[Obtaining of the affinity purified antibodies against survivin for the structure functional study of the protein].

Tumor-associated protein survivin is the bifunctional protein which can participate either in cell division regulation or in apoptosis inhibition depending on its localization and structure state. The aim of this work was to obtain monospecific antibodies useful for investigation of protein structure and functional features. Six affinity purified antibodies directed to different protein regions were obtained. The ability of antibodies obtained to detect survivin in tumor cells and breast cancer tissues was studied. It was shown that antibodies to (1-22) and (95-105) survivin fragments have the highest specific activity. In western-blot antibodies to (1-22) region predominantly binds with survivin-containing complex, which may be the survivin dimer as we suppose, while antibodies to (95-105) region detects only monomeric form of the protein. Breast cancer tissues study demonstrated that survivin monomer presents only in the tumor core tissues, while survivin-containing complex is expressed both in tumor core and tumor periphery tissues. It was shown that antibodies to (1-22) fragment detect predominantly nuclear survivin, which participates in mitosis regulation, while antibodies to (95-105) fragment gave nucleoplasm and cytoplasm staining at all stages of cell cycle. Thereby antibodies obtained are the useful tool for structure-functional study of survivin.

[Overexpression of the nucleolar protein SURF-6 in mouse fibroblasts NIH/3T3 leads to stabilisation of intragenic transcribed spacers of the pre-rRNA].

SURF-6 is an evolutionary conserved nucleolar protein that is required for maintenance of cell viability, but its functional significance in mammals still remains illusive. In the present work we examined effects of SURF-6 overexpression in mouse NIH/3T3 fibroblasts transfected with two plasmids. The plasmid pUHrT62-1 encodes a tetracycline-dependant trans-activator, the protein rtTA, the plasmid pBI-SURF6--the genes of EGFP (enhanced green fluorescent protein) and of mouse SURF-6 which expression was controlled by the rtTA-responsive bi-directorial promoter. Western blot analysis showed that the SURF-6 level was severely augmented in cells transfected with pUHrT62-1 and pBI-SURF6 and incubated with the inducer--doxycycline opposed to the transfected but not-induced cells. The increase of SURF-6 was observed in 24 and 48 h after adding the inducer doxycycline. Dot-hybridization of isolated RNA with biotinilated oligonucleotide probes to various regions of mouse primarily pre-rRNA transcripts showed that overexpression of SURF-6 enhanced levels of the second intragenic transcribed spacer ITS2 in about seven folds and of the 5' external transcribed spacer 5'ETS in two folds. Amounts of fragments corresponding to 18S, 5.8S and 28S rRNA remained almost unchanged. These observations for the first time demonstrated that mammalian SURF-6 helps to stabilize or prevents premature cleavage of the pre-rRNA intragenic transcribed spacers, particularly of ITS2, similar to its homologue in S. cerevisiae the protein Rrp14. Today metazoan proteins that play a similar role in ribosome biogenesis, are not described.

[Dynamics and mechanisms of the nucleolus reorganization during mitosis].

The nucleolus is the largest and most dynamic nuclear domain in the vast majority of eukaryotic cells. The main and universal nucleolar function is participation in ribosome biogenesis, including ribosomal DNA (rDNA) transcription, pre-rRNA processing and ribosome subunit assembly. Furthermore, the nucleolus and its proteins also participate in cell cycle regulation, apoptosis and cell aging. These nucleolar functions are realized predominantly in interphase and, apparently, are abolished during mitosis, when the nucleolus disassembles. In this review, literature and our own data on the dynamics and mechanisms of the nucleolus disassembly and reassembly during mitosis in animal and plant cells are summarized. Particular attention is paid to the results obtained by analysis of the nucleolar dynamics in living cells and to modeling of the premature assembly of nucleolus upon various experimental conditions.

[Assembly of nucleolus-derived foci in various cultured mammalian cells during mitosis].

During mitosis, rebuilding of the nucleolus is a step-wise process that, above all, includes an assembly of nucleolus-derived foci (NDF) in the cytoplasm of telophase cells. In this study, we performed a comparative analysis of NDF formation in mitotic cells of various mammalian cell cultures, such as green monkey CV1 cells, human HeLa cells, mouse 3T3 cells, and pig PK cells, both in control and following inhibition of rRNA synthesis by actinomycin D or by an adenosine analogue, DRB. The results obtained show that in all examined cell types NDF are formed shortly after or simultaneously with the onset of chromosome segregation to the poles of the mitotic spindle. However, an efficiency of NDF assembly, i.e. the number of NDF per anaphase or telophase cell, and the portion of anaphase and telophase cells containing NDF vary in different cell cultures, being most prominent in CVI and HeLa cells. In these cells, the vast majority of NDF accumulate several proteins of the mature nucleolus, such as B23/nucleophopmin, C23/nucleotin, fibrillarin, and, to a lesser extent, Nop52. The rRNA harbored by NDF is synthesized several hours prior mitosis, and plays an essential role the maintenance of NDF structural integrity. Starting from early stages of the assembly onwards, the NDF are predominantly located in the area occupied by aster microtubules of the mitotic spindle.

[Premature assembly of nucleolus-derived foci induced by a reversible hypotonic shock in metaphase CV1 and HeLa cells].

The assembly of nucleolus-derived foci (NDF) in the cytoplasm of telophase cells is an early stage of nucleolus reassembly during mitosis. In current literature, significant attention is paid to the molecular composition of NDF and their participation in reassembly of the mature nucleolus. However, very little is known about mechanisms controlling the NDF formation. The authors have demonstrated for the first time that a reversible action of low ionic strength buffers (lypotonic shock treatment) on living mitotic human HeLa and green monkey CV1 cells triggers a premature assembly of NDF at metaphase. Like the true NDF, i. e., those assembled in telophase mitosis, NDF prematurally induced at metaphase contain RNA and proteins required for rRNA processing (fibrillarin, B23/nucliophosmin, C23/nucleolin), but lack UBF, an auxiliary factor of RNA polymerase I. We have assumed that a reversible action of hypotonic shock on metaphase cells may result in temporal increase in intracellular [Ca2+](i) that, in its turn, may induce a premature assembly of NDF under isotonic conditions. The structural integrity of the mitotic spindle apparently plays an essential role in the response of metaphase cells to hypotonic shock treatments.

[Activation of transcription of ribosome genes following human embryo fibroblast infection with cytomegalovirus in vitro]. / Aktivatsiia transkriptsii ribosomnykh genov pri tsitomegalovirusnoi infektsii fibroblastov émbriona cheloveka in vitro.

Cytomegalovirus (CMV) infection of human diploid embryo fibroblasts in vitro causes a massive cell death on the 3-4th day of infection with a high primary infection coefficient (1-5 U/cell). Cytopathological effects of viral infection on the 3-4th day includes diminishes of the cell size, changes in their form, compaction of the nuclear chromatin, and disorganization and inactivation of the nucleolus. However, the early stages of the viral infection progression (the 1st-2nd day) are accompanied by unequivocal activation of rDNA (ribosomal gene) and the bulk of chromatin transcription. There are several features to support this conclusion: In the early CMV-infected cell 1) the nucleolar size is increased; 2) the number of intranucleolar foci binding the specific RNA-polymerase I transcription factor (UBF) is augmented; 3) the Ag-NOR staining is enhanced; 4) 3H-uridine incorporation to the nucleoli is activated; 5) the ultrastructure of the nucleolus is changed. Altogether, these data argue in favor of activation of rDNA transcription in human fibroblasts in vitro at the initial stages of infection.