Nanohybride Materials Based on Magnetite-Gold Nanoparticles for Diagnostics of Prostate Cancer: Synthesis and In Vitro Testing.

We synthesized a fluorescence conjugate and modified magnetite-gold nanoparticles carrying prostate specific membrane antigen (PSMA) as the ligand. Analysis of their binding to human prostate cancer cell lines PC-3 (PSMA-) and LNCaP (PSMA+) showed selective interaction of the synthesized conjugate and modified nanoparticles with LNCaP cells. These findings suggest that these nanoparticles can be used in tissue-specific magnetic-resonance imaging.

[Cohesion inside eu- and heterochromatin in human cells].

It is considered that sister chromatids are held together immediately after replication by special protein complex--cohesin that consists of Smc1--Smc3 core dimer and two additional subunits, Scc1 and Scc3. This process is called cohesion. We have characterized binding of cohesin complex to early- and late-replicated chromatin at different stages of the cell cycle in human cells HeLa and HT1080 using superresolution microscopy (based on Structural ilumination microscopy--SIM) and immunoelectron microscopy. It has been shown that cohesins do not play important role in cohesion of heterochromatic domains, but they provide cohesion and organization of subdomains in euchromatic regions.

[Resolution of spatial constraints during replication of peripheral chromatin].

Tight association of peripheral chromatin with nuclear lamina unavoidably creates topological constraints during replication. Additional complications are associated with high stability of lamina meshwork, which may hinder an access of replication factors to the sites of DNA synthesis in highly condensed template with limited mobility. In the current work we studied structural organization and dynamics of lamina as a function of replicative status of associated peripheral heterochromatin. The studies of molecular mobility of laminas at various stages of S-phase in vivo and using super-resolution microscopy showed no correlation between lamina dynamics and replicative status of attached heterochromatin. These data support the hypothesis that lamina-chromatin interactions during S-phase are regulated at the level of adapter proteins. Ultrastructural studies have demonstrated that temporal break of lamina-chromatin connections during replication does not cause noticeable spatial separation of replicating domains from nuclear periphery.

[High level of expression and nuclear localization of RNA, transcribed from genome region, containing repetitive elements].

The non-coding and repetitive sequences constitute a great amount of higher eukaryotes genomes, but the elucidation of its role and mechanisms of action is now at the very beginning. Here we found, that internal telomeric repeats in Danio rerio are colocalized with some repetitive elements, namely, hAT and EnSpm repeats, which are highly represented in vertebrate genome. While investigating one of genome regions, containing two pairs of such repeats in close proximity we found, that it is transcribed. RNA-dependent structures, containing this sequence, were revealed in D. rerio fibroblast nuclei, which may serve as evidence of functional relevance of repetitive elements in genomes or of their transcripts.

Proliferative potential of multipotent mesenchymal stromal cells from human bone marrow.

We studied the capacity of multipotent mesenchymal stromal cells isolated from human bone marrow (BM) to long-term passaging, cloning, and re-cloning. Initial multipotent mesenchymal stromal cells and cells after gene labeling were studied. Multipotent mesenchymal stromal cells were obtained from donors (13-59 years) and cultured for 7 passages. Third generation lentivector was used for delivery of green fluorescent protein marker gene. The procedure of infection revealed reduced proliferative potential of multipotent mesenchymal stromal cells from elder donors. Hierarchy of precursor cells differing by their proliferative potential was demonstrated in the culture of multipotent mesenchymal stromal cells. Three categories of multipotent mesenchymal stromal cells were identified: mature cells incapable of proliferation (75.7±2.4% population) and cells with low and high proliferative potential (17.6±2.1 and 6.7±0.3%, respectively). The relative content of these cells insignificantly differed from passage to passage. The efficiency of cloning also remains stable, but re-cloning capacity sharply decreased after passage 3 and completely disappeared in multipotent mesenchymal stromal cells after cryopreservation. Thus, cultured multipotent mesenchymal stromal cells represent a heterogeneous and hierarchically organized population and the characteristics of this population depend of the duration of culturing and age of BM donor. This should be taken into account when using multipotent mesenchymal stromal cells in clinical practice.

Characteristics of mesenchymal stromal precursor cells labeled with lentiviral vector in long-term bone marrow culture.

Mouse mesenchymal stromal precursor cells were labeled with lentiviral vector in long-term bone marrow culture. We studied the fate of labeled cells in the stromal sublayer of the long-term bone marrow culture and in ectopic hemopoiesis foci formed from the labeled cultures. The incidence of labeled polypotent fibroblast CFU in sublayers of long-term bone marrow culture and in ectopic hemopoiesis foci formed from these sublayers under the renal capsule of syngeneic mice was also analyzed. It was shown that the marker gene was present in about 40% cells of the stromal sublayer and 30% fibroblast CFU and that effective gene transfer did not affect the total production of hemopoietic cells. The size of ectopic hemopoietic foci formed after implantation of labeled sublayers of the long-term bone marrow culture under the renal capsule did not differ from the control. Differentiated cells of the osseous shell in these foci carried the marker gene in 40% cases. Analysis of fibroblast CFU in these foci showed that despite the total concentration of fibroblast CFU was comparable to that in the bone marrow, the concentration of labeled fibroblast CFU was about 6%, which suggests that one more class of precursors probably exists in the hierarchy of stromal cells presumably between mesenchymal stem cells and fibroblast CFU. Our findings demonstrate the capacities of mesenchymal stem cells to self-maintenance and differentiation without losing the marker gene integrated into the genome.