[Nuclear delivery of oligonucleotides via nanocomposites based on TiO2 nanoparticles and polylysine].

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 TiO2 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.

[TiO2-DNA nanocomposites capable of penetrating into cells].

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.

[Characteristics of the cytomixis channel formation in Nicotiana tabacum L. pollen mother cells].

The cytomictic channels in pollen mother cells of tobacco can be formed by two distinct ways: on the basis of plasmodesmata and de novo without any relation to ones. Cytomictic channels formation it was shown to be possible on the basis of single plasmodesma. It is not unlikely that special electron-dense bodies involve de novo formation of the channels. Also the role of cytomictic channels in the pollen development regulation is discussed.

[Investigation of the impact of cowpox virus BTB/kelch gene deletion on some characteristics of infection in vitro].

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.

An examination of the ability of titanium dioxide nanoparticles and its conjugates with oligonucleotides to penetrate into eucariotis cells.

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].

[Comparative study of the effectiveness of HBcAg presentation to the immune system using attenuated Salmonella strains, serovars S.enteritidis and S.typhimurium]. / Sravnitel'noe issledovanie éffektivnosti predstavleniia immunoi sisteme HBcAg s pomoshch'iu attenuirovannykh shtammov Salmonella serovarov S.enteritidis i S.typhimurium.

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.

[Isolation and study of recombinant strains of Salmonella typhimurium SL 7207, producing HBcAG and HBcAG-HBs]. / Poluchenie i issledovanie rekombinantnykh shtammov Salmonella typhimurium SL 7207, produtsiruiushchikh HBcAg i HBcAg-HBs.

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.

[Ultrastructure of bacteroid-containing tissue of Pisum sativum L. peas having various regulatory mechanisms of nodulation]. / Ul'struktura bakteroidsoderzhashchei tkani linii gorokha Pisum sativum L., imeiushchikh raznye reguliatornye mekhanizmy kluben'koobrazovaniia.

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.

[Effect of cryopreservation on the morphology of blastocysts of the ferret Mustela eversmanni]. / Vliianie kriokonservatsii na morfologiiu blastotsist svetlogo khor'ka Mustela eversmanni.

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.

Scheduled perturbation in DNA during in vitro differentiation of mouse embryo-derived cells.

Studies of sister chromatid exchanges (SCE) and recombination rate of certain minisatellite DNAs have demonstrated that their levels are considerably higher during the preimplantation stage than in latest developmental stages of embryos. It appeared likely that single-strand DNA breaks (SSB) may be relevant to both events during early development. With this in mind, we estimated SSB during in vitro retinoic acid (RA)-induced and spontaneous differentiation of mouse teratocarcinoma (EC) and embryonic stem (ES) cells. Using the method of nucleoid sedimentation and single-cell DNA electrophoresis, we have observed a dramatic increase in the SSB during the first 2-4 mitoses after beginning of differentiation of EC cells, followed by a gradual return to the basal level characteristic of undifferentiated cells. The increase in the SSB was manifested as the appearance of mass nucleoids with slow sedimentation rates, as well as the low-weight mass fragments in DNA patterns of most cells. We concluded that not less than half of genomic DNA has been nicked at the early steps of differentiation. The decrease in SSB level was observed in spite of continuing differentiation, as judged by embryonic antigens and morphological criteria. Also, the increase in the SCE level coincided with that of SSB, possibly being its consequence. The scheduled "surge" of SSB may be the earliest event in commencing differentiation at steps without a phenotypic manifestation.

[Visualization of DNA segments, interacting with reactive oligonucleotide derivatives in interphase nuclei and metaphase chromosomes]. / Vizualizatsiia uchastkov DNK, vzaimodeistvuiushchikh s reaktsionnosposobnymi oligonukleotidov, v sostave interfaznykh iader i metafaznykh khromosom.

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.

Oral manifestations of alpha-mannosidosis: report of a case with ultrastructural findings.

Alpha-mannosidosis is a rare storage disease with distinct biochemical, clinical, histological and ultrastructural features. The oro-facial findings in a 26-year-old man are described. Gingival and oral mucosal hyperplasias were studied using histology and transmission electron microscopy (TEM). The TEM findings were comparable to those of other tissues examined in previous reports, consisting of histiocytic cells containing storage vacuoles with fine reticulo-granular material.

A molecular and cytogenetic analysis of lambda 20p7 fragment DNA from the proximal beta-heterochromatin of Drosophila melanogaster.

A DNA fragment from the Drosophila melanogaster genome, cloned in lambda 20p7, was derived independently from clones lambda 20 and lambda L [Baiborodin et al., Genetika 29 (1993) 403-416; Sharakhov et al., Genetika 29 (1993) 392-402]. In situ hybridization of lambda 20p7 DNA to the chromosomes of D. melanogaster demonstrated preferential hybridization of the fragment to the chromocenter of polytene chromosomes and to pericentric heterochromatin of chromosomes II, IV and X at the metaphase plate. Copy number per haploid genome for lambda 20p7 was estimated as approximately 200. Based on Southern blotting, the major portion of this moderate repeat was localized in the region of a 5.5-kb HindIII digest. In situ hybridization to polytene chromosomes from strain fs(2)B trophocytes revealed that repeats homologous to lambda 20p7 are located in the proximal heterochromatin which undergoes structural reorganization during tissue differentiation. The nucleotide sequence of two segments of the clone lambda 20p7, Dm0.9 and Dm270, was determined. Sequence analysis of the 300-bp Dm0.9 clone revealed that it contains 21-bp and 30-bp d(GT/CA) sequences, a 12-bp AT box, recognition sites for nuclear factors NFI and SpI, and a set of inverted repeats. Clone Dm270 contains an open reading frame (ORF). The deduced amino acid (aa) sequence shares homology with the gag-like gene from type-I (R1) ribosomal DNA insertion and may code for a polypeptide of 10 kDa. The Dm270 sequence was found to contain two direct repeats showing homology to the human CENP-B box.

[Drosophila beta-heterochromatin: molecular organization and function. Cloning and molecular biological analysis of the lambda 20 DNA fragment from Drosophila melanogaster beta-heterochromatin]. / beta-Geterokhromatin Drosophila: molekuliarnaia organizatsiia i funktsionirovanie. Klonirovanie i molekuliarno-biologicheskii analiz lamda 20 fragmenta DNK iz beta-geterokhromatina Drosophila melanogaster.

To isolate the DNA sequences specific for the pericentric heterochromatin of Drosophila we used two CREST-autoimmune sera which bind in the Western-blot analysis the nuclear antigens of 30 kDa, 43 kDa and 45 kDa molecular weight. Cloning of the DNA fragments associated with these CREST-specific proteins of Drosophila resulted in obtaining 8 clones. One of them, lambda 20, hybridized mainly to the chromomcenter of polytene chromosomes. The further analysis indicated that the lambda 20 DNA might belong to the proximal beta-heterochromatin of the polytene chromosomes of D. melanogaster.

[The detection of the location of glucose-6-phosphate dehydrogenase in the fibroblasts of voles and mice and in rat myoblasts by using monoclonal antibodies against glucose-6-phosphate dehydrogenase]. / Vyiavlenie lokalizatsii gliukozo-6-fosfatdegidrogenazy v fibroblastakh polevok i myshi i v mioblastakh krysy s pomoshch'iu monoklonal'nykh antitel protiv gliukozo-6-fosfatdegidrogenazy.

Monoclonal antibodies (MAs) were produced against glucose-6-phosphate dehydrogenase (G6PD) of two vole species--Microtus arvalis and M. subarvalis. The binding level of the MAs to G6PD in both species were almost the same, which suggested that these MAs may be specific for the antigenic determinants common to G6PD of these species. The MAs produced against the vole G6PD were used for its intracellular localization. The patterns obtained after staining cells with the use of MAs against G6PD were the same as those obtained after staining with the use of antibodies against F-actin. There was a good conformity between the results of light and electron microscopic immunoenzyme analyses with regard to the binding of MAs produced to the actin microfilaments. It is concluded that G6PD is closely associated with actin microfilaments of the cell cytoskeleton.

[The behavior of the X chromosomes in intra- and interspecific hybrid cells of murine teratocarcinoma PCC4azal]. / Povedenie X khromosom vo vnutri- i mezhvidovykh gibridakh kletok teratokartsinomy myshi PCC4azal.

We have demonstrated that X chromosomes are reactivated in hybrids obtained by fusion of mouse PCC4azaI teratocarcinoma cells (XO, 39HPRT-) with splenocytes from mouse females heterozygous in Hprt gene. These hybrids are capable of spontaneous differentiation. We also obtained similar interspecies hybrids of PCC4azaI cells with bone marrow cells of the American mink. The majority of such hybrids remained undifferentiated, however, after long-term cultivation at high cell density they differentiated into epithelial- or fibroblast-like cells similarly to PCC4azaI cells. Two hybrids had the autosomal complement of the mouse and two X chromosomes (mouse and mink); both X chromosomes were active. These X chromosomes were not inactivated during differentiation in vitro.

A tissue-specific puff (Balbiani ring a) in Chironomus thummi may contain a gene encoding a 67-kDa protein which exhibits non-tissue-specific expression.

A 2.3-kb genomic clone has been isolated from the region where the tissue-specific puff, Balbiani ring a (BRa), is found on chromosome IV of the special lobe of Chironomus thummi salivary gland cells. The clone was characterized by nucleotide sequence analysis. Two clusters of direct tandem repeats were identified, as well as large and small open reading frames (ORFs). The large ORF was fused to an Escherichia coli lacZ gene. Antibodies against the beta-galactosidase/ORF fusion protein reacted selectively on Western blots with a 67-kDa protein. Western-blot analysis and immunoelectron microscopy showed that this protein was distributed in the cells of all larval tissues examined. We concluded that BRa, a tissue-specific puff, whose activity correlates with the synthesis of 160-kDa secretory protein [Kolesnikov et al., Chromosoma 83 (1981) 661-677], may also contain a gene which is not expressed in a tissue-specific manner.