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E S Nadezhdina

Publications and source records attributed to E S Nadezhdina.

At least 19 recordsLinked to original sources
PubMed2003

Enzymatic activity of protein kinase LOSK: possible regulatory role of the structural domain.

LOSK (LOng Ste20-like Kinase) protein kinases of mammals belong to a recently identified family of GCK kinases which are involved in the induction of apoptosis. LOSK have an N-terminal acidic catalytic domain and a long C-terminal basic structural domain which is cleaved off in cells by caspases during apoptosis. To study the LOSK enzymatic activity and its dependence on the structural domain, two preparations of this protein kinase were prepared: a natural full-length protein immunoprecipitated from CHO-K1 cultured cells and a recombinant N-terminal catalytic fragment synthesized in E. coli. Both preparations displayed the ability for autophosphorylation and the ability for phosphorylation of MBP and of H1 histone, and their activities were comparable. H1 histone was a better substrate for LOSK than casein and ATP was a better substrate than other nucleotides. The pH dependence of the activity of the immunoprecipitated protein was more pronounced than the pH dependence of its recombinant fragment deprived of the C-terminal domain. The catalytic and the structural domains of LOSK can interact through electrostatic forces; therefore, effects were studied of various polyions at the concentration of 0.1 mg/ml on the activity. Heparin, protamine sulfate, and poly(L-Lys) decreased tenfold the ability of the full-length kinase to phosphorylate H1 histone. Heparin did not affect the activity of the recombinant fragment, whereas protamine sulfate and poly(L-Lys) had a slight effect. Moreover, protamine increased fourfold the autophosphorylation of the immunoprecipitated protein kinase. These data suggest that the structural C-terminal domain of LOSK should be involved in the regulation of its protein kinase activity: the LOSK protein kinase with C-terminal domain cleaved off could significantly less depend on conditions in the cell than the full-size enzyme.

Adenosine Triphosphate↗
PubMed2001

Transcription factors NF-kappaB and AP-1/c-fos in cell response to nocodazole.

The effects of nocodazole (an agent causing degradation of microtubules in vitro) on the content of transcription factors NF-kappaB and AP-1/c-fos in the nuclei of cultured cells CHO-K1 and HeLa, as well as on the activity of these factors in the nuclei of T-cell hybridoma 2B4 were studied. Immunoblotting, immunofluorescence, and gel retardation techniques revealed that in all cell lines studied nocodazole induced rapid and considerable activation (an increased content in the nuclei) of transcription factor AP-1/c-fos. In 2B4 cells, nocodazole had no effect on the activity of NF-kappaB, but in CHO-K1 and HeLa cells it caused a transient decrease, sometimes followed by an increase in the content of subunit p50. Besides, nocodazole inhibited proteolytic degradation of NF-kappaB in CHO-K1 and HeLa cells and caused a short-term decrease in the content of subunit p65 in CHO-K1 cells. The results suggest that nocodazole may interfere with the genome expression, and these alterations should be taken into consideration, whatever experimental studies this substance is used in.

Animals↗
PubMed1999

Exclusively juvenile centrioles in Xenopus laevis oocytes injected with preparations of mature centrioles.

Activated oocytes of Xenopus laevis were injected with centriole preparations isolated either from spermatozoa of loach fish Misgurnus fossilis or from rat liver. These injections induced the development of cytasters in the ooplasm and egg cleavage. Electron microscopic study of cytasters was made at the stage that corresponded to interphase between first and second cleavage divisions. This study revealed in cytasters singleton centrioles surrounded by pericentriolar material and numerous microtubules. Surprisingly, the ultrastructure of centrioles in cytasters corresponded to that of juvenile, newly formed vertebrate centrioles, whereas the injected preparations contained only adult mature centrioles. We suggested that xenogenic centrioles injected to Xenopus laevis oocytes could dissolve after formation of centrioles made from molecules of oocyte origin. A special mechanism that eliminates male centrioles after egg fertilization is speculated.

Animals↗
PubMed1998

Antibodies against EMC virus RNA-VPg recognize Tyr-(5'P-->O)-pU and immunostain infected cells.

Covalent complexes of nucleic acids and proteins are widespread among viruses. Covalent complexes of RNA and proteins are proposed to exist in eukaryotic cells. The goal of this work was to obtain specific antibodies to the covalent linkage unit (CLU) between virus RNA and protein to search cellular RNA-protein complexes. Antibodies were generated by direct immunization of a rabbit with the BSA-coupled EMC virus RNA-VPg complex. By a dot-blot immunoassay and immunofluorescent microscopy it was found that the antibodies specifically recognize both EMC virus RNA-VPg and synthetic CLU-containing compounds. Thus, a fraction of the antibodies was directed to CLU.

Animals↗
PubMed1997

A major 170 kDa protein associated with bovine adrenal medulla microtubules: a member of the centrosomin family?

Microtubules isolated from bovine adrenal medulla cells contain a major 170 kDa protein (p170). p170 is heat-labile and is associated with microtubules in an ATP-insensitive manner. This protein was purified to near homogeneity using FPLC. A preparation containing purified p170 caused bundling of microtubules. By microsequencing of p170, two polypeptides were identified which appeared to be identical to a recently sequenced p167 centrosomin-related protein. Polyclonal affinity-purified anti-p170 antibody was found to immunostain microtubules and to recognize the 170 kDa polypeptide in culture cells. We suggest that p170 is a new member of a centrosomin family and is a new structural protein associated with microtubules in some cell types.

Adrenal Medulla↗
PubMed1997

Chinese hamster protein homologous to human putative protein kinase KIAA0204 is associated with nuclei, microtubules and centrosomes in CHO-K1 cells.

Monoclonal antibody raised against a preparation of loach fish sperm centrosomes was used for screening of cDNA expressing library of Chinese hamster CHO-K1 cells. Two positive clones appeared to encode 628 amino acid protein fragment that was 72% identical to human KIAA0204 protein, i.e. putative protein kinase. Polyclonal antibodies raised against products of cDNA expression in E. coli recognized 210-kDa polypeptide in CHO-K1 cells and immunostained nuclear speckles, centrosomes and microtubules in these cells. The 210-kDa polypeptide (named MAK-L) co-sedimented with exogenous microtubules. Thus, one more protein kinase seems to be associated with the microtubule network in vertebrate cells.

Animals↗
PubMed1996

[Centrosome morphogenesis in the early development of mice: an immunofluorescence study using antibodies to the centriole antigen].

The distribution of a centrosomal antigen, recognized by RN-C6 monoclonal antibody (monAB. RN-C6), was studied. The monAB. RN-C6 was directed against the common phosphoepitope in several high molecular cell proteins. In the mouse embryonal fibroblasts the monAB. RN-C6 stained discrete spots in the interphase nuclei, centrioles throughout the cell cycle and the midbody in mitosis. In the oocytes and the early mouse embryos, the monAB. RN-C6 stained only the nuclei, the staining of pronuclei in zygote being absent. In the mouse spermatozoon, the monAB. RN-C6 recognized the centriole and axoneme. Nevertheless, the stained sperm axoneme was revealed in the cytoplasm of early embryos up to the morula stage. No staining was observed in the mitotic spindle or in the midbody, or in any dots in the cytoplasm of oocytes/early embryos. On the blastocyst stage, the monAB. RN-C6 stained first the cytoplasmic dots in some cells, in the spindle poles and midbody. The appearance of stained dots and mitotic figures coincides with that of centrioles revealed at the ultrastructural level in some cells. Thus, during centrosome formation a given centriolar antigen is found only in the centriole, never being accumulated previously in the cytoplasm or in cell organelles.

Animals↗
PubMed1996

[Centrosomal proteins].

The review summarizes recent data on structure and function of nearly two dozen centrosomal proteins of the animal cells; centrosome is an organelle that organizes cellular microtubules. Most of these data were obtained by molecular biology techniques including screening of phage expression cDNA libraries with centrosome-specific antibodies, bacterial expression of recombinant proteins recognized by these antibodies, analysis of multiple non-allelic gene of cytoskeletal proteins, etc. new classification of centrosomal proteins is suggested based on their possible centrosomal function comprising proteins of microtubule polymerization templates, structural proteins of pericentriolar material, microtubule severing proteins, microtubule-dependent transport-catalyzing motor proteins, regulatory proteins (specific protein kinases, phosphatases, etc), and centriolar proteins.

Animals↗
PubMed1994

[An electron microscopic study of centriole and centrosome morphogenesis in the early development of the mouse].

Using ultrathin section electron microscopy, the precise time of the appearance of centrioles and centrosomes and their morphogenesis in early mouse development was followed. It turns out that in murine morulae and early blastocyst stage centrosomes, centrioles or other microtubule organizing centres are absent; the network of microtubules in interphase cells is diffuse. In 4-day blastocyst four nascent centrioles were found in two cells of trophectoderm in addition to one nascent centriole in the only cell of the inner cell mass. The nascent centrioles were not connected with microtubules. In the late blastocyst (more than 4.5-days of development) among the cells of inner cell mass, cells without centrioles were found, with nascent centrioles but not connected with microtubules; sometimes cells with true centrosomes were seen, having double centrioles and microtubule organizing centres associated with them. Thus, the centrosomes appear asynchronously in different cell types of blastocyst at the preimplantation stage. The centrosome development begins from a self-assembly de novo of the nascent centrioles without any association with microtubules.

Animals↗
PubMed1987

[Reorganization of the system of intermediate filaments and detection of the organization centers after centrifugation of cells attached to a substrate].

Cultured pig kidney epithelial cells were centrifuged at 20,000 gav so that the centrifugation force was oriented parallel to the substrate, fixed and processed for indirect immunofluorescent staining with tubulin and vimentin antibodies. After a 2 hour centrifugation vimentin filaments aggregated in the centripetal parts of the cells (probably, because of their association with floating lipid vesicles). Microtubule-organizing centers were found near the centripetal poles of the nuclei, which migrated in the direction of the centrifugal force. The distribution of the cytoplasmic microtubules did not change during centrifugation. The staining of the cultures one hour after centrifugation revealed vimentin-containing spots with radiating intermediate filaments in most of the cells. These spots were localized near the cell nuclei; double immunofluorescent staining with tubulin and vimentin antibodies showed that their position was identical to that of the microtubule-organizing centers. Similar foci of vimentin filaments were seen in the cells after a 3-4 hour centrifugation. Probably, these structures participate in organizing the intermediate filament cytoskeleton in cells.

Animals↗
PubMed1986

The centriolar rim. The structure that maintains the configuration of centrioles and basal bodies in the absence of their microtubules.

Preparations of centrioles from bovine spleen were incubated in solutions of NaCl, MgCl2, HCl, NaOH, EDTA and heparin. Their effects on the centrioles were studied by electron microscopy of ultrathin sections. It was found that the microtubules of centriolar cylinders gradually disintegrate at a higher than physiological ionic strength and at a pH value lower than 3.5 and higher than 8.5. After microtubule extraction, a closely apposed rim or sheath of dense centriolar matrix remains which has the same dimensions of length and width as the original centriole. Some other centriolar structures, including the pericentriolar satellites and certain structures in the cylinders (hub) are also preserved. The basal bodies of fish spermatozoa revealed similar structures, including the centriolar rim and hub, after microtubule extraction. Thus, the microtubule triplets are not involved in maintaining the structure of the centriolar cylinder; this role is rather carried out by amorphous material--the matrix, surrounding the microtubules.

Animals↗
PubMed1986

Identification of a 34-kD polypeptide as a light chain of microtubule-associated protein-1 (MAP-1) and its association with a MAP-1 peptide that binds to microtubules.

We examined the association of a 34-kD light chain component to the heavy chains of MAP-1 using a monoclonal antibody that specifically binds the 34-kD component and labels neuronal microtubules in a specific and saturable manner. Immunoprecipitation of MAP-1 heavy chains together with the 34-kD component by the antibody indicates that the 34-kD polypeptide forms a complex with MAP-1 heavy chains. Both major isoforms of MAP-1 heavy chains (MAP-1A and MAP-1B) were found in the immunoprecipitate. Digestion of MAP-1 with alpha-chymotrypsin and analysis of the chymotryptic peptides reveals a 120-kD fragment of the MAP-1 heavy chain that binds to microtubules and is precipitable with the 34-kD light chain antibody, suggesting that the 34-kD light chain also binds to this domain of the molecule. Since microtubules that contain the 120-kD fragment lack the long lateral projections characteristic of microtubules with intact MAP-1, the 34-kD light chains may be localized at or near the microtubule surface.

Animals↗
PubMed1986

[A cytoskeletal protein with a molecular weight of 100 kD is a component of the endosomes participating in receptor-mediated endocytosis].

Our previous paper (Rodionov et al., 1985) reported production of monoclonal antibodies RN-17 reacting in cultured fibroblasts with a protein having a molecular weight of 100 kD. Immunofluorescence and immunoelectron microscopy showed that this protein was a component of microtubules, intermediate filaments and coated vesicles. We challenged a possibility whether these coated vesicles containing the 100 kD protein may take part in the receptor-mediated endocytosis. alpha 2-Macroglobulin conjugated with fluorescein isothiocyanate or 20 nm colloidal gold particles was used as a marker of the receptor-mediated endocytosis. Mouse embryo fibroblasts or Swiss 3T3 cells were incubated with labeled alpha 2 M, fixed and "stained" with DN-17 antibody, and the distribution of alpha 2 M and 100 kD protein was examined within the same cells. In both cell lines the endocytic vesicles contained 100 kD protein and alpha 2 M. Therefore 100 kD protein is a component of endocytic vesicles. Probably this protein mediates microtubule-dependent transport of endocytic vesicles in the cells.

Animals↗