INCREASED FORMATION OF PHOSPHORYLATED H2AX FOCI IN NUCLEI OF CELLS INFECTED BY HEPATITIS B AND B+D VIRUSES.

Liver cirrhosis and hepatocellular carcinoma are the most common outcomes of chronic hepatitis B. Hepatitis B virus (HBV) induces transformation and cell death in chronic hepatitis B (CHB). DNA double strand breaks (DSBs) represent the most dangerous type of genome damage. It was shown previously that generation of phosphorylated histone H2AX foci is a reliable marker of DSBs. The aim of this study was to analyse generation of yH2AX foci in HBV and hepatitis D virus (HDV) infection in vitro and in liver biopsies of patients with CHB and CHB with delta-agent (CHD). Human hepatoma cell line HepG2-1.1merHBV with activated HBV life cycle was used to perform real-time PCR for analysis of pregenomic RNA, HBV DNA, HBV cccDNA and for immunocytochemical analysis of yH2AX. Liver biopsies from CHB and CHD patients were analyzed to confirm the results. HBV induces multiple discrete yH2AX foci in HepG2-1.1merHBV cells in vitro and in biopsies of CHB and CHB+D patients. The ratio of hepatocytes w/o yH2AX foci is significantly lower (49,9+/-12,3% vs. 85,5+/-0,9%, p.

[Overexpression of DNA-methyltransferases in persistency of cccDNA pool in chronic hepatitis B]. / Rol' giperékspressii DNK-metiltransferaz v persistentsii kol'tsevoi kovalentno zamknutoi DNK pri khronicheskom gepatite V.

AIM: To define the role of DNA-methyltransferases of type 1 and type 3A in hepatitis B viral cycle. MATERIAL AND METHODS: Human hepatoma cells HepG2 with stable expression of 1.1-mer HBV genome were transfected with vectors encoding DNA-methyltransferase 1 (DNMT1), DNA-methyltransferase 3A (DNMT3A) or were co-transfected with these vectors. Total HBV DNA copy number, relative expression of pregenomic RNA (pgRNA), S-protein-encoding RNA (S-RNA) and cccDNA were analyzed by quantitative and semi-quantitative real-time PCR-analysis with TaqMan probes for assessment of DNMTs-mediated effects on HBV. RESULTS: DNMT1 and DNMT3A suppress HBV transcription and replication, though to different magnitude. cccDNA pool is enlarged statistically significantly ≈2-fold (P<0.005) after transfection of DNMT3A, but is unaltered under DNMT1 treatment. CONCLUSION: DNMT3A regulates the size of cccDNA pool and is important for persistency of HBV infection.

[Regeneration and fibrosis of corneal tissues].

In this review, the features of the regeneration of corneal tissue and its disorders leading to the development of fibrosis are considered. The data on the presence of stem (clonogenic) cell pool in the corneal tissues (epithelium, endothelium, stroma) are given; these cells can serve as a source for regeneration of the tissues at injury or various diseases. The main steps of regeneration of corneal tissues and their disorders that lead to outstripping proliferation of myofibroblasts and secretion of extracellular matrix in the wound area and eventually cause the formation of connective tissue scar and corneal opacity are considered. Particular attention is given to the successes of translational medicine in the treatment of corneal tissue fibrosis. The methods of cell therapy aimed at the restoration of stem cell pool of corneal tissues are the most promising. Gene therapy provides more opportunities; one of its main objectives is the suppression of the myofibroblast proliferation responsible for the development of fibrosis.

[Localization of crystallins in Muellerian cells in the grass frog retina]. / Lokalizatsiia kristallinov v miullerovskikh kletkakh setchatki travianoi liagushki.

Cell localization of 23 kDa- and 35 kDa-crystallins in the retina of adult common frogs Rana temporaria L. was studied using indirect immunofluorescence. Intense specific fluorescence of both crystallins was observed all over the retina, in both periphery and central area. It was localized in elongated radially oriented cells, whose bodies were located in the inner nuclear layer. These cells gave many fluorescing processes in the same layer and main processes in the outer nuclear and ganglion layers, one in each. The processes formed a strong network of fibers around the photoreceptor and ganglion cells. Intense fluorescence was also observed in the layer of nerve fibers and adjoining inner limiting membrane. The distribution and morphology of crystalline-containing cells mostly coincides with what is known for the Muller cells of vertebrate eye. The identity of the cells we described and Muller cells was also confirmed using the antiserum to glial fibrillary acidic protein.

[The 23-kDa polypeptide from Common Frog lens: isolation, characteristics and cellular localization]. / 23 kDa-polipeptid khrustalika glaza travianoi liagushki: vydelenie, svoistva, kletochnaia lokalizatsiia.

The major water-soluble polypeptide with molecular weight of approximately 23 kDa (the 23-kDa polypeptide) was identified in the lens of common frog Rana temporaria L. According to the gel filtration data, the peptide is a part of an oligomeric protein with molecular weight of more than 300 kDa (alpha-crystallin fraction). A highly pure fraction of the 23-kDa polypeptide was isolated by two-step ion-exchange chromatography and SDS electrophoresis and the specific antibodies were obtained. Immunohistochemistry showed the presence of the 23-kDa polypeptide in the cytoplasm of lens epithelial cells (including its central region) and in the zones neighbouring the plasma membranes in cortical fibers. The 23-kDa polypeptide was not found in the lens central zone (nucleus). It was also present in the retina (in the cells of inner nuclear layers), but not in the other tissues and organs of adult frog. Immunochemical analysis showed that the 23-kDa polypeptide was different from all known crystallins of frogs and other animals (bull, mouse, rat, and chicken). The nature of the 23-kDa polypeptide and the relation of its expression with lens cell differentiation are discussed.

Developmental patterns of crystallin expression during lens fiber differentiation in amphibians.

Data on activation of crystallin synthesis during lens fiber (LF) formation in amphibians are summarized to point out the questions particularly interesting in the context of lens cell lineage-specific expression programming under different developmental conditions. LFs are known to differentiate throughout life along the same pathway that includes at least five compartments. Using the amphibian eye lens as a model, we have studied how crystallins are expressed in the course of: (1) embryonic LF formation, (2) LF differentiation in adults, and (3) LF transdifferentiation from other (non-lens) eye tissues. Our experiments showed that synthesis of crystallins during morphologically similar LF differentiation in embryonic and adult amphibian lens has different spatial-temporal patterns (i.e., is apparently activated according to different programs). Certain results obtained in our studies suggest the absence of any direct relationship between the capacity of adult newt iris cells to transdifferentiate into LFs and crystallin synthesis (<> to such transdifferentiation) in them. Crystallins appear at the advanced stages of iris transdifferentiation into the lens and dynamics of their synthesis in the <> lens resembles that in the embryonic lens, although a new lens rudiment develops from the adult iris epithelium. Data on alternative patterns of the crystallin gene activation are summarized and compared with recent observations on spatial-temporal expression of Pax genes, which play an essential role in lens cell commitment and crystallin synthesis. On this basis, it is suggested that ontogenetic and tissue- or cell-specific changes in Pax gene expression may result in altered programs for activation of crystallin genes in embryonic, adult, and regenerating lens.

[The crystalline lens as a model for studying the cellular and molecular mechanisms of differentiation in the adult organism]. / Khrustalik kak model' dlia izucheniia kletochnykh i molekuliarnykh mekhanizmov differentsirovki vo vzroslom organizme.

This review summarizes the published and authors own data about characteristics of expression of tissue-specific lens proteins (crystallins) during differentiation of lens epithelial cells into lens fibers in adult mammals, birds, reptiles, and amphibians. Information is analyzed about the presence, synthesis, and localization of different crystallins in lens epithelium and fiber cells of lens cortex and nucleus which correspond to sequential stages of lens cell differentiation. The data available suggest that morphologically similar differentiation of lens epithelial cells into fibers in different classes of vertebrates is accompanied by different programs for activation (and/or increase) of tissue-specific protein synthesis.

[The synthesis and localization of crystallins in different cell compartments of the crystalline lens in adult frogs: immunoautoradiographic and immunofluorescent research]. / Sintez i lokalizatsiia kristallinov v razlichnykh kletochnykh kompartmentakh khrustalika glaza vzroslykh liagushek: immunoavtoradiograficheskoe i immunofluorestsentnoe issledovanie.

The purpose of this study was to analyze immunochemically the synthesis and distribution of tissue-specific proteins, i.e., alpha-, beta- gamma- and rho-crystallins, in morphologically distinct regions of the frog (Rana temporaria L.) lens which consist of cells at various stages of differentiation, maturation and aging. Five such cell compartments can be distinguished in the lens: (1) central zone of lens epithelium (stem/clonogenic cells); (2) equatorial epithelial cells (differentiating cells); (3) lens fibers of the outer cortex (post-mitotic differentiated cells); (4) lens fibers of the deep cortex (cells without nuclei at terminal stage of differentiation); and (5) cells of the lens "nucleus" (cells formed during embryogenesis). Intact lenses and isolated lens epithelium were cultured in vitro in the presence of 35S-methionine. Then lens epithelium, outer and deep cortex, and lens nucleus were extracted with buffered saline and extracts used for immunoautoradiography. Distribution of crystallins in paraffin sections of the whole lens or isolated lens epithelium was studied using indirect immunofluorescence. Synthesis of alpha-crystallins was observed in lens epithelium and cortex, but not in lens nucleus. According to immunohistochemistry, these proteins were absent from central part of the lens epithelium: positive fluorescence was observed only in elongating cells at its periphery and in lens fibers. The data on beta-crystallins are similar except that synthesis of these proteins (traces) was detected also in lens nucleus. Synthesis of gamma-crystallins was detected in lens cortex and nucleus (traces) but not in epithelium. Immunohistochemistry showed that these proteins are absent from all regions of lens epithelium and found only in fiber cells of cortex and nucleus. Rho-crystallin was synthesized in all cell compartments of the adult lens, and all lens cells contained this protein. Our results show that cells of central lens epithelium do not contain alpha- beta- or gamma-crystallins (or the rate of their synthesis is insignificant). While cells are moving towards lens equator and elongating, synthesis of alpha- and beta-crystallins is activated. Gamma-crystallins are synthesized later, first in young lens fibers near lens equator. During embryonic development in amphibia, in contrast, gamma- and beta-crystallins are detected at earlier stages than alpha- and rho-crystallins (Mikhailov et al., 1988). These data suggest that different mechanisms are involved in differentiation on lens fibers from embryonic precursor cells, on one hand, and from epithelial stem cells of adult lens, on the other.

[Concanavalin-binding proteins and cytokeratins in different tissues of the early amphibian gastrula (Rana temporaria, Xenopus laevis)]. / Konkanavalin-sviazyvaiushchie belki i tsitokeratiny raznykh tkanei rannei gastruly amfibii (Rana temporaria, Xenopus laevis).

Concanavalin A (con A), a lectin which specifically interacts with aD-mannose and aD-glucose, has a neutralizing effect on the explants of the early gastrula ectoderm of several amphibian species. Consequently, it was interesting to study con A-binding protein spectrum of the ectoderm and compare it to those of other early gastrula tissues. Animal pole ectoderm (APE), dorsal blastopore lip (DBL) and vegetal pole endoderm (VPE) were dissected from early gastrulae of Rana temporaria and Xenopus laevis. The extracts were subjected to SDS-PAGE with subsequent immunoelectroblotting on nitrocellulose membranes. The blots were sequentially treated with con A solution, horseradish peroxidase and diaminobenzidine. Spectra of the con A-binding glycoproteins were similar in APE, DBL and VPE of R. temporaria. Ten-twelve fractions with the molecular weight in the range from 30 to 150 kDa were stained in each blot. Fractions with the molecular weight of 150, 125, 104, 94 and 42 kDa showed more prominent lectin binding. Con A-binding protein spectra remained unchanged after freezing-thawing of the studied extracts, as well as after blots were treated with neuraminidase or sulphuric acid in order to remove sialic acid residues; the only exception was 42 kDa fraction. At the same time, a-methyl-D-mannoside pyranoside completely blocked con A binding by fractions of the studied extracts. In histological sections of R. temporaria early gastrula, all cells bound FITC-labelled con A. Similar data were obtained with tissues of X. laevis early gastrula. While electrophoretic pattern of X. laevis tissues drastically differed from that of R. temporaria, there were no significant differences between con A-binding protein spectra of X. laevis APE, DBL or VPE. Thus, all studied tissues of the amphibian early gastrula contain similar set of con A-binding proteins; however, only APE is capable of neutralization in response to con A action. These data favor our earlier assumption (see Mikhailov et al., 1989) that con A reception and transmission of the corresponding signal do not determine the characteristics of the target cells response. APE, DBL and VPE extracts were assayed also for the presence of a protein similar to cytokeratin No. 8 characteristic of simple epithelia of mammals. Experiments were performed using immunoelectroblotting with monoclonal antibodies (mAB) against cytokeratin No. 8 from rat colon (mAB E2 and E7 kindly supplied by Dr. G. A. Bannikov). In R. temporaria embryos, cytokeratin 8 was detected in APE, but not in DBL or VPE. In X. laevis gastrulae all the tissues studied contained this cytokeratin.

[A 35-kDa polypeptide of the crystalline lens in the common frog: its biochemical properties, tissue specificity and appearance in the developmental process]. / 35 kDa-polipeptid khrustalika glaza travianoi liagushki: biokhimicheskie svoistva, tkanevaia spetsifichnost', vozniknovenie v protsesse razvitiia.

The vertebrate lens contains so-called taxon-specific water-soluble proteins. One of them is p-crystallin with a molecular weight of 35 kDa characteristic of Ranidae family. We have identified a polypeptide with a molecular weight of 35 kDa in the eye lens of Rana temporaria which: (1) can be extracted from the lens by aqueous salt solutions, (2) has a molecular mass of 36.1 +/- 0.4 kDa (by SDS-electrophoresis) and 37 kDa (by gel filtration), (3) is heterogeneous in terms of isoelectric point (pI 6.5-8.0), (4) binds to heparin-agarose, (5) denatures in response to freezing-thawing, lyophilization and in solutions with low ionic strength. Thus, major biochemical parameters of this polypeptide differ from that of amphibian alpha, beta- and gamma-crystallins. In addition to lens, 35 kDa polypeptide was detected by immunoelectroblotting in retina, testes, liver, kidney, spleen, stomach, intestine and lungs. Its level (as percentage of water-soluble protein) is 1.1 +/- 1.4% in the lens, 1.6 +/- 0.7% in retina. 0.05% in testes and liver and 0.01% or less in other organs. Thus, despite its wide tissue distribution, 53 kDa polypeptide is expressed predominantly in lens and retina. We studied the time-course of appearance and accumulation of this polypeptide in tissues where it is expressed at high or low levels. 35 kDa polypeptide was detected for the first time during larval development: (1) in the lens (some time after the mouth opening; stages 33-34 according to Dabagian and Sleptsova, 1975), (2) in the retina (by the time of anus opening; stages 36-37), (3) in the liver (at the stage of elongated hind limb bud; stages 40-41). Definitive expression level of this protein was achieved in the lens by the beginning of metamorphosis and in the retina and liver during first months of development. Hence, during the whole period of larval development 35 kDa polypeptide content of the lens exceeds that of retina or liver. A more substantial evidence is required to confirm the identity of studied polypeptide with rho-crystallin.

[cAMP-independent protein kinase from amphibian lens: identification, organ distribution and substrates of phosphorylation]. / cAMP-nezavisimaia proteinkinaza iz khrustalikov amfibii: identifikatsiia, analiz raspredeleniia v organe i substraty fosforilirovaniia.

Eye lens extracts of the frog Rana temporaria contain a cAMP-independent protein kinase which is quantitatively adsorbed on immobilized RNA at physiological salt concentrations. The enzyme activity is maximal in the lenticular cortex, medium in the epithelium and minimal in the lens nuclei. Crude preparations of RNA-binding protein kinase from the epithelium, cortex and nuclei of the eye lens were prepared by affinity chromatography on poly(U)-Sepharose. It was found that these preparations contain no active forms of phosphatases, ATPases or proteases which may interfere with the results of phosphorylation experiments on exogenous and endogenous substrates. The protein kinase under study catalyzes the binding of phosphate groups to threonine and serine residues in casein molecules, does not phosphorylate histones and utilizes GTP alongside with ATP as phosphate donors. Heparin and RNA used at low concentrations inhibit the protein kinase activity. The data obtained allow the identification of lenticular RNA-binding protein kinase(s) as a casein kinase type II. It was shown that incubation of RNA-binding proteins from epithelium and lenticular cortex with [gamma-32P]ATP results in the label incorporation into six to seven polypeptide chains with Mr of 27-130 kDa. Poly(U) and heparin inhibit the self-phosphorylation reaction, cAMP has no stimulating effect on this process, while Ca2+ ions inhibit the self-phosphorylation of RNA-binding proteins.

[Casein kinase type II during lens fiber differentiation in amphibians]. / Kazeinovaia kinaza tipa II v protsesse differentsirovki volokon khrustalika amfibii.

cAMP-independent protein kinase activity of casein type was found in Rana temporaria eye lens. The highest activity was observed in "cortex" lens fibres, and decreased two-fold in lens epithelium. Minimum activity was found in lens "nucleus" fibres. Thus, protein kinase activity is characteristic of metabolically active differentiating lens cells. Enzyme fraction showed almost complete binding to the immobilized RNA. The enzyme was inhibited by heparine, phosphorylated casein (but not histones). It could use either ATP or GTP as a source of phosphate, and caused modification of serine and threonine residues in casein molecule. The protein kinase from lens epithelium and cortex was purified 6,000-7,000-fold and was identified as a type II casein kinase.