Biomedical subjects

A P Voronin

Publications and source records attributed to A P Voronin.

14 recordsLinked to original sources

PubMed2006

Telomere and TRF2/MTBP localization in respect to satellite DNA during the cell cycle of mouse cell line L929.

The mouse Mus musculus chromosomes are all acrocentric; each centromere (CEN) is adjacent to a telomere. The aim of the current work is to find out if at least half of the mouse telomeres (Tel) always follow satellite DNA sequences and if membrane telomere binding protein TRF2/MTBP is always in association with the Tel during the cell cycle. FISH, immunoFISH and confocal microscopy were used. During the cell cycle, Tel undergo extensive movement and rearrangement. Most Tel tend to aggregate into large conglomerates in G0/G1. Aggregates colocalize with major satellite (MaSat) and minor satellite (MiSat) to a lesser extent. Tel aggregates are embedded into the MaSat granules at G0/G1. A number of single Tel signals underline the nuclear envelope. In prometaphase, during the metaphase plate formation, half of the Tel, together with CEN, are arranged in a circle and half of the long arms form four clusters. Most of the Tel hybridization signals are colocalized with TRF2/MTBP throughout all stages of the cell cycle, although it is possible to find some telomeres that are not covered with the protein. A prominent shift of TRF2/MTBP signals in respect to the Tel signals is visible in the prophase. The biochemical features of TRF2/MTBP make it possible for the protein to be responsible for Tel clustering.

Animals↗

PubMed2003

Structure-specific DNA-binding proteins as the foundation for three-dimensional chromatin organization.

Any functions of tandem repetitive sequences need proteins that specifically bind to them. Telomere-binding TRF2/MTBP attaches telomeres to the nuclear envelope in interphase due to its rod-domain-like motif. Interphase nuclei organized as a number of sponge-like ruffly round chromosome territories that could be rotated from outside. SAF-A/hnRNP-U and p68-helicase are proteins suitable to do that. Their location in the interchromosome territory space, ATPase domains, and the ability to be bound by satellite DNAs (satDNA) make them part of the wires used to help chromosome territory rotates. In case of active transcription p68-helicase can be involved in the formation of local "gene expression matrices" and due to its satDNA-binding specificity cause the rearrangement of the local chromosome territory. The marks of chromatin rearrangement, which have to be heritable, could be provided by SAF-A/hnRNP-U. During telophase unfolding the proper chromatin arrangement is restored according to these marks. The structural specificity of both proteins to the satDNAs provides a regulative but relatively stable mode of binding. The structural specificity of protein binding could help to find the "magic" centromeric sequence. With future investigations of proteins with the structural specificity of binding during early embryogenesis, when heterochromatin formation goes on, the molecular mechanisms of the "gene gating" hypothesis (Blobel, 1985) will be confirmed.

Animals↗

PubMed2003

Telomere-binding TRF2/MTBP localization during mouse spermatogenesis and cell cycle of the mouse cells L929.

Observations of the organization and distribution of telomeres (Tel) in somatic tissues still remain controversial. The Tel topography revealed by modern microscopy shows them to be associated with the nuclear envelope (NE) in a wide variety of eukaryotic cells, although not at the Rabl orientation (peripheral position at one pole of the nucleus at prophase). We used two cell types that have different nuclear architectures. The cell line L929 shows lack of any rigid Tel architecture in the nucleus. In contrast, spermatozoa have a precise architecture established during spermiogenesis. We observed Tel and membrane Tel binding protein (MTBP/TRF2) position by immunoFISH in L929 cells and by immunofluorescence and immunogold electron microscopy, using antibodies against Membrane Tel Binding Protein (MTBP/TRF2), during different stages of spermiogenesis. At all stages of the L929 cell cycle, MTBP/TRF2 is co-localized with Tel. The only Tel order found in this cell type is similar to the Rabl-orientation, probably due to fast divisions. In the mouse pachytene spermatocytes, the membrane structures abut on the synaptonemal complex (SC) attachment sites contain MTBP/TRF2. In fully formed spermatozoa and during spermiogenesis, apart from the expected MTBP/TRF2 position at the nuclear periphery, MTBP/TRF2 unexpectedly localized at the acrosomal membrane that is adjacent to the nucleus. The difference in the MTBP/TRF2 distribution in the oocyte and spermatozoa leads to the suggestion that the MTBP/TRF2 location might reflect preparation for fertilization events. The Tel distribution is not static in cultured cells throughout the cell cycle or during spermatogenesis. When the Tel are attached to the NE, as during SC formation, MTBP/TRF2 is the member of the protein complex, which appears to be responsible for this attachment.

Animals↗

PubMed2000

Nuclear envelope associated protein that binds telomeric DNAs.

Rana temporaria oocytes at the 6th diplotene stage of maturation contain a special structure, the karyosphere capsule, with chromosomes covered and detached from the nuclear envelope (NE), though at the previous stage the telomeres were attached to the membrane, as characteristic of germ cells. The DNA-protein complexes from band shift assays with proteins extracted from oocyte NEs and telomeric DNA fragment (T(2)G(4))(130) were isolated and injected into a guinea pig. In the present paper the only protein of 70 kDa recognized by antibody (AB) in the NE is named the Membrane Telomere Binding Protein (MTBP). Western blots with guinea pig AB and AB against telobox peptide from TRF2 show that protein of 60 kDa (probably TRF1) belongs to the chromatin, but MTBP (TRF2 according to immunoprecipitation) belongs to the NE. In the somatic cell nuclei both proteins are present and recognized by AB against telobox peptide, but AB raised recognize only MTBP/TRF2 due to the epitope different from telobox. Combined in situ hybridization with the vertebrate telomeric DNA sequences (T(2)AG(3))(135) and immunocytochemistry with the MTBP AB showed them to be colocalized within the mouse nucleus. As it was shown by immunofluorescense of NE spread, MTBP is organized in a distinct pattern that looks like a network made of double-dots. Electron microscope immunogold staining with both ABs showed that the protein is localized on the outer surface of the oocyte NE within cup-like structures attached to the membrane. This is the first clear evidence of a protein, which could be responsible for the attachment of telomeres to the nuclear membrane.

Animals↗

PubMed1998

Effects of exogenous stress protein 70 on the functional properties of human promonocytes through binding to cell surface and internalization.

The presence of antibodies against the major stress protein, Hsp70, in patients with autoimmune diseases led us to hypothesize that Hsp70 may occur extracellularly, and could exert chaperoning and regulatory effects on various cells. We examined the action of pure Hsp/Hsc70 on the main physiological functions of human promonocytic U-937 cells. The protein was isolated from calf muscle and was shown to be a mixture of inducible Hsp70 (60%) and constitutive Hsc70 (40%) isoforms. It was observed that the addition of the protein up-regulated two major monocyte/macrophage differentiation markers, CD11c and CD23, by 20-35%, while it had no effect on CD14. The experiments performed to investigate the influence of Hsp/Hsc70 on the reaction of U-937 cells to differentiation stimuli demonstrated that the addition of the protein prior to PMA was able to inhibit binding of proper transcription factors to double-symmetry and cAMP-response elements of the c-fos early response gene promoter. Administration of exogenous Hsp/Hsc70 prior to treatment with the tumor necrosis factor-alpha significantly lowered the number of apoptotic and necrotic cells. In no case did the control protein, ovalbumin, taken in the same concentration give a comparable effect on U-937 cells. Since the Hsp/Hsc70 effects occurred within the first hour of co-incubation, and therefore they might be explained by its interaction with the cell surface, we assayed binding of the biotinylated protein to U-937 cells by immunoenzyme assay, flow cytometry and indirect immunofluorescence. Using these three techniques we were able to detect Hsp/Hsc70 bound to cells after a 20 min incubation. According to flow cytometry data, at this time 32% of cells were positively stained with streptavidin-FITC. Immunofluorescence studies demonstrated Hsp/Hsc70 bound to the cell surface after a 20 min incubation followed by induction of patch and cap-like structures. One hour later, the majority of the protein had been internalized by U-937 cells.

Animals↗

PubMed1996

Exogenous heat shock protein hsp70 activates potassium channels in U937 cells.

With the use of patch clamp technique, the effect of exogenous heat shock protein hsp70 on ion channel properties in the plasma membrane of human promonocyte U937 cells has been examined. Cell-attached experiments showed that the addition of 30-100 micrograms/ml hsp70 to the pipette solution resulted in an activation of outward currents through potassium-selective channels of 9 pS unitary conductance. The activity of K(+)-selective channels did not depend on membrane voltage and could be controlled by the intracellular free calcium concentration as revealed in inside-out recordings. K+ channels with similar conductance and kinetic behaviour were found in normal cell-attached patches very rarely. Outside-out experiments showed that the addition of hsp70 to the external solution induced a channel-like stepwise increase of inward current which may provide cation entry from the extracellular medium. The interaction of extracellular hsp70 with the membrane surface of the native cell and of the excised fragment was found to be different. The results suggest that hsp70-induced activation of Ca-dependent K channels in monocyte-macrophage cells may be due to a local increase of free Ca2+ concentration just near the inner membrane side.

Calcium↗

PubMed1986