoing mitosis, the signal was localized in the cytoplasm with zones of higher intensity around chromatin. In some cells, at the beginning of interphase (G-1 phase), fluorescence was not detected at all. The latter may be explained by the brief moment in the cell cycle when oligonucleotides delivered in the nanocomposite cannot be taken up by cells. The studied nanocomposites are prone to aggregation. The degree of aggregation increases with the storage time up to complete loss of the ability of the nanocomposites to penetrate the cells.

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.

In the present paper, we report on the series of experiments where multiple regimens of CP and dsDNA injections were tested for targeting the ascites form of murine Krebs-2 cancer in situ. We show that combining CP with cross-linked human and salmon dsDNA results in a synergistic toxicity for ascites-bearing mice, an observation supported by the histopathology analysis of organs and tissues of experimental animals. In contrast, using a composite mixture of native and cross-linked human and salmon DNA after CP injections leads to a significant increase in average lifespan of the treated mice. Further, we demonstrate that repeated rounds of CP+dsDNA injections result in dramatic anticancer effect. The timing of injections is chosen so that they target the cells that were insensitive to the previous treatments as they were in the G2/M phase. 3-4 rounds of injections are needed to eliminate the subpopulation of tumor-initiating cancer stem cells. Our experiments identified the regimen when complete resorption of the primary Krebs-2 ascites occurs in all of the treated animals, followed by a remarkable remission period lasting 7-9 days. Yet, this regimen does not prevent secondary site metastases (either solid or ascites form) from developing, which is likely caused by the migration of ascites cells into adjacent tissues or by incomplete eradication of cancer stem cells. To address these and other questions, we expanded the study and performed histopathology analysis, which indicated that secondary metastases is not the only cause of death. In fact, many animals displayed unfolding systemic inflammatory reaction which was culminated by multiple organ failure. Thus, we developed the concept for treating ascites form of Krebs-2 cancer, which allows elimination of the primary ascites.

The synergic action of the cytostatic drug cyclophosphamide (CP) and fragmented exogenous DNA causes illness and death in mice (Dolgova et al ., 2011–2013). The observed «delayed death» effect was most clearly pronounced when the DNA preparation was administered 18 to 30 hours after CP treatment. This time span is designated as «death window». It was found that injections of exogenous DNA result in sustained increase in bone marrow cell (BMC) apoptosis, which occurs throughout the time of DNA administration (18–30 hours). Exogenous DNA, both allogeneic and belonging to various taxa induces BMCs apoptosis. Plasmid DNA has the greatest effect on apoptosis induction. The analysis of reduction and restoration of BMC subpopulations as the mice progressed to death revealed a virtually complete loss of the 12–20-mkm fraction of the cell population (about 3–4 % vs. 35–40 % in the control), which corresponds to the maximum leukopenia on day 3 after CP treatment. However, the relative amount of CD34+ hematopoietic stem cells (HSCs) from day 15 and till the end of the observation constituted 1,2–1,4 %, which corresponds to the wild-type range. Comparison of BMC smears from the sternal bone marrow of the CP and CP+DNA groups of mice indicates that the BMC populations isolated from CP+DNA animals lack young committed lymphopoiesis progenitor cells. Moreover, the affected mice had immature blast cell types in their blood, which was never observed in healthy or CP-treated mice. Pathological and morphological analyses show that starting from posttreatment day 9, mice that received CP+DNA preparations displayed pronounced morphological changes in their lungs, liver, pancreas, central and peripheral immune system organs, and brain. Most of the pathological changes observed are consistent with severe inflammatory response. This suggestion is proven by structural equivalents of functional involution of lymphoid organs, such as thymus, spleen, and lymph nodes. We speculate that the death of treated animals resulted from multiple organ dysfunctions caused by accidental involution of lymphoid organs and the systemic inflammatory response syndrome, both associated with injections of fragmented exogenous DNA into experimental animals within the «death window», which corresponds to the final step in the repair of the majority of CP-induced double-strand breaks.

The synergistic action of the cytostatic drug cyclophosphamide (CP) and fragmented exogenous DNA causes illness and death in mice (Dolgova et al., 2011–2013). The observed “delayed death” effect was most clearly pronounced when the DNA preparation was administered 18 to 30 h after CP treatment. This time span was termed the “death window.”

Methods of noncovalent immobilization of DNA fragments on titanium dioxide nanoparticles (TiO 2 ) were developed to design TiO 2 ∼DNA nanocomposites, which were capable of penetrating through cell membranes. TiO 2 nanoparticles of different forms (amorphous, anatase, brookite) with enhanced agglomeration stability were synthesized. The particles were characterized by X-ray diffraction, small-angle X-ray scattering, infrared spectroscopy and atomic force microscopy. Three approaches to the preparation of nanocomposites are described: 1) sorption of polylysine-containing oligonucleotides onto TiO 2 nanoparticles, 2) the electrostatic binding of oligonucleotides to TiO 2 nanoparticles bearing immobilized polylysine, and 3) sorption of oligonucleotides on TiO 2 nanoparticles in the presence of cetyltrimethylammonium bromide (cetavlon). All three methods provide an efficient and stable immobilization of DNA fragments on nanoparticles that leads to nanocomposites with a capacity of up to 40 nmol/mg for an oligonucleotide. DNA fragments in nanocomposites were shown to retain their ability to form complementary complexes. It was demonstrated by confocal laser microscopy that the proposed nanocomposites penetrated into cells without transfection agents and other methods of exposure.

Methods of noncovalent immobilization of DNA fragments onto titanium dioxide nanoparticles (TiO2) were developed, which led to TiO2-DNA nanocomposites capable of penetrating through cell membranes. TiO2 nanoparticles of different forms (amorphous, anatase, brookit) with enhanced agglomeration stability were synthesized. The particles were characterized by X-ray diffraction, small angle X-ray scattering, infrared spectroscopy and atomic force microscopy. Three approaches to the preparation of nanocomposites are described: (1) sorption of polylysine-containing oligonucleotides onto TiO2-nanoparticles, (2) the electrostatic binding of oligonucleotides to TiO2 nanoparticles bearing immobilized polylysine, and (3) sorption of oligonucleotides on TiO2 nanoparticles in the presence of cetavlon. All three methods provide an efficient and stable immobilization of DNA fragments onto nanoparticles, which leads to nanocomposites with a density for an oligonucleotide up to 40 nmol/mg. It is shown that DNA fragments in nanocomposites retain their ability to form complementary complexes and can be delivered into cells without transfection agents and other methods of exposure.

Different types of bleomycins are used extensively in cancer therapy. Antibiotics scarcely penetrate through cell membranes, leading to the necessity of using high concentrations of the drug and, therefore, overall body toxicity. To study the penetration of an antibiotic into cells, the fluorescein residue (Flu) was introduced in bleomycin A5 (Blm) to form Blm Flu . It was shown by confocal fluorescent microscopy that Blm Flu in the presence of titanium dioxide nanoparticles (TiO 2 , 3–5 nm), in contrast to free Blm Flu , efficiently penetrate the cell cytoplasm. After being delivered into cells in the presence of TiO 2 , Blm appeared to be 7–10 times more efficient than free Blm in the fragmentation of intracellular nucleic acids. The results suggest that nanoparticles are very promising to promote the delivery of antibiotics into cells, which will allow reducing their therapeutic dose.

Electron-microscopic analysis of cytomictic channels formation in the pollen mother cells in tobacco at the stage of meiosis prophase I of anthers has been conducted. The cytomictic channels in the pollen mother cells in tobacco have been established to be formed under the basis of both single plasmodesmata and de novo with the involvement of specific electron-dense bodies. The role of cytomictic channels in microsporogenesis regulation is discussed.

In this study we examine the possibility that TiO2 nanoparticles and their conjugates can penetrate into cultivated cells without any special transfection procedures. Oligonucleotides and their derivates were conjugated with the TiO2 nanoparticles, which were obtained as colloidal solutions at a concentration of TiO2 0.3M by TiCl4 hydrolysis. The electronic microscopy of various cell cultures (KCT, Vero, and MDCK) treated with nanoparticle solutions (20 µg/µl) showed that nanoparticles could enter the cells and accumulate in the vacuoles and phagosomes and form inclusions in cytoplasm. Thus, we demonstrated the penetration of TiO2 nanoparticles and their oligonucleotide conjugates into intracellular space without any auxiliary operations. Most other researches used electroporation techniques for similar purposes [1, 2, 5].

The biological properties of cowpox virus (CPXV) mutants with target deletion of 4 of the 6 BTB/kelch genes (D11L, C18L, G3L, and A56R) were examined in CV-1 cell cultures. There were changes in mutant temperature sensitivity and a reduction in a viral cytopathic effect. The mutant-infected culture yielded a smaller number of cells with actin-related long cellular protrusions (63 of 300 cells) as compared with wild CPXV (127 of 300). The length of the protrusions was 20-60 and 40-120 microm, respectively). Confocal microscopy revealed the formation of large globed structures containing both actin and CPXV antigens in the cells infected with quadruple mutants. These globed structures were recognized as incomplete protrusions. The findings show that the formation of long protrusions in the cells infected with wild type CPXV represents a type of specific viral potency related to the activity of BTB/kelch genes whose deletion results in cellular insufficiency to form full-fledged protrusions.

We carried out an in vitro histological and TEM investigation of the organization of the interna of Peltogasterella gracilis (Crustacea: Rhizocephala), a parasite of the hermit crab. The colonial interna of P. gracilis includes a reproductive system with multiple nuclei (externa rudiments) and a trophic system of absorbing lampbrush and transportation canals. The nucleus forms a cluster of embryonic stem cells. In the distal parts of the trophic system, there are cells that function in the absorption, processing, and storage of trophic substances. Cells filled with trophic resources disintegrate (evidently by apoptosis), releasing trophic substances and cell remnants into the canal cavity formed through this process. The parasitic phase of the P. gracilis life cycle is characterized by the loss of the basic morphological features and complete pattern of organization of the arthropods and by chaos and fractalization in the interna.

In this work the results of obtaining HBcAg-producing attenuated Salmonella strains, serovars S. enteritidis and S. typhimurium, and their comparative study is presented. As revealed in this study, attenuated S. enteritidis strain E-23 and S. typhimurium strain T-10, producing HBcAg, induce cell-mediated and humoral immune response to HBcAg after injected into anovals. After injection S. typhimurium strain T-10 induces a much higher titer of specific antibodies than S. enteritidis strain E-23. The level of specific antibodies induced by recombinant HBcAg seems to correlate with the capacity of salmonellae for survival inside macroorganisms.

The infected root nodule cells of Pisum sativum cvs. Torsdag, Rondo and its supernodulating mutant nod3 have been investigated by transmission electron microscopy and morphometrically. Torsdag and nod3 developed effective nodules, when grown with or without nitrates in the growth medium. The nodules developed by Rondo were ineffective in the presence of nitrates, and otherwise effective. An obvious similarity in the fine structure of bacteroid tissue of root nodules has been observed in Torgsdag (Nod5) and the supernodulating mutant nod3, both forms being nitrate-tolerant, but nodulation being controlled by different genetic systems. The statistical processing results showed significant differences in the respective morphometric parameters of nodule cells between the plants grown according to either scheme: with and without nitrates. Combined nitrogen is likely to affect the ratio of symbionts in the infected nodule cells of cultivars with nitrate-tolerant nodulation.

Recombinant strains producing hepatitis B virus (HBcAg) core protein and chimeric core protein exposing on its surface the major immunogenic epitope of HBsAg (HBcAg-HBs) were constructed on the base of attenuated S. typhimurium SL 7202 strain. The resultant Salmonella strains produced proteins which were capable of self-assembly into virus-like particles and showed antigenic properties of both core and surface hepatitis B proteins. A single rectal immunization with recombinant S. typhimurium induced humoral and cellular immune response to HBcAg and HBsAg. Specific anti-HBcAg were detected in animal sera and intestinal tissues, which indicated the formation of specific mucosal immunity.

In this study, we have undertaken an attempt to use the technique of cryopreservation of a mustelid embryo-sing Mustela eversmanni as the experimental model. Ferret blastocysts were frozen with glycerol or DMSO. Characteristic feature of blastocyst morphology, as well as changes in their ultrastructure after freezing and cryopreservation have been examined by techniques of light and electron microscopy. When glycerol was used, damage of ferret blastocysts induced by freezing was more significant than with DMSO as a cryoprotector. In all cases, structural alterations in inner cell mass after cryopreservation were much more significant than in the cells of the trophoblast. We discuss possible use of cryopreservation of embryos for the conservation of Mustelid species.

Reaction of (pdT)16 derivatives, bearing 4-(N-2-chloroethyl-N-methylamino)benzylphosphamide group on its 5' end and biotin on its 3' end with DNA in interphase nuclei and metaphase chromosomes has been investigated by fluorescence and electron microscopy. The result obtained evidence that in interphase nuclei DNA in active chromatin (nucleolus) is the most available for specific modification. In metaphase chromosomes the modified DNA regions are situated on the surface of chromosome.