[Ultrastructural and X-ray spectral analysis of cells U-937 during apoptosis process induced by hypertony].

The results of this work concerning ultrastructural changes of U-937 cells in a state of apoptosis are largely in consistent with the same information available in the literature. However, we have got the original data on the ultrastructural changes of cell organelles and immune localization and distribution of proteasomes. It has been demonstrated that Golgi apparatus is located close to the plasma membrane in the case of apoptosis induced by incubating the cells in a hypertonic suchrose solution (200-400 mM). The fact can be considered as an indirect indication of depolymerization of cytoskeletal elements, in particular, MTs maintaining Golgi apparatus in a cell centre. In the later stages of apoptosis, the distances between Golgi cisterna are significantly increased. It can be explained by hydrolysis of golgins binding cisterna between each other. Mitochondria are not significantly changed in these cells. They have regularly disposed crista and sufficiently dense matrix with a few vacuoles. Proteasomes as rod-shaped osmiophilic particles (12 x 30 nm) have been revealed during each apoptosis stage both in nuclei and cytopl;asm of cells studied. The particles form aggregates of different densitities and sizes unlimited by membrane. It has been proposed that the particle aggregates revealed in the work are analogous to "processing bodies" or aggresomes described in the literature. They can be detected in cells under conditions of suppressed nucleus transcriptional processes in the nucleus and participate in storing and degradation of various mRNAs, RNP and proteins. The changes of intracellular contents of Na and K in a single cell during apoptosis induced by osmotic shock have been revealed using method of X-ray microanalysis. It has been demonstrated the increase in the ratio of intracellular contents Na+/K+ in the most of apoptotic cells in comparing with control cells.

[Investigation of the kinetics of insulin amyloid fibrils formation].

Investigation of the structure of ordered protein aggregates--amyloid fibrils, the influence of the native structure of the protein and the environment on the process of fibrillation is currently the subject of intensive research. The present work is devoted to the study of the kinetics of insulin amyloid fibrils formation at low pH values (which are produced at many stages of the isolation and purification of the protein) using a fluorescent probe thioflavin T (ThT). It has been shown that the increase of fluorescence intensity of ThT during the formation of amyloid fibrils is described by a sigmoidal curve, in which 3 areas can be distinguished: the lag phase, the growth and the plateau, which characterize the various stages of fibril formation. Despite the variation in the length of the lag phase at the same experimental conditions (pH and temperature), we have found its reduction with stirring the solution and seeding. Data obtained using electron microscopy showed that the formed fibrils are long, linear filament having a diameter of -20 nm. With increasing incubation time fibril diameter did not change while their length increases to 2-3 µm, which was accompanied by a significant increase in the number of aggregates of fibrils. All the experimental data shows that, regardless of the kinetics of the formation of amyloid fibrils, their properties after the fibrillation process are identical. The results of this work together with the previously studies of insulin amyloid fibrils might be important for clarification the mechanism of their formation, as well as for the treatment of amyloidosis associated with the aggregation of insulin.

[The role of chromatoid bodies and cytoskeleton for differentiation of rat spermatozoids].

The ultrastructural and immunocytochemical study of rat male germ cells on different developing stages has been made. The investigation of morphological changes of spermatogenic cells has demonstrated the presence of tight connections between chromatoid bodies (CBs) and other cell organelles, particularly with the nucleus and Golgi apparatus; has revealed the association of manchette noncentrosomal microtubules (MT) with spermatid perinuclear ring plasma membrane (PM) in the zone of the adhesion intercellular contact--zonula adhaerens (ZA). The comparison of the results obtained in this work with available literary data has given possibility to analyze expected pathways of noncentrosomal MT nucleation in the late spermatids. This paper puts the supposition that noncentrosomal MTs are nucleated on the sites of perinuclear ring ZA. The immunocytochemical analysis discovered two novel proteins for these cells--BASP1 and MARCKS. It has been shown that these proteins present in the CBs in the early spermatids. During the spermatozoid differentiation these proteins are revealed along the outer dense fibers (ODFs) of the sperm tail. BASP1 and MARCKS are supposed to involve in the processes of calcium accumulation in the CBs and ODFs. Calcium ions seem to play the significant role in RNA processing and protein synthesis in spermatids. Calcium is also necessary for the mobility of sperms which is mainly determined by ODFs.

[The effect of red pigment of Saccharomyces cerevisiae on insulin fibril formation in vitro].

The effect of the yeast red pigment, the result of polymerization of AIR, and of its low molecular weight derivative (presumably devoid of phosphoribosyl moiety) on the formation of amyloid fibrils in vitro was studied. Both the red pigment and its derivative, the result of acid hydrolysis of the original pigment, were shown to diminish the intensity of amyloid bound Thioflavine T fluorescence. Correlation between the decrease of the intensity of Thioflavine T fluorescence and the concentration of both forms of the red pigment was demonstrated. Both forms were also able to compete with Thioflavine T for amyloid fibrils. Electron microscopy permitted to visualize a drop of fibril size in the case of red pigments presence during their formation.

[Oligomeric forms, functions and cellular localization of alpha-crystallin type protein from Acholeplasma laidlawii].

Alpha-Crystallin type heat shock protein (alpha-HSP) IbpA from Acholeplasma laidlawii was expressed in Escherichia coil and isolated from cell extract on Ni-sepharose column. Recombinant IbpA, like other alpha-HSPs, spontaneously formed oligomeres in vitro. High resolution electron microscopy revealed regular structures with 15 nm in diameter. Evaluation of molecular mass of IbpA oligomers was performed by gel filtration. Most of oligomers consist of 24 subunits. Recombinant IbpA prevents heat denaturation of soluble proteins in cell extract of E. coli and displays a mild positive effect on thermotolerance of E. coli cells during severe heat shock. We investigated a localization of IbpA in A. laidlawii cell by immunocytochemistry. We suppose that IbpA may protect various intracellular structures from damage during heat shock.

[Localization of the division protein FtsZ in mycoplasma cells Mycoplasma hominis].

Localization of the protein FtsZ in Mycoplasma hominis cells was determined. Ultra thin sections were treated by rabbit polyclonal antibodies against FtsZ M. hominis: a conjugate of protein A with colloidal gold particles was used instead of secondary antibodies. Considerable polymorphism of cells was seen on electron microscopy pictures of M. hominis cells, which is typical for mycoplasmas. Among a wide variety of cell shapes we distinguished dumbbell-shaped dividing cells, and the cells connected with each other with the aid of thin membrane tubules (former constrictions). Dominants distribution of the label in the constriction area of dividing M. hominis cells and in the area of the thin membrane tubules was observed. We revealed the cross septum in the mycoplasma cells for the first time, as well as the gold labeling of this structure. Furthermore, in some rounded and oval cells colloidal gold particles labeled the whole plasma membrane in ring-shaped manner. Probably, the label in these cases marks a submembrane contractile ring (Z-ring). The facts mentioned above confirm that FtsZ of M. hominis plays an active role in the mycoplasma cytokinesis. In a series of cases spiral-like distribution of gold particles was observed. Probably, FtsZ protofilaments in M. hominis cells can form spiral structures similar to Z-spirals of Bacillus subtilis and Escherichia coli. Its presence in mycoplasma cells may be considered as an important argument in favour of model of Z-ring assembling through reorganization of Z-spirals. FtsZ also may participate in maintenance of mycoplasma cell shape (membrane localization).

[Arginin-vasopressin-induced alterations in the structure of MDCK cells].

Alterations in the structural organization of MDCK cells under arginin-vasopressin (AVP) action were studied by electron and fluorescent microscopy. Electron-microscopical evidence was obtained that MDCK cells within monolayer form structurally distinct apical and basolateral surfaces separated by well-developed intercellular contact zones. It was proved that AVP specifically bound to the receptors on the basolateral surface of the cells, and was internalized from the surface in 10-15 min. AVP action resulted in fragmentation of Golgi apparatus and swelling of Golgi cisterns caused by initiation of osmotic water flow across the monolayer. Significant depolymerization of cells actin cytoskeleton was revealed under AVP or forskolin (an adenylate cyclase activator) exposure. Functional role and regulatory mechanisms of described structural alterations are discussed.

[Golgi apparatus in parasitic protists (review of the literature)].

This review summarizes modem data on Golgi apparatus of parasitic protists and demonstrates how the parasitic lifestyle determines functional and structural peculiarities of secretory systems in unrelated groups of unicellular parasites, in comparison to ones of "model systems", mammalian and yeast cells. The review covers the most well-studied protists, predominantly of high medical importance, belonging to following taxons: Parabasalia (Trichomonas), Diplomonada (Giardia), Entamoebidae (Entamoeba), parasitic Alveolata of the phyllum Apicomplexa (Toxoplasma and Plasmodium), and Kinetoplastida (Trypanosoma and Leishmania). Numerous recent publications demonstrated that studies on intracellular traffic in the mentioned above parasites essentially advanced our knowledge of Golgi function, traditionally based on research of cultured mammalian and yeast cells. Morphology of Golgi organelle in eukaryotes from various taxonomic groups has been compared. Within three of total six the highest taxons of Eukatyota (Adl et al., 2005) there exist at minimum eight groups represented by species lacking Golgi dictiosomes. However, biochemical and (or) molecular (genomic) evidences indicate that the organelle with functions of Golgi was present in every studied so far lineage of eukaryotes. Loss of Golgi organelle is a secondary event, which has been proven by identification of Golgi genes in the genomes of Golgi-lacking lineages. This loss might have occurred independently several times in the course of evolution. Neither the number of stacks, nor the size of the organelle correlates with intensity of secretion, or the position of the species on the evolutionary tree (in terms of presumably early/lately diverged lineages).

[Structural-functional organization of Golgi apparatus].

This review is dedicated to the structure and function of Golgi apparatus (GA). It summarizes contemporary data published in numerous experimental papers and in several reviews. Possible ways of intra-Golgi transport of proteins, existent models of structural and functional organization of Golgi organelle, as well as the issues of its biogenesis, posttranslational modification and sorting of proteins and lipids, and mechanisms of their traffic-king are discussed. Special attention is paid to the role of coatomer proteins (COPI, COPII and clathrin), fusion proteins (SNAREs), and small GTPases (ARF, SARI) in the secretory pathway. In addition, the phenomena of ultrastructural alterations of GA due to various functional conditions and physiological stimuli are specifically accented. We included in this review our original data on a probable involvement of GA in water transport, and on the organization of atypical GA in microsporidia--intracellular parasitic protists.

[Structural-functional analysis of diffusion in glucose absorption by rat small intestine enterocytes].

To elucidate mechanisms providing transport of sugars across intestinal epithelium, on taking into account the current hypotheses (active transport, participation of paracellular transport and passive component of transcellular transport), it was important to reveal structural changes of tight junctions and distribution of the carriers of facilitated diffusion of GLUT2 and protein kinase C during absorption of glucose. On using confocal and electron microscopy, ultrastructural and immunocytochemical studies of enterocytes after perfusion of isolated rat small intestine fragment with 75 mM glucose (chronic experiment) have shown: 1) fluorescent labels of transporter GLUT2 and PKCbetaII are located in the apical area of enterocytes situated at the upper half of the villus. Antibodies against GLUT2, conjugated with gold, are revealed at the microvilli or apical membrane and in the area of terminal network; 2) no ultrastructural changes of the tight junction are detected on ultrathin sections and freeze--fracture replics. At the same time, fluorescent and gold labels against actin are concentrated in the vicinity of the lateral membrane in the tight junction area. The results obtained can serve a confirmation of a hypothesis that at high glucose concentrations GLUT2 participates in its transfer across the apical membrane.

[Structural-functional organization of Golgi apparatus].

This review is dedicated to the structure and function of Golgi apparatus (GA). It summarizes contemporary data published in numerous experimental papers and in several reviews. Possible ways of intra-Golgi transport of proteins, existent models of structural and functional organization of Golgi organelle, as well as the issues of its biogenesis, posttranslational modification and sorting of proteins and lipids, and mechanisms of their trafficking are discussed. Special attention is paid to the role of coatomer proteins (COPI, COPII and clathrin), fusion proteins (SNAREs), and small GTPases (ARF, SARI) in the secretory pathway. In addition, the phenomena of ultrastructural alterations of GA due to various functional conditions and physiological stimuli are specifically accented. We included in this review our original data on a probable involvement of GA in water transport, and on the organization of atypical GA in microsporidia--intracellular parasitic protists.

[Immunocytochemical localization of vasopressin at its absorption by cells of rat small intestine]. / Immunotsitokhimicheskaia lokalizatsiia vazopressina pri ego vsasyvanii kletkami épiteliia tonkoi kishki krysy.

Morpho-physiological characteristics of the transport of cyclic nonapeptide arginine vasopressin (AVP) across the rat intestinal epithelium was studied in experiments in vitro. A partial absorption of physiologically active AVP was followed when filling the isolated intestinal lumen by hormone solution. By methods of immunoelectron and immunofluorescence confocal microscopy, using polyclonal anti-AVP antibodies, cytoplasmic localization of AVP label was shown in enterocytes. The AVP label was also observed in the intercellular space in the basal area of epithelium. No label was revealed in the intercellular junctions, and no predominant label accumulation was found in any cytoplasmic structures of the epithelial cells. The obtained results are considered as evidence for the transcellular pathway of partial AVP absorption in rat small intestine.

[Structural and functional analysis of glucose adsorption at high maltose concentrations in the rat small intestine in vivo]. / Strukturno-funktsional'nyi analys mekhanizmov vsasyvaniia gliukozy pri vysokikh kontsentratsiiakh mal'tozy v tonkoi kishke krys in vivo.

To elucidate the mechanism of glucose absorption at high substrate concentrations, we studied structural and ultrastructural peculiarities of enterocytes arranged at different levels along the intestinal villus. The preparations were obtained from an isolated segment of the rat small intestine after its perfusion with maltose solutions with both low (25 mM) and high (100 mM) concentrations, respectively. Under conditions of chronic experiment at high substrate concentration, an enlargement of intercellular clefts, indicating glucose absorption, occurred in deeper areas of the villus. Besides, also in chronic experiment, we studied kinetics of maltose hydrolysis and derived glucose absorption in the isolated segment of the rat small intestine after its perfusion with maltose at superhigh (up to 200 mM) initial concentrations. Based on these data, a conclusion is made that active transport is the main mechanism of absorption of glucose derived from maltose hydrolysis, operating both at low disaccharide concentrations, and in the range of its superhigh (up to 200 mM) concentrations.

[Microtubule dynamics in epithelial cells]. / Dinamika microtrubochek épitelial'nykh kletok.

Microtubules (MTs) are necessary components of all eukaryotic cells. They fulfill various functions being involved in cell division, ciliar and flagellar beating, cell shape maintaining, organelle distribution in the cell, organization of other cytoskeletal elements. Dynamic features of MTs have been commonly studied in vitro or on undiffirentiated cultured cells by means of molecular and ultrastructural methods. It is generally accepted that the phenomenon of dynamic instability is the major mechanism of MT turnover in the cell. MTs radiate from the centrosome and take part in the distribution of cell organelles. In addition, epithelial, nerve, and skeletal muscle cells contain non-centrosomal MTs. A few hypothesis of their origin have been so far put forward. According to the capture-release hypothesis, MTs are first nucleated on the a centrosome, then release to be driven in various parts of the cell by molecular motors. Some alternative mechanisms of non-centrosomal MT formation are also proposed in literature. For example, the nucleation sites were reported not only in centrosomes but also in other parts of cells, such as the apical membranes of epithelial cells, the nuclear membrane of muscle cells, pigment granule aggregates of melanophores. On studying frog urinary bladder and large intestine epithelial cells the authors observed in these cells numerous non-centrosomal MTs. This makes epithelial cells, good models for analysing structural and dynamic features of non-centrosomal MTs in differentiated cells. For the urinary bladder the pool of specific granules may serve as MT organizing centers. Non-cenrosomal MTs of these cells have big diameters (35-38 nm) and form bundles oriented in the apical-basal axis of the cell. In addition, non-centrosomal MTs of these cells may participate in the transport of specific granules and giant vacuoles that appear under stimulated water flows through the cell.

[Electron microscopic study of colonic epithelial cells from the grass frog Rana temporaria under different intensity of water absorption]. / Elektronno-mikroskopicheskoe issledovanie kletok épiteliia tolsto kishki travianoi liagushki Rana temporaria pri razlichnoi intensivnosti vsasyvaniia vody.

Three cell types have been revealed in the epithelium of the frog large intestine: granular, mitochondria-rich, and mucosal cells. Under a low water permeability (0.12 +/- 0.10 mkl/(min.cm2)) the distribution of intramembrane particles (IMP) in the apical cell membrane was the same as in the most cell plasma membranes studied with freeze-fracture method. Under rising osmotic permeability and water absorption (0.43 +/- 0.05 mkl/(min.cm2)) the IMP distribution did not change. In these conditions, the quantity of fusion sites between granule membranes and the apical membrane increased, and the intercellular spaces in basolateral epithelial region were diluted. A a low water permeability, in addition to usual microtubules, bundles of noncentrosomal microtubules with associated osmiophilic globules were revealed. A comparative analysis has been made of the present evidence and previously obtained data on the frog urinary bladder epithelium.

[Aquaporins of plasma membranes of epithelial cells]. / Akvaporiny plazmaticheskikh membran épitelial'nykh kletok.

The early 90s have brought us a discovery of a new class of membrane proteins--aquaporins with a function of transmembrane water channels. Being genetically closed proteins aquaporins are members of a large family of channel-forming proteins called MIPs (major intrinsic proteins). All aquaporins, except AQP4, are mercury-sensitive. Many aquaporins have been cloned and identified. Polyclonal antibodies grown against some of them promoted numerous studies of aquaporin localization and distribution in animal and plant tissues. Up to the present, 10 and 2 aquaporins have been described in mammalian and amphibian epithelial tissues, respectively. One of described aquaporins, AQP2, whose localization is confined to kidney collecting duct principal cells, has been found to be a hormone-depending water channel. The insertion of apical vesicles bearing AQP2 was shown to be regulated by vasopressin, meanwhile all other aquaporins are inserted into the plasma membrane constitutively. There is a vast evidence showing that the integrity of microtubules is necessary for both pathways of aquaporin insertion. AQP2 is important for normal kidney functioning and AQP2 mutations cause water-balance disorders. On the contrary, the AQP1 mutations are not accompanied by any evident clinical pathology. This review is focused on a discussion of the data so far available on aquaporin distribution in different animal tissues.

[Current concept of structure and function of the Golgi apparatus. On the 100-anniversary of the discovery by Camillo Golgi]. / Sovremennye predstavleniia o strukture i funktsii plastinchatogo kompleksa. K 100-letiiu otkrytiia Kamillo Gol'dzhi.

The paper is a brief review of the current data on the structure and function of the Golgi apparatus since its discovery till the recent investigations, including the works published in 1997. Apart from reviewing the electron microscopy level of the structure of the Golgi apparatus, the data are considered on its molecular and supramolecular organization. The paper analyses critically the proposed mechanisms of the intracellular transport of proteins and their processing and modifications in the Golgi apparatus both in terms of the vesicular theory and in a model based on the gradual maturation of the cis-cistern and its transformation to the trans-cistern (i.e. its propagation from one pole to the other, a so-called "progression"). Experimental data are described, which disagree with the current models of the intracellular transport. Based on the literature and authors' own data, a modified model of the intracellular transport is proposed. This model eliminates, to a degree, the contradictions present in the models discussed above.

[Analysis of rat enterocyte ultrastructure during glucose absorption]. / Analiz ul'trastruktury énterotsitov krysy pri vsasyvanii gliukozy.

Electronmicroscopic study of rat enterocytes under glucose load (10-40 mM) has shown some changes of their structure: aggregations of intramembrane particles of the apical membrane in the microvilli region, the dilitation of lateral intercellular spaces below tight junction, the condensation of actin near tight and intermediate junctions. The presence of these changes and almost absolute absence of destructions in tight junctions organization indicate that the main pathway of the isotonic fluid containing glucose across leaky epithelium of rat small intestine is a transcellular one.

[The ultrastructural characteristics of the epithelial cells in the frog bladder under the action of vasopressin and in a vasopressin-independent increase in permeability for water]. / Osobennosti ul'trastruktury kletok épiteliia mochevogo puzyria liagushki pri deistvii vazopressina i pri vazopressin-nezavisimom uvelichenii pronitsaemosti dlia vody.

In experiments on isolated frog urinary bladders it has been found that the low basal level of water permeability in the absence of arginine-vasopressin (AVP) could be significantly increased when the serosal solution was changed several times every 15 min for a fresh Ringer solution. The electron microscopic study of these cells by the freeze-fracture technique showed that the enhancement of water permeability by AVP-independent manner was related to the appearance of intramembranous particle aggregates in luminal membrane of granular cell, that are usually observed only under the action of AVP. The immunocytochemical experiments with monoclonal antibodies against actin revealed the similarity in intracellular actin distribution under the action of AVP and AVP-independent increase of water permeability.

[An ultrastructural study of the apical cytoskeleton of the epithelial cells in the frog bladder with an ADH-dependent and an ADH-independent increase in osmotic permeability]. / Issledovanie ul'trastruktury apikal'nogo tsitoskeleta kletok épiteliia mochevogo puzyria liagushki pri ADG-zavisimom i ADG-nezavisimom uvelichenii osmoticheskoi pronitsaemosti.

Immunocytochemical methods of electron and confocal microscopy were applied for studying the primembrane actin cytoskeleton in the frog urinary bladder granular cells, following the two actions: under the increased vasopressin-induced water permeability, and following autacoid removal by multiple changes of the Ringer solution around the serosa. In both cases similar changes have been revealed in the structure of the apical cytoskeleton and, in addition, a decrease in the density of its actin filament distribution was noticed.