Transcriptional Levels of Intercellular Junction Proteins in an Alveolar Epithelial Cell Line Exposed to Irradiation or Bleomycin.

We performed a comparative study of the effects of X-ray irradiation and bleomycin on the mRNA levels of E-cadherin and tight junction proteins (claudin-3, claudin-4, claudin-18, ZO-2, and occludin) in an alveolar epithelial cell line L2. Irradiation decreased claudin-4 levels and increased occludin levels, while the levels of other mRNAs remained unchanged. Bleomycin increased the expression levels of all proteins examined except claudin-3. Irradiation and bleomycin have different effects on the expression level of intercellular junction proteins, indicating different reactions triggered in alveolar epithelial cells and a great prospects of further comparative studies.

[Prolactin-releasing peptide increases rat anxiety].

BACKGROUND: Prolactin-releasing peptide(Prl-RP), in addition to stimulating the production of prolactin, interacts with various parts of the central nervous system, participating in the implementation of many functions that are reflected in behavior. AIM: The effect of Prl-RP on the anxiety of white Wistar rats was studied since there were no data in the literature on the relationship between Prl-RP and anxiety. MATERIALS AND METHODS: Anxiety was assessed in two tests. In the elevated plus-maze (EPM), the time spent in the open arms and the number of edge reactions were recorded. In the social preference test, the time spent near a stranger, near a familiar individual, and in neutral territory were recorded. RESULTS: The administration of Prl-RP at a dose of 10-10 M with a volume of 10 µl in each nostril reduced the time spent by the animals in the open arms of the EPM, and the number of edge reactions. For testing the social interaction, animals were pre-selected for high or low levels of anxiety in the EPM. In rats with initially low levels of anxiety, Prl-RP reduced the time spent near a stranger, indicating an increase in anxiety levels. The behavior of rats with initially high levels of anxiety did not change after application of the Prl-RP. CONCLUSION: The results of our experiments indicate that the intranasal administration of Prl-RP increases the anxiety of rats.

[COMPARATIVE ANALYSIS OF TIGHT JUNCTIONS OF EPITHELIUM OF RATS JEJUNUM UNDER THE EFFECT OF LIPOPOLYSACCHARIDE AND CHOLERA TOXIN].

AIM: Comparative study of tight junctions and ultrastructure alterations of enterocytes of mucous membranes of jejunum of rats under the effect of lipopolysaccharides and cholera toxin. MATERIALS AND METHODS: Lipopolysaccharides (Sigma-Aldrich, Germany) and cholera toxin (Sigma-Aldrich, Germany) were used. The study was carried out in Wistar line rats. Effect of lipopolysaccharides and cholera toxin on epitheliocytes was carried out by a method of withdrawal of segments of rat jejunum and their incubation with the specified substances. Comparative analysis of ultrathin sections of enterocytes of jejunum of rats and tight junctions between them was carried out in control and under the effect of lipopolysaccharides and cholera toxin. RESULTS: Effect of lipopolysaccharides on ultrastructure of enterocytes of rat jejunum manifested in the change of cell form as a result of increase of intercellular space without destruction of tight junctions. Disappearance of desmosomes, increase of nuclei and more pronounced ER were noted in some epitheliocytes. Effect of cholerogen on epitheliocytes of mucous membrane of rat jejunum by a number of signs is similar to the effect of lipopolysaccharides, that manifested in an alteration of ultrastructure of cell, the form of those also transformed as a result of an increase of intercellular space, this process was not accompanied by destruction of tight junctions. Disappearance of folding of the lateral region of plasmatic membrane of cells and a reduction of a number of microvilli was observed under the effect of cholera toxin. CONCLUSION: A similar character of effect of lipopolysaccharides and cholera toxins on ultrastructure of cells and region of tight junctions of enterocytes of rat jejunum was detected, both substances caused an increase of intercellular space without the destruction of tight junctions.

Claudin expression in follicle-associated epithelium of rat Peyer's patches defines a major restriction of the paracellular pathway.

AIM: Members of the tight junction protein family of claudins have been demonstrated to specifically determine paracellular permeability of the intestinal epithelium. In small intestinal mucosa, which is generally considered to be a leaky epithelium, Peyer's patches are a primary part of the immune system. The aim of this study was to analyse the tight junctional barrier of follicle-associated epithelium covering Peyer's patches (lymphoid follicles). METHODS: Employing small intestinal tissue specimens of male Wistar rats, electrophysiological analyses including the Ussing chamber technique, marker flux measurements and one-path impedance spectroscopy were performed. Morphometry of HE-stained tissue sections was taken into account. Claudin expression and localization was analysed by immunoblotting and confocal laser scanning immunofluorescence microscopy. RESULTS: Almost twofold higher parameters of epithelial and transepithelial tissue resistance and a markedly lower permeability for the paracellular permeability markers 4 and 20 kDa FITC-dextran were detected in follicle-associated epithelium compared to neighbouring villous epithelium. Analysis of claudin expression and localization revealed a stronger expression of major sealing proteins in follicle-associated epithelium, including claudin-1, claudin-4, claudin-5 and claudin-8. Therefore, the specific expression and localization of claudins is in accordance with barrier properties of follicle-associated epithelium vs. neighbouring villous epithelium. CONCLUSION: We demonstrate that follicle-associated epithelium is specialized to ensure maximum restriction of the epithelial paracellular pathway in Peyer's patches by selective sealing of tight junctions. This results in an exclusive transcellular pathway of epithelial cells as the limiting and mandatory route for a controlled presentation of antigens to the underlying lymphocytes under physiological conditions.

[Intranasal application of a thyrotropin-releasing hormone attenuates state-anxiety of the rats].

During the experiments we investigated the influence of thyrotropin-releasing hormone (TRH) on the changes in the behaviour of white rat (Rattus norvegicus) males (n = 90), induced by light uncontrolled impact. After two-week procedure of handling male rats were tested using elevated plus-maze to determine the basal level of anxiety, locomotor and investigative activity and emotionality. A month later we investigated the influence of intranasal administration of TRH-solution (10(-10) M) in a volume of 20 mkl on the anxiety-level increase, induced by stress: short electric foot-shocks. Four hours later animals were tested in the elevated plus-maze. In vehicle-treated animals we detected the increase of anxiety and emotionality level and the decrease of locomotor and investigative activity. In contrast there was no increase of anxiety and emotionality in TRH-treated rats. As to the locomotor and investigative activity, it decreased in TRH-treated animals as well as inthe vehicle-treated. It shows the specific influence on the level of anxiety, which doesn't affect other components of stress-induced behavioral changes Present results suggest TRH as a potential anxiolitic.

[Claudins as tight junction proteins: the molecular element of paracellular transport].

Epithelium is a key structure of tissue barriers ensuring a creation of electrochemical and osmotic gradients. There are transcellular and paracellular types of transepithelium transport of molecules and ions. Epithelial layer permeability for paracellular transport as well as restriction of lateral integrative protein diffusion in a plasma membrane is determined by apical intercellular complex including tight junctions. Integrative proteins of the claudin family are basic molecular components of tight junctions. Properties of single claudins and their complexes define the differences in a degree of epithelial permeability. The subfamily of claudins forming charge- and size-selective pores provides selective paracellular diffusion. The subfamily of claudins increasing epithelial impermeability strengthens epithelial barrier features.

[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.

[Effect of cholerogen and protamine on tight junctions of rat enterocytes and colonocytes].

AIM: Study of tight junction state and ultrastructure changes of rat jejunum enterocytes and colon colonocytes under the effect of cholerogen and protamine. MATERIALS AND METHODS: Cholerogen (cholera toxin, Sigma-Aldrich, Germany) and protamine sulfate (Russia) were used in the study. The study was carried out in Wistar line rats. Effect of cholera toxin and protamine on rat intestine epitheliocytes was carried out by incubating intestine segments in the respective solutions. Ultrastructure changes caused by cholerogen and protamine in rat enterocytes and colonocytes were assessed based on ultrathin section analysis by transmission electron microscopy of the cells themselves and tight junctions between them compared with control. RESULTS: Effect of cholerogen on intestine mucous membrane epitheliocytes manifested in changes of cell ultrastructure, the form of which transformed as a result of increase of intercellular space without the destruction of tight junctions. Disappearance of cell plasma membrane lateral area folding and decrease of number of microvilli was noted. Enlargement of nuclei was noted only in individual cells. Effect of protamine on epithelial cell layer ultrastructure differed significantly from the effect of cholerogen. Increase of cell plasma membrane lateral area folding and significant enlargement of nuclei that moved to the central part of cells reaching its apical end were characteristic effects for protamine. Surface of a part of epitheliocytes lost microvilli with simultaneous destruction of tight junction structure. Protamine induced increase of folding only in colon without affecting jejunum. At the same time both of these substances caused increase of intercellular space in jejenum and colon epithelium. CONCLUSION: Differences in ultrathin structure of rat small intestine and colon epitheliocyte tight junctions under the effect of cholerogen and protamine were revealed.

[Barrier properties and tight junction protein expression along the longitudinal axis of rat intestine].

The tight junction (TJ) protein family of claudins is a major determinant of barrier properties in a wide variety of epithelia. Aim of the study was to compare epithelial barrier properties with the presence of TJ proteins in exactly defined intestinal segments. Transepithelial resistance (R(t)) of duodenum, jejunum, ileum and colon tissue preparations was measured in Ussing chambers. In parallel, expression of TJ proteins was analyzed by Western blots. Colon was characterized by higher R(t) than more proximal segments. However, among the small intestinal segments, R(t) was highest in duodenum and lowest in ileum. Along the intestine different claudins were detected by Western blotting with different signal intensities. Colon showed strongest signals for sealing claudins in accordance with R(t), whereas predominant expression of permeability-mediating claudins was observed in small intestinal segments. Along the intestine, claudins show a marked segment-specific expression which is in accordance with respective barrier properties.

Effect of interleukin-1ß on the expression of tight junction proteins in the culture of HaCaT keratinocytes.

We studied the effect of IL-1ß on the expression of tight junction proteins (occludin and claudins) in cultured HaCaT keratinocytes and changes of transepithelial resistance. Addition of IL-1ß had little effect on transepithelial resistance, increased the expression of claudin-1, and did not modify the expression of occludin. In other tissues, IL-1ß also increases claudin-1 expression, but significantly decreases occludin expression. These changes are accompanied by the reduction of transepithelial resistance. The IL-1ß-induced increase in the expression of claudin-1 in cultured HaCaT keratinocytes simulates the appearance of claudin-1 at the early stage of skin wound healing. It is accompanied by an increase in IL-1ß concentration in the wound fluid.

[The tight junction proteins expression in the epithelium of the small intestines in a rat].

Proteins' expression of tight junction proteins in rat small intestine was analyzed by means of immunohistochemistry and Western blotting. Claudins -1, -2, -3, -4, -5, -7 and occludin were analyzed. Confocal laser-scanning microscopy revealed differences in the subcellular distributions of these proteins. Claudins-1, -2, -3, -4, -5, and -7 were localized within the tight junction complex and were also detectable in subjunctional regions. Distributed subcellular localization of these proteins may reflect their different roles during formation of epithelial cell layers as well as in paracellular transport of ions in the small intestine.

Interaction of ceruloplasmin, lactoferrin, and myeloperoxidase.

When lactoferrin (LF) and myeloperoxidase (MPO) are added to ceruloplasmin (CP), a CP-LF-MPO triple complex forms. The complex is formed under physiological conditions, but also in the course of SDS-free PAGE. Polyclonal antibodies to both LF and MPO displace the respective proteins from the CP-LF-MPO complex. Similar replacement is performed by a PACAP38 fragment (amino acids 29-38) and protamine that bind to CP. Interaction of LF and MPO with CP-Sepharose is blocked at ionic strength above 0.3 M NaCl and at pH below 4.1 (LF) and 3.9 (MPO). Two peptides (amino acids 50-109 and 929-1012) were isolated by affinity chromatography from a preparation of CP after its spontaneous proteolytic cleavage. These peptides are able to displace CP from its complexes with LF and MPO. Both human and canine MPO could form a complex when mixed with CP from seven mammalian species. Upon intravenous injection of human MPO into rats, the rat CP-human MPO complex could be detected in plasma. Patients with inflammation were examined and CP-LF, CP-MPO, and CP-LF-MPO complexes were revealed in 80 samples of blood serum and in nine exudates from purulent foci. These complexes were also found in 45 samples of serum and pleural fluid obtained from patients with pleurisies of various etiology.

[A new method of monitoring the psycho-emotional condition in patients during balanced anesthesia based on regional blockades]. / Novyi metod monitoringa psikhoémotsional'nogo sostoianiia patsientov vo vremia sbalansirovannoi anestezii na osnove regionarnykh blokad.

The potentialities of a skin-galvanic reaction (SGR) were studied, to evaluate the psycho-emotional status, in 9 sedated (midazole, thiopental-sodium, propofol) classes I and II ASA patients operated (10 interventions) under regional block. An original software was designed to analyze the SGR; it detects the signs of psycho-emotional discomfort (including positional discomfort) in drug-induced depression of consciousness as well as the uncontrollable moments of falling-asleep and awakening.

[Inclusion of H3-leucine into the mouse mammary gland tissue within various intervals following offspring suckling]. / Vkliuchenie H3-leitsina v tkan' molochnoi zhelezy myshei cherez razlichnye intervaly posle prekrashcheniia kormleniia potomstva.

In lactating mice, intensity of synthetic processes within different intervals following the suckling cessation, was studied. The study has shown that increasing of the interval following the suckling cessation and the related accumulation of secrete in the alveolas cavities entails diminishing of the H3-leucine inclusion into the tissue and reflects a reduction in intensity of the synthesis processes of the de novo protein molecules in the mammary gland.

[Effect of prostaglandin F2alpha on cells of the mammary gland alveoli in mice]. / Issledovanie vliiania prostaglandina F2al'fa na kletki al'veol molochnoi zhelezy myshei.

In studies on lactating laboratory mice an influence of prostaglandin F2 alpha on the value of transepithelial potential difference and resistance of the alveolar secretory epithelium in the mammary gland, was studied. Prostaglandin F2 alpha did not affect initial level of transepithelial potential difference and resistance in alveoles. In all experiments with the preliminary application of prostaglandin F2 alpha in different concentrations (1 x 10(-5)-1 x 10(-11) M) was registered a reliable increase in an amplitude (before 31 +/- 2%) and duration (before 43 +/- 3%) to return reactions on oxytocin. Prostaglandin F2 alpha caused a reduction of transepithelial resistance in the alveolar secretory epithelium of first phase to return reaction on oxytocin on 28 +/- 22%. The data obtained indicate a possibility of participation of prostaglandin F2 alpha in development of certain stages of shaping a composition of milk.