[Immunocytochemical visualization of P185(HER2) receptor by antibody fused with dibarnase and conjugate of barstar with colloidal gold].

We studied the localization of transmembrane receptor P185(HER2) in SKOV-3 and BT-474 cancer cells by fluorescence, confocal and electron immunomicroscopy. P185(HER2) is a marker of breast and ovarian tumors, it is considered as a target for anticancer therapy. It is extremely important to choose a universal immunicytotoxic agent applicable, first, to study the distribution of P185(HER2) in cancer cells, secondly, to remove P185(HER2) from the cell surface and, thirdly, to eliminate target cells. In this work for visualization of P185HER2 We prOposed immunocytotoxic system, consisting of the monoclonal miniantibody 4D5 scFv to extracellular P185E domain fused with two molecules of barnase (ribonuclease from Bacillus amyloliquefaciens) and of its specific inhibitor barstar. Fluorescence microscopy has showed that the module 4D5 scFv-dibarnase:barstar efficiently identified P185(HER2) on the surface of cancer cells. It was revealed by confocal microscopy that interaction with 4D5 scFv-dibarnase lead to internalization of P185(HER2). The localization of P185(HER) in human ovarian carcinoma cells SKOV-3 and breast carcinoma cells BT-474 was compared by electron microscopy using 4D5 scFv-dibarnase:barstar-Au and 4D5 scFv-dibarnase-Au complexes. P185(HER) distributed on the cell surface unequally with preferential localization on protrusions or close to their bases and in contacts between protrusions and cell membrane. At 37 degrees C, P185(HER2) internalized through coated pits and vesicles and concentrated in the endosomes and multivesicular bodies in the cells of both cell lines, as well as in lysosomes in cells BT-474.

[Study of the positional relationship of nucleolar chromatin and nucleolar compartments in somatic nuclei OPF the ciliate Didinium nasutum].

We showed earlier that nucleoli in interphase ciliates Didinium nasutum, appearing on single ultrathin sections as individual structures, actually are parts of more complex network-like structures in which fibrillar component is located on periphery, and granular--in the central part of a nucleolus. It is known, that nucleolar organizers in D. nasutum are represented by chromatin bodies connected with nucleoli. In this work we used 3D reconstruction on the basis of serial ultrathin sections to study localization of chromatin bodies which by morphological criteria might correspond to nucleolar organizers. Our data showed, that all such chromatin bodies settled down outside of nucleoli, near the periphery of fibrillar component. Even those chromatin bodies which on single sections looked completely surrounded by fibrillar nucleolar component, actually settled down in fibrillar component cavities open to nucleoplasm. Analysis of distribution of nucleolar chromatin bodies allowed us to conclude that activity in different parts of interphase complex network-like nucleoli of D. nasutum is approximately the same.

[Three-dimensional organization of nucleoli of Didinium nasutum (Ciliata)].

A comparative study of nucleolar organization in the somatic nuclei of the ciliate Didinium nasutum was carried out using 3D reconstruction on the basis of serial ultrathin sections. Recently fed interphase ciliates, starved interphase ciliates and cysts were studied. The nucleoli at the interphase stage were shown to have a complex architecture: the fibrillar component forms a complicated network, the granular component is located inside of it. It was shown that nucleoli, which look like individual structures in single sections, are in fact parts of branched nucleolar networks. A 30-h starvation doesn't lead to disintegration of these networks. However in the starved cells the granular component becomes more dense and vacuolized. In the fed ciliates there are many holes in the fibrillar component, whereas in starved ones the fibrillar component is virtually devoid of them. These holes can be proposed to ensure the transport of newly synthesized rRNP. The nucleolar networks didn't occur in D. nasutum cysts. Nucleoli in the cysts look like small individual structures, mainly consisting of fibrogranular component.

[Immunocytochemical study of the hcs2 gene products distribution in the command neurons of grape snail]. / Immunotsitokhimicheskoe izuchenie lokalizatsii produktov gena hcs2 v komandnykh neironakh vinogradnoi ulitki.

In the present study the cellular and subcellular distribution of putative protein products of hcs2 gene in the giant command neurons of parietal ganglia of the terrestrial snail Helix lucorum L. were investigated using light- and electron-microscopic immunocytochemistry. The product of hcs2 gene is a hybrid precursor protein belongs to the EF-hand family of the Ca(2+)-binding proteins, whose processed products are neuropeptides. By use of polyclonal antibodys against a synthetic CNP3, CNP4 and C-terminus peptide immunoreactivity was observed in the cytoplasmic secretory granules. The colloidal gold density in the granules for CNP3-4 neuropeptides was twice one for the Ca(2+)-binding protein. These immunocytochemical results point to a specific connection between putative protein products of hcs2 gene and the cell secretory apparatus, that correspond to our early expressed hypothesis that products of hcs2 gene act as neuromodulators or neurotransmitters.

[Dynamics of structural changes of Paramecium caudatum and Bursaria truncatella macronuclear chromatin and nucleoli under hypotonic conditions]. / Dinamika ul'trastrukturnykh izmenenii khromatina i iadryshek makronukleusa infuzorii Paramecium caudatum i Bursaria truncatella pri gipotonicheskoi obrabotke.

Dynamics of structural changes of nucleoli, complex nucleolar aggregates and chromatin bodies in macronuclei (Ma) of ciliates Paramecium candatum and Bursaria truncatella under hypotonic conditions was studied. It was shown that after a 3 min hypotonic treatment nuclei swelled and became highly vacuolated. 3D-reconstruction showed that such nucleoli were formed by nucleolonema-like threads about 100-200 nm in thickness. Intranucleolar chromatin bodies decompacted, but remained bound with fibrillar component of the nucleolus by thin fibres about 10 nm thick. After 6 min hypotonic treatment the nucleolar material loosened and had a "gauze", or network-like appearence. After 10 min hypotonic treatment nucleoli dissociated completely. It was shown that a transition of chromatin bodies from completely compact to partially and fully decompacted state occurred cooperatively in different regions of Ma. In particular, chromatin bodies in the central part of complex nucleolar aggregates decompacted much faster than those in the Ma karyoplasm. It evidences for a specific, well-ordered chromatin organization in Ma. Prolonged hypotonic treatment led to a complete dissociation of Ma components; fibres 6-10 nm thick were solely observed in such preparations. Such fibres may represent remnant structures of the nuclear matrix. Dynamics of Ma chromatin bodies decompaction correlates well with that of chromomeres in the nuclei of higher eukaryotes. Our data confirm that chromatin 100-200 nm bodies in the ciliate Ma are analogues of chromomeres--looped discrete chromatin domains, observed in the nuclei of higher eukaryotes.

[Formation of chromoneme-like fibrils induced in infusorian somatic nuclei by magnesium ions]. / Formirovanie khromonemopodobnykh fibrill v somaticheskikh iadrakh infuzorii pod deistviem ionov magniia.

A study was made of the effect of Mg2+ on higher-order chromatin structure in marconuclei (Ma) of infusoria Paramecium aurelia and Bursaria truncatella. In infusorian Ma, inactive chromatin is commonly packed in chromatin bodies sized 60-200 nm. When isolated chromatin or Ma were treated with Mg2+ (about 3 mM), chromatin bodies arranged into fibrils 100-300 nm in diameter, which resembled higher eukaryotic chromonemes. The formation dynamics of chromoneme-like fibrils was described. The results testified to the similarity of chromatin bodies to chromomeres of higher eukaryotes. Structurally intact central chromomere cores proved to be essential for the formation of chromoneme-like fibrils. Chromatin organization in infusoria was shown to follow the discrete-level model (nucleosomes-nucleomeres-chromomeres-chromonemes) assumed for higher eukaryotes.

[Comparative study of localization of tryptophanyl-tRNA-synthetase and components of high molecular weight aminoacyl-tRNA-synthetase complex in animal cells]. / Sravnitel'noe izuchenie lokalizatsii triptofanil-tRNK-sintetazy i komponentov vysokomolekuliarnogo aminoatsil-tRNK-sintetaznogo kpmpleksa v kletkakh zhivotnykh.

A comparative study on the localization of cytosolic Trp-tRNA synthetase (TrpRS), aminoacyl-tRNA synthetases associated in a multienzyme complex (Glu-tRNA synthetase (GluRS), and Arg-tRNA synthetase (ArgRS)) and polypeptides p37 and p43 from the multienzyme complex was carried out on ultrathin sections of cultured rabbit cells RK-1 by means of immunogold technique. It is shown that GluRS, ArgRS, and polypeptide p43 have approximately the same distribution in the cell as TrpRS. The data obtained evidences in favour of a multienzyme structure of most (or, may be all) aminoacyl-tRNA synthetases in intact cells. A statistical analysis of enzyme distribution in different cell organelles showed nonrandom, compartmentalized distribution of studied synthetases in the mammalian cell. Aminoacyl-tRNA synthetases were found in the cell nucleus in the vicinity of interchromatin granules and in the regions of diffused chromatin. This fact points to a role which these proteins may play in active chromatin functions (transcription, processing, transfer of gene products, etc.) and needs special attention. Detection of ArgRS and GluRS in the nucleus allows one to suggest that either multienzyme synthetase complexes are present not only in the cytoplasm, but also in the nucleus, or these enzymes can dissociate from the complex and pass to the nucleus as individual proteins.

[Immunoelectron-microscopic determination of the localization of tryptophanyl-tRNA synthetase in eubacterial cells of Escherichia coli and Methanococcus halophilus archaebacteria]. / Immunoélektronno-mikroskopicheskoe opredelenie lokalizatsii triptofanil-tRNK-sintetazy v kletkakh éubakterii Escherichia coli i arkhebakterii Methanococcus halophilus.

Localization of tryptophanyl-tRNA-synthetase (TRS) was studied in halophilic archaebacterium Methanococcus halophilus and eubacterium E. coli. Ultrathin sections of the cells, fixed with glutaraldehyde and embedded in "Lowicryl K4M" at -35 degrees C, were treated with colloidal gold complexes containing monoclonal antibodies Aml against TRS. The latter bind specifically to TRS isolated both from eucaryotes, archae- and eubacteria. According to the label distribution three zones in M. halophilus and E. coli can be distinguished: (i) about 75% of the whole amount of gold particles are localized in the cytoplasm, the distribution of label being more or less homogeneous; (ii) cytoplasmic regions, adjacent to nucleoid, are intensively labelled (about 20% of the whole amount of label); (iii) very few gold particles (not more than 10% of the whole amount) are present in the nucleoid. The data obtained show, that the distribution of TRS in the nucleoid and cytoplasm of archaebacterium M. halophilus is close to the distribution of TRS, found in E. coli. It supports our previous conclusion that the structural organization of transcription-translation apparatus in methanogen and halophilic archaebacteria is similar to that in eubacteria.

[Detection of viral proteins during synthesis of defective phage of Proteus mirabilis D52 using the immunocolloid gold method]. / Vyiavlenie virusnykh belkov v protsesse sinteza defektnogo faga Proteus mitabilis D52 metodom immunokolloidnogo zolota.

We have applied the method of immunocolloidal gold in order to label the ribonuclear proteins of prokaryotic cells on isolated bacterial chromatosomes. In the process of protein synthesis it was possible to visualize a definite protein of defective phage D52 of Proteus mirabilis.