Filatov Mv

Exosomes are natural carriers of exogenous siRNA to human cells in vitro

BackgroundExosomes are nano-sized vesicles of endocytic origin that are involved in cell-to-cell communication including shuttle RNA, mainly mRNA and microRNA. As exosomes naturally carry RNA between cells, these particles might be useful in gene cancer therapy to deliver therapeutic short interfering RNA (siRNA) to the target cells. Despite the promise of RNA interference (RNAi) for use in therapy, several technical obstacles must be overcome. Exogenous siRNA is prone to degradation, has a limited ability to cross cell membranes and may induce an immune response. Naturally occurring RNA carriers, such as exosomes, might provide an untapped source of effective delivery strategies.ResultsThis study demonstrates that exosomes can deliver siRNA to recipient cells in vitro. The different strategies were used to introduce siRNAs into human exosomes of various origins. The delivery of fluorescently labeled siRNA via exosomes to cells was confirmed using confocal microscopy and flow cytometry. Two different siRNAs against RAD51 and RAD52 were used to transfect into the exosomes for therapeutic delivery into target cells. The exosome-delivered siRNAs were effective at causing post-transcriptional gene silencing in recipient cells. Moreover, the exosome-delivered siRNA against RAD51 was functional and caused the massive reproductive cell death of recipient cancer cells.ConclusionsThe results strongly suggest that exosomes effectively delivered the siRNA into the target cells. The therapeutic potential of exosome-mediated siRNA delivery was demonstrated in vitro by the strong knockdown of RAD51, a prospective therapeutic target for cancer cells. The results give an additional evidence of the ability to use human exosomes as vectors in cancer therapy, including RNAi-based gene therapy.

Fractal nature of chromatin organization in interphase chicken erythrocyte nuclei: DNA structure exhibits biphasic fractal properties

Arrangement of chromatin in intact chicken erythrocyte nuclei was investigated by small angle neutron scattering. The scattering spectra have revealed that on the scales between 15 nm and 1.5 μm the interior of the nucleus exhibited properties of a mass fractal. The fractal dimension of the protein component of cell nucleus held constant at approximately 2.5, while the DNA organization was biphasic, with the fractal dimension slightly higher than 2 on the scales smaller than 300 nm and approaching 3 on the larger scales.

Overexpression of bacterial RecA protein stimulates homologous recombination in somatic mammalian cells.

The pairing of homologous molecules and strand exchange is a key event in homologous recombination promoted by RecA protein in Escherichia coli. Structural homologs of RecA are widely distributed in eukaryotes including mouse and man. As has been shown, human HsRad51 protein is not only structural but also functional homolog of RecA. The question arises whether the bacterial functional homolog of Rad51 can function in mammalian cells and increase the frequency of the homologous recombination. To investigate possible effects of bacterial RecA protein on the frequency of homologous recombination in mammalian cells, the E. coli RecA protein fused with a nuclear location signal from the large T antigen of simian virus 40 was overexpressed in the mouse F9 teratocarcinoma cells. We found that the frequency of gene targeting at the hprt locus was 10-fold increased in the mouse cells expressing the nucleus-targeted RecA protein. Southern blot analysis of individual clones that were generated by targeting recombination revealed predicted type of alterations in hprt gene. The data indicate that the bacterial nucleus-targeted RecA protein can stimulate homologous recombination in mammalian cells.

Amylolytic activity of IgG and sIgA immunoglobulins from human milk.

BACKGROUND New natural amylolytic abzymes (Abs) for catalytically active antibodies from human milk have been identified and investigated. METHODS The amylolytic activity of human milk autoantibodies was studied by TLC and HPLC techniques analyzing the hydrolysis of maltooligosaccharides with different degrees of polymerization and of 4-nitrophenyl 4,6-O-ethylidene-alpha-D-maltoheptaoside (EPS). IgG and sIgA fractions were isolated from human milk by affinity chromatography. After SDS-PAGE preparation of native IgG and sIgA and their renaturation, the amylolytic activity was in-gel assayed. RESULTS All electrophoretically homogeneous preparations of IgG and its Fab fragments as well as sIgA antibodies possessed alpha-amylolytic activity. The specific activities of these catalytic antibodies varied in the range from 1.83 up to 3.33 kat/kg, which is about one order of magnitude higher than that for IgGs from the sera of cancer patients. IgG and sIgA fractions showed Michaelis constants for hydrolysis of 4-nitrophenyl 4,6-O-ethylidene-alpha-D-maltoheptaoside in the range of 10(-4) M/l. Fractions of autoantibodies from different donors exhibited different modes of action in hydrolysis of maltooligosaccharides, maltose and p-nitrophenyl-alpha-D-glucopyranose. CONCLUSIONS IgG antibodies, their Fab fragments, and sIgA fractions isolated from human milk of healthy women possessed amylolytic activity in the hydrolysis of maltooligosaccharides and several artificial substrates.

Structural hierarchy of chromatin in chicken erythrocyte nuclei based on small-angle neutron scattering: Fractal nature of the large-scale chromatin organization

The chromatin organization in chicken erythrocyte nuclei was studied by small-angle neutron scattering in the scattering-vector range from 1.5 × 10−1 to 10−4 Å−1 with the use of the contrast-variation technique. This scattering-vector range corresponds to linear dimensions from 4 nm to 6 μm and covers the whole hierarchy of chromatin structures, from the nucleosomal structure to the entire nucleus. The results of the present study allowed the following conclusions to be drawn: (1) both the chromatin-protein structure and the structure of the nucleic acid component in chicken erythrocyte nuclei have mass-fractal properties, (2) the structure of the protein component of chromatin exhibits a fractal behavior on scales extending over two orders of magnitude, from the nucleosomal size to the size of an entire nucleus, and (3) the structure of the nucleic acid component of chromatin in chicken erythrocyte nuclei is likewise of a fractal nature and has two levels of organization or two phases with the crossover point at about 300–400 nm.

Active dissociation of Hoechst 33342 from DNA in living mammalian cells.

The fluorescent dye Hoechst 33342, which binds tightly to DNA in the minor groove, can be effectively extracted from the DNA in vivo due to an energy-dependent process. It is the dissociation of the dye from DNA rather than its removal from a cell that has a critical role in this process. The dissociation can be suppressed by the topoisomerase-2 inhibitors novobiocin, ellipticine and etoposide. Breaks in the DNA also inhibit the process. The dissociation of the dye requires that DNA remain intact along a region of at least several thousand base pairs. It is proposed that DNA in mammalian cells is a dynamic, conformationally nonstable system and that topologically closed loops consisting of several thousand base pairs constantly appear and disappear in chromatin.

Camptothecin enhances random integration of transfected DNA into the genome of mammalian cells

In order to study the involvement of DNA topoisomerase I (top1) in recombination, we examined the effect of the anti-neoplastic drug camptothecin, which selectively poisons top1 by trapping top1-cleavable complexes on integration of exogenic vector into the genome of mammalian cells. We transfected mouse F9 teratocarcinoma cells as well as Chinese hamster V79 cells with a plasmid carrying a selectable neo gene treated with camptothecin, and determined the frequency of neo+ (G418(R)) colonies. We found that treatment with camptothecin for as short a time as 4 h after electroporation resulted in a 4- to 33-fold stimulation of plasmid integration into the recipient genome via non-homologous recombination. These results imply that top1-cleavable complexes trapped by camptothecin could be potentially recombinogenic structures and could stimulate non-homologous recombination in vivo, promoting the integration of transfected plasmids into mammalian genome.

Ceruloplasmin decreases respiratory burst reaction during pregnancy

Abstract Testing of pregnant women reveals weakening of neutrophil-mediated effector functions, such as reactive oxygen species generation. This study provides data confirming the phenomenon, gained through application of the flow cytometry technique. Key factors influencing neutrophil functional activity in blood plasma of pregnant women have not been detected so far. At the same time, concentration of ceruloplasmin – a copper-containing glycoprotein – is known to increase in blood significantly during pregnancy. We observed the negative correlation between ceruloplasmin concentration in blood plasma of pregnant women and the intensity of respiratory burst of neutrophils. Fractionation of plasma using gel-filtration revealed that ceruloplasmin-containing fraction demonstrated suppression of the respiratory burst reaction. Partial elimination of ceruloplasmin from the blood of pregnant women, performed with the help of specific antibodies and followed by immunoprecipitation, leads to an increased respiratory burst reaction. On the contrary, addition of ceruloplasmin to blood samples of healthy donors noticeably decreases the respiratory burst reaction. The results presented prove that change in ceruloplasmin level in plasma is necessary and sufficient for modulating the ability of neutrophils to produce reactive oxygen species during pregnancy.

Methods for registration of spontaneous DNA instability in mammalian cells

A phenomenon of spontaneous DNA instability displays itself as the low level of repair DNA synthesis that takes place during any cell cycle phases. However, there is a problem in detection of very low intensive repair DNA synthesis. This paper suggests two approaches to detect the spontaneous DNA instability. The first method involves a blockade of the DNA gaps sealing by a combination of inhibitors, hydroxyurea and arabinofuranosyl cytosine. An accumulation of single strand gaps leads to production of DNA double strand breaks and results to reproductive inactivation of cells. It was shown that registration of both these events by different methods (such as viscoelastometry of DNA, orthogonal pulse electrophoresis or comet assay for double strand breaks as well as effectiveness of colony growth for cell inactivation) may be used as suitable measure of the spontaneous DNA instability. The second approach bases on photolysis of bromodeoxyuridine incorporated into repair DNA patches during the spontaneous repair DNA synthesis. Long wave UV irradiation of cells containing bromodeoxyuridine labeled DNA stained with Hoechst 33342 causes their inactivation. Experimental results presented confirm that both methods actually detect the spontaneous DNA instability. It takes note of the spontaneous DNA instability varies for cells from different tissues and species and increases during aging.

Purification and in vitro analysis of exosomes secreted by malignantly transformed human cells

Exosomes are natural nanoparticles secreted by different cells and capable of carrying protein markers and genetic information, thus participating in cellular communication. There is good reason to think that quantitative and qualitative characterization of these microparticles produced by different tissues in normal and pathological states can give valuable diagnostic and prognostic information and be a biomarker of different diseases, including oncological ones. Elaboration of the purification of exosomes and their proteome analysis was the aim of the present work. An original approach to enhancing exosome production in cultured transformed human cells was developed. The data obtained allowed us to detect exosomes in cultural conditioned samples and control the quality of produced exosomes at all stages of their purification. Electrophoretic analysis of proteins obtained from exosomes of different origins shows differences in protein profiles. Proteins from exosomes of glioblastoma cell lines were separated by two-dimensional electrophoresis. Protein profiles were further analyzed by densitometry and mass spectrometry, which allowed more than 30 proteins, including specific tumor markers, to be identified.