[The interaction between SGLT1 or GLUT2 glucose transporter and the cytoskeleton in the enterocyte as well as Caco2 cell during hexose absorption].

The distribution of cytoskeleton elements (microtubules and actin filaments) and SGLT1 or GLUT2 glucose transporter in enterocyte of rat intestine and Caco2 cell during hexose absorption has been considered. The alteration of SGLT1 and GLUT2 transporter distribution in absorptive cell of intestine villus depending on maltose concentration has been determined using the confocal microscope. The colocalization of the transporters and actin has been revealed. The increase of vesicles number close to microtubules in the apical part of cell during absorption of high hexoze concentration has been found by electron microscope. The fact together with the transporter and actin as well as actin and α-tubulin colocalizations can prove the participation of cytoskeleton elements in glucose transporter movement to apical membrane of the cells studied.

[Influence of protamine on expression of tight junction proteins in Caco-2].

Action of polycation protein protamine on the expression of tight junction proteins (claudins-1, -2, -3 and occludin) which contribute to paracellular transport function was investigated on cellular models of tight (MDCK I cell line) and leaky (Caco-2 cell line) epithelium. The expression of claudins-1,-3 and occludin was observed in both cell lines by methods of immunocytochemistry. Influence of protamine (100 microg/ml; 30 min; apical) on fluorescence intensity of claudins-1, -3 was different in MDCK I and Caco-2 cells. Addition ofprotamine to the incubation medium of Caco-2 cells resulted in significant increase of claudin-3 expression by 45 % (p <0.01) in comparison with control, whereas claudin-1 and occludin expression did not alter. On the contrary, in MDCK I cells protamine induced the significant decrease ofclaudin-1 and -3 expression by 25 % (p <0.001) and 15 % (p < 0.01) respectively, whereas occludin expression did not alter. It was confirmed by the methods of confocal laser scanning microscopy that protamine alter the expression of claudins-1, -3 directly in the tight junctions. Our results suggest that charged chyme components may alter paracellular permeability of epithelium.

[Responses of peptide hydrolases of the small and large intestines in rats on the administration of antibiotics].

Effects of antibiotics on the structure and functional state of the intestine are not clear. We investigated some structural parameters of the small and large intestine, and activities of two intestinal peptide hydrolases in rats after administration of ampicillin and metronidazole during 3 and 5 days. After 3 days of antibiotic administration a decrease in the weight of mucosa in the small intestine, accompanied with a reduction in the villous height and width in this part of the intestine, and in the weight ofmucosa in the colon occured. At the same time the number of goblet cells in the small intestinal epithelium was increased. Specific activities of aminopeptidase M, and glycyl-L-leucine dipeptidase (micromol/min per g) in the mucosa of the small intestine were increased, and the total activities (micromol/min calculated per a part of the intestine) of the same enzymes did not change. The administration of antibiotics for 5 days resulted in increase of specific activity ofaminopeptidase M in the mucosa of the proximal part of the small intestine. In the chyme of the small intestine and colon, activities of the same enzymes (micromol/min calculated per a part of the intestine) were increased on the third and fifth days of the antibiotic administration. Thus, the application ofampicillin and metronidazole within 3-5 days causes a disturbance of the structural and functional parameters in the small and large intestines, which is most pronounced on the third day of the drug administration.

[Caco 2 cell culture as intestinal epithelium model for hexose transport studying].

Distribution of SGLT1 and GLUT2 hexose transporters as well as that of fibrillar actin and tight junction proteins in cultured Caco2 cells incubated in medium with different hexose concentrations has been considered. Glucose absorption by the cells from incubation medium has been determined. Fibrillar actin was concentrated in the microvilli and closely to tight junction. The actin distribution was not dependent on the glucose concentration. There was no SGLT1 association with brush border actin and the transporter localization was not dependent on the concentration of hexose. GLUT2 was localized in the basal part of Caco2 cells after low concentration hexose load (2.5 mM). The transporter was colocalized with microvilli actin in the apical part of the cells after high concentration hexose load (25 mM). The tight junction proteins, occludin and claudin 1, 3, 4 were not dependent on glucose concentration. Claudin 2 was not detected in Caco2 cells. Caco2 cell culture can be used as a model for studying of hexose transport in small intestine epithelium.

[Comparative analysis of SGLT1 and GLUT2 transporter distribution in rat small intestine enterocytes and Caco2 cells during hexose absorption].

SGLT1 and GLUT2 hexose transporter distribution into enterocytes of small intestine isolated loop and Caco2 cell culture after absorption of high and low hexose concentrations has been considered. SGLT1 was found along intestine villus edge in isolated loop. After high concentration hexose load GLUT2 appeared to be situated in the apical parts of enterocytes. It is evident that GLUT2 participates in hexose transport across apical membrane. Cultured Caco2 cells form microvilli and cell junction complex typical for enterocyte. Glucose and galactose absorption by the cells from incubation medium has been observed. SGLT1 transporter is situated in the apical parts and around the nuclens of Caco2 cells and combined into globules. After low concentration hexose load, CLUT2 transporter is localized in the basal parts of Caco2 cells. Caco2 cell culture can be used as a model for studying of hexose transport in small intestine epithelium.

[Towards the computational three-dimensional histology. Application of computer models to the reconstruction of the three-dimensional structure of the biological tissue with the sensory cochlear acoustic epithelium in birds as an example].

This article presents new efficient method of 3-D structure reconstruction of epithelia. In contrast to existing empirical approaches, this method is based on the use of the families of topological and geometrical models of tissue structure, obtained from the axiomatic theory of biological tissue structure, with the database of model sections obtained by computer simulation. This methodology involves comparison of tissue sections obtained with those taken from the database of theoretically predicted models and the selection of the model, the sections of which best fit the real ones. This approach significantly reduces the amount of necessary tissue sections and eliminates the need for their precise superposition. As a result, new method improves resolution and and the efficiency of the studies of the spatial organization of epithelia, at the same time simplifying them. This approach is oriented primarily on examination of tissue topology as the most important aspect of its histoarchitectonics. Application of the models also facilitates the evaluation of geometrical properties of the tissue and even allows to predict their transformations, thus creating the real prerequisite for transition to the 3-D histology.

[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.

[The role of facilitated diffusion in glucose transport across the apical membrane of enterocytes].

In chronic experiments on Wistar rats, glucose and galactose absorption in the isolated loop of the small intestine considerably decreased in presence of both phloridzine am phloritine (inhibitors of the glucose transporters SGLT1 and GLUT2). The load of the isolated loop with glucose or galactose solutions scarcely influenced the absorption of 2-deoxi-D-glucose (substrate for GLUT2). According to the immunocytochemical analysis by means of confocal microscopy, after the load of the isolated loop with glucose (75 mM) the labels to GLUT2 and proteinkinase C (PKC betalI) were concentrated mainly in the apical part of the enterocytes, whereas after the load with the Ringer solution--in the basal part of the enterocytes. It was shown on the mathematical model that the part of the facilitated diffusion in the total glucose absorption was considerably lesser in comparison with the active transport mediated by SGLT1. Thus the findings support the hypothesis about a recruitment of the transporter GLUT2 into the apical membrane of the enterocytes and its involvement in glucose transfer across this membrane. However, under natural conditions, the active transport is the main mechanism of glucose absorption, whereas the facilitated diffusion plays a certain role only at high carbohydrate loads.

[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.

[The 3-dimensional organization of the sensory epithelium of the cochlea in birds]. / Trekhmernaia organizatsiia sensornogo épiteliia ulitki ptits.

Using authors' original approach to studying histoarchitecture of cellular layers three-dimensional structure of bird helix sensory epithelium was studied theoretically and experimentally. The composition of its elementary morphofunctional unit, AB3 (one sensory cell per three supporting cells) was established. Three dimensional model of this unit was worked out that described shape and arrangement of cells at apical, basal and intermediate levels of the layer. The presence of its translation symmetry was demonstrated. Comparison of theoretical model and results of morphological research of sensory epithelium allowed to conclude that the model corresponds with the real tissue. The model assists in three-dimensional reconstruction of sensory epithelium. And allows to deal with minimum number of sections and also gives an opportunity to forecast its developmental changes.

[Three-dimensional histoarchitectonics of the larval epidermis of the grass frog]. / Trekhmernaia gistoarkhitektonika épidermisa lichinki travnoi liagushki.

We have conducted theoretical and experimental study of the three-dimensional structure of epidermis in the larva of the grass frog. Three-dimensional modeling of cell shape and mutual arrangement yielded several variants of cell-to-cell connections at the apical, basal, and intermediary levels of the cell sheet. Concepts of the normal and inverted orientation of the epithelial sheet have been introduced, and arguments for the existence of translation symmetry in this sheet were presented. At least two topological variants of three-dimensional organization can be found in the larval epidermis of the grass frog. Comparison of theoretical concepts and experimental results allowed us to conclude that the models developed by us are in good agreement with actual existing patterns of the epithelial structure; these models contribute to better reconstruction of their three-dimensional structure.

[The translation symmetry in cell mosaics of amphibian embryonic ectoderm as a manifestation of their modular structure]. / Translatsionnaia simmetriia kletochnykh mozaik éktodermy zarodysha amfibii kak proiavlenie ikh modul'nogo stroeniia.

A theoretical proposition that the cell mosaics possess translation symmetry has been experimentally confirmed by scanning electron microscopy of the common frog embryonic ectoderm. This symmetry is a manifestation of the tissues modular structure and it provides new data for quantitative estimation of composition and structure of tissue morphofunctional element as well as for diagnostics and prediction of tissue development.