[PRODUCT OF THE BMI1--A KEY COMPONENT OF POLYCOMB--POSITIVELY REGULATES ADIPOCYTE DIFFERENTIATION OF MOUSE MESENCHYMAL STEM CELLS].

Bmil is a key component of Polycomb (PcG), which in mammals controls the basic functions of mammalian somatic stem cells (SSC) such as self-renewal and differentiation. Bmi1 supports SSC via transcriptional suppression of genes associated with cell cycle and differentiation. The most studied target genes of Bmi1 are the genes of Ink4 locus, CdkI p16(Ink4a) and p1(Arf), suppression of which due to activating mutations of the BMI1 results in formation of cancer stem cells (CSC) and carcinomas in various tissues. In contrast, inactivation of BMI1 results in cell cycle arrest and cell senescence. Although clinical phenomena of hypo- and hyperactivation of BMI1 are well known, its targets and mechanisms of regulation of tissue specific SSC are still obscure. The goal of this study was to evaluate the regulatory role of BMI1 in adipocyte differentiation (AD) of mouse mesenchymal stem cells (MSC). Induction of AD in mouse MSC of the C3H10T1/2 cell line was associated with an increase in the expression levels of BMI1, the genes of pRb family (RB, p130) and demethylase UTX, but not methyltransferase EZH2, whose products regulate the methylation levels of H3K27. It was observed earlier that H3K27me3 may play the role of the epigenetic switch by promoting AD of human MSC via activating expression of the PPARγ2, the master gene of AD (Hemming et al., 2014). Here we show that inactivation of BMI1 using specific siRNA slows and decreases the levels of AD, but does not abolish it. This is associated with a complete inhibition of the expression of adipogenic marker genes--PPARγ2, ADIPOQ and a decrease in the expression of RB, p130, but not UTX. The results obtained give evidence that the epigenetic mechanism regulating AD differentiation in mouse and human MSC is different.

[Threshold rat neonatal cardiomyocyte response to gradual cryptosporidial infection severity increase].

Infectious gastroenteritis is one of the common causes of tachyarrythmia, malabsorbtion and growth retardation in children. Our recent studies have indicated that neonatal.cryptosporidial gastroenteritis is associated with long-term cardiomyocyte abnormalities. The aim of the present study was to find out how neonatal cryptosporidiosis of various severities affects cardiac anatomy and cardiomyocyte polyploidization, remodeling and HIF-1α expression. Using real-time PCR, cytometry, immunohistochemistry, image analysis and interatrial septum visual examination, we revealed that gradual increase in cryptosporidial invasion was associated with threshold changes. At weak parasitic infection, interatrial septum was entire and there was no statistically significant change in cardiomyocytes. At moderate and severe infection, all changes in cardiac anatomy and cardiomyocytes were statistically significant and demonstrated approximately similar degree. Compared to control, heart were atrophied and elongated, interatrial septum contained a small window (patentforamrn ovale), and cardiomyocytes lost protein, became elongated, thin and accumulated additional genomes. Also we found HIF-1α mRNA hyperexpression. Notable, the threshold response to gradual stimulus is an important criterion of development programming since such a response is commonly a consequence of abnormal anatomic structure formation and cell differentiation failure. Our results can be interesting for physicians because they indicate that even moderate cryptosporidiosis can be dangerous for neonatal heart and can trigger neonatal programming of cardiovascular pathology. Also, our results for the first time demonstrate the association between gastroenteritis, patent foramen ovale and cardiomyocyte malfunction.

[Changes in the heart of neonatal rats after cryptosporidial gastroenteritis of different degrees of severity].

Disturbances at the childhood age increase risk of the appearance of cardiovascular diseases decades later. The nature of this interconnection called ontogenetic programming is not completely understood. Valuable sources of knowledge about mechanisms of ontogenetic programming are data of interspecies study of biology of the body life cycles and of heart physiological capabilities. Taken into account the interspecies differences, these data allow finding the correct direction of experimental investigations. Results of studies of almost 100 homoiothermal species have shown the slow growth and a high loading on the heart at postnatal development to decrease its aerobic capability in adults. Basing on these data, we suggested that the neonatal gastroenteritis causing tachyarrhythmia, malabsorption, and the growth deceleration might lead to pathological changes in the heart. Our task was to evaluate the effect of cryptosporidial gastroenteritis of different degrees of severity on heart of the neonatal rats. By using methods of Real-Time PCR, immunocytochemistry, image analysis, and study of interatrial septum, we have established that a gradual increase of intensity of infestation with Cryptosporidium parvum oocytes causes sharp changes corresponding to the "all or nothing" response. At a weak infestation the interatrial septum was close (like in control), while significant changes in expression of isoforms of heavy chains of alpha- and beta-myosin were absent. At the intermediate and severe infestation, in the interatrial septum the foramen ovale was visualized and there were observed cardiac atrophy and a strong shift of ration of expression of myosin heavy chains toward the low-velocity beta chain. Thus, by disturbing the frequency-strength ratios and causing outflow of resources from the formed heart, the neonatal gastroenteritis produces pathological changes of the organ molecular and anatomical structures. Our results can be interest to evolutionary biologists and physicians, as they show importance of knowledge of evolutionary-comparative investigations for the search for novel risk factors of heart diseases and demonstrate interconnection between gastroenteritis, pathology of interatrial septum, and a change of composition of the main contractile proteins in cardiomyocytes.

[Rat cardiomyocyte remodeling after neonatal cryptosporidiosis. II. Elongation, excessive polyploidization and HIF-1alpha overexpression].

Retrospective epidemyological studies evidence that infant diseases leave survivors with an increased susceptibility to cardiovascular diseases in later life. At the same time, the mechanisms of this link remain poorly understood. Based on medical statistics reporting that infectious gastroenteritis is the most common cause of maladies in babies, infants and children, we analysed the effects of moderate cryptosporidial gastroenteritis on the heart and ventricular cardiomyocyte remodelling in rats of the first month of life. The disease was challenged by a worldwide human protozoic pathogen Cryptosporidium parvum (Apicomplexa, Sporozoa). The main symptoms manifested in the growth retardation moderate diarrhea. Using real-time PCR, cytophotometry, confocal microscopy and image analysis, we indicated that cryptosporidiosis was associated, with the atrophy heart and the elongation, narrowing, protein content decrease and hyperpolyploidization of cardiomyocytes and the moderate overexpression of hypoxia inducible factor 1alpha (HIF-1alpha) mRNA. Cardiomyocyte shape remodeling and heart atrophy presented in all age groups. The severity of these changes, hovewer, declined gradually from younger to older groups. In contrast, hyperpolyploidization and HIF-1alpha mRNA overexpression were registered mainly among animals aged between 6 and 13 days, and were barely detected and non-significant in older age groups. In the rat the time period covering 6-13 days after birth is known to coincide with the intensive cardiomyocyte polyploidization and the switch from proliferation to hypertrophy. Thus, our data indicate that neonatal cryptosporidiosis may be potential cardiovascular diseases risk factor and that one of the critical time windows for the growing heart covers the time period when cardiomyocyte undergo polyploidization.

[Phosphorylation of histone H2AX in human lymphocytes as a possible marker of effective cellular response to ionizing radiation].

DNA double-strand breaks (DSBs) which occurs in cells after ionizing radiation (IR) or chemical agents are the most dangerous lesions in eukariotic cells, which leads to cell death or chromosome abberations and cancer. One of the earliest response of cells to DSBs formation is phosphorylation by 139 serine of core variant histone H2AX in megabase chromatin domains around DSB (gamma-H2AX), which amplify signal and makes it possible to identify even one DSB in genome. Effective formation of gamma-H2AX is very important for maintenance of genome stability. Here, using immunofluorescent and Western blotting techniques, we studied dynamics of gamma-H2AX formation in human lymphocytes of various individuals irradiated ex vivo. We have found that dynamics of gamma-H2AX formation in lymphocytes differ between individuals but have similar kinetics and statistically is independent on people age.

[NADP increases the level of histone H2AX phosphorylation in mouse heart cells after ionizing radiation].

Phosphorylation of replaceable histone H2AX occurs in megabase chromatin domains around DNA double-strand breaks (DSBs), and this modification called gamma-H2AX can be used as an effective marker for DSBs repair and DNA damage response. Using Western blotting and immunohistochemistry techniques we have studied here the influence of exogenous nicotinamide adenine dinucleotide phosphate (NADP) which could potentially increase the intracellular level of NAD+ and on the level of gamma-H2AX formation in mouse heart cells after ionizing radiation (IR). We have found that injection of NAD+ in different doses immediately after IR causes an increased level of gamma-H2AX in mouse heart cells 20 min after IR at the dose of 3 Gy compared to control mice after IR exposure. It indicates that it could be a relationship between intracellular NAD+ content and DNA damage response in vivo.

[Cardiomyocyte myosin heavy chain composition change after cryptosporidial gastroenteritis].

Cardiovascular diseases are the most common case of human death in developed countries. Thus, the discovering of their new risk factors is of primary importance. Based on epidemiology studies, vertebrate life-history traits comparison and cross-species cardiomyocyte transcriptome analysis, we suggest that one of these factors could be infectious gastroenteritis. This disease outflows recourses from cardio-vascular system and triggers pathological stimuli, like tachyarrhythmia, inflammation, malapsorption and energy depletion thereby disturbing cardiomyocyte metabolism and function. To test this hypothesis, we challenged gastroenteritis in neonatal rats with widespread human parasite Cryptosporidium parvum (Apicomplexa, Sporozoa). The results obtained by the methods of immunocytochemistry, quantitative morphometry and real-time PCR, indicate that moderate cryptosporidiosis lasting four days induces dramatic shift in myosin isoform expression ration toward isoform beta (with low ATPase activity) both at mRNA (by 1.7-4.5 folds) and protein (by 2.5-6 folds) levels. Antithetical manner of this shift and coherence between changes in mRNA and protein suggest that cryptosporidiosis affects all main steps of a complex myosin heavy chain regulatory network. Since the overexpression of myosin heavy chain beta (showing several times lower ATPase activity than myosin heavy chain alfa) is a generally accepted marker of human cardiac failure, we can consider cryptosporidial gastroenteritis as a new risk factor of cardiac contractile ability impairment. Our data can be interesting for clinicians.

[Toxicity assessment of the vegetable emulsifier- the aqueous extract from the common soapwort roots].

Within the limits of experimental observation the assessment of acute and subacute safety of an aqueous extract from Acanthophyllum roots with a solids content of 7%, used as an emulsifier has been manufactured by production of emulsion products. Acute toxicity (LD50) AEAR, probed on mice-male; has allowed to refer to an emulsifier to a class of nontoxic substance. The assessmentof subacute toxicity of an extract (injection in a food allowance within 30 days in a recommended dose--5 ml on 100 g of a standard food allowance) was spent on laboratory rats. Extent of toxicity of a specimen judged on the general state of animals, a body mass dynamics, internals masses, to biochemical parameters of blood plasma, parameters of a peripheric blood, a morphological pattern of an internals. Comparative analysis of results allows to expel toxic effect AEAR in the recommended dose on an organism of animals.

[Hypolipidemic activity of a water extract from roots carline thistle].

In the researches which have been lead on white not purebred rats--mans on model nutritional hyperlipoproteinemia it fixed hypolipidemic activity of a water extract from roots carline thistle with the content of dry solvends of 7% which.

BM-derived cells restore expression of peroxiredoxin V in the airways following acute naphthalene injury in mice.

BACKGROUND: Naphthalene-induced respiratory tract toxicity in mice is characterized by specific and rapid loss of the Clara cell population, which is restored only after several days. The sources of restoration of this cell population remain unclear. We investigated whether BM-derived cells participated in the process of epithelial restoration following naphthalene toxicity compared with bacterial infection. We further investigated the role of BM-derived cells in restoration of expression of peroxiredoxin V (PRXV), one of the major proteins of antioxidant defense, specifically expressed in the bronchial epithelium. METHODS: We transplanted GFP-tagged BM cells into 5 Gy-irradiated C57BL/6 recipients. Following 1 month of recovery, experimental animals were subjected to 250 mg/kg naphthalene i.p. An additional group of animals received intratracheal instillation of Escherichia coli to induce acute bacterial inflammation. Animals were killed at 1-12 days after naphthalene and analyzed immunohistochemically. RESULTS: Recipients' cells of bronchial epithelium demonstrated significantly reduced levels of PRXV expression following naphthalene. In animals with acute bacterial inflammation, PRXV levels were not reduced in epithelium and participation of BM-derived cells in epithelial restoration was minimal. Following naphthalene, GFP(+) cells were present in large numbers in lung parenchyma and epithelium of conducting airways starting at 1 day following injury. GFP(+) progeny of BM cells was the major source of PRXV in the epithelium. DISCUSSION: These data suggest that BM-derived cells may provide a source of antioxidant protection of airways by expression of PRXV in a model of acute epithelial respiratory tract toxicity.

[The BCL-xL and ACR-1 genes promote differentiation and reduce apoptosis in muscle fibers of mdx mice]. / Geny BCL-xL i ACR-1 sposobstvuiut differentsirovke i umen'shaiut gibel' myshechnykh volokon myshei mdx.

The effects of the human BCL-xL and ACR-1 genes on dystrophin expression in cross-striated muscle fibers (CSMF) and on CSMF viability were studied in mdx mice after ballistic cotransfection with the human dystrophin minigene. In control mice, the proportion of dystrophin-positive (D(+)) and dying CSMF were 2.1 +/- 0.1 and 2.1 +/- 0.3%, respectively. Introduction of the dystrophin minigene (20 micrograms of the pSG5dys plasmid) increased the proportions of D(+) and dying CSMF to 5.6 +/- 1.4% and 4.5 +/- 0.9%, respectively. When pSG5dys was introduced along with the pSFFV-Neo plasmid carrying the BCL-xL gene (10 micrograms of each plasmid per shot), the death of CSMF decreased to 3.7 +/- 1% and the proportion of D(+) CSMF significantly (P < 0.05) increased to 12.2 +/- 2.2%. Contransfection with the dystrophin minigene and the BCL-xL gene at 20 micrograms of each plasmid per shot did not stimulate generation of D(+) CSMF, but did reduce the CSMF death to 1.5 +/- 0.3%. Introduction of pSG5dys along with the pRc-CMV-10.1 plasmid containing the ACR-1 gene (10 micrograms of each plasmid per shot) reduced the proportion of D(+) CSMF to 1.1 +/- 0.5% and significantly reduced the proportion of dying CSMF to 0.9 +/- 0.3% as compared with the proportions observed in intact mice or in mice subjected to transfection with pSG5dys. Introduction of the pSG5dys plasmid substantially reduced the proportion of CSMF with peripheral nuclei, suggesting disturbed CSMF differentiation. After cotransfection with the human-dystrophin minigene, the BCL-xL and ACR-1 genes did not affect the extent of CSMF differentiation as compared with that observed in the case of the dystrophin minigene alone. Thus, ballistic transfection of mdx mice with the human dystrophin gene used along with the BCL-xL or ACR-1 gene was shown to suppress the death of muscle fibers and to expedite dystrophin synthesis and cell differentiation.

Effects of Siberian ginseng extract and ipriflavone on the development of glucocorticoid-induced osteoporosis.

Siberian ginseng extract produced a protective effect during experimental steroid-induced osteoporosis, which was comparable with the influence of ipriflavone.

[Gene hACR-1 suppresses apoptosis of striated muscle fibres of m. quadriceps femoris after ballistic transfection of mdx mice]. / Gen hACR-1 podavliaet apoptoz poperechnopolosatykh myshechnykh volokon m.quadriceps femoris myshei mdx pri ballisticheskoi transfektsii.

Human minidystrophin gene (pSG5dys plasmid) and hACR-1 gene (pRc-CMV-10.1 plasmid) were cotransfected by means of "gene-gun" to M. quadriceps femoris of mdx mice. Effects of transfection on dystrophin expression and survival of striated muscle fibres (SMF) were studied on the 21st day after shots. In the control mdx dystrophin-positive muscular fibers [D(+)] SMF and destroyed SMF made 2.1 +/- 0.1 and 2.1 +/- 0.3%, respectively. In mice transfected with pSG5dys plasmid (20 mkg of DNA per mouse), the shares of D(+) SMF and dead SMF raised, respectively, up to 5.6 +/- 1.4 and 4.5 +/- 0.9%. Transfection of mice with pRc-CMV-10.1 (DNA dose is 20 mkg per mouse) reduced the levels of apoptosis in SMF and D(+) SMF level to 1.6 +/- 0.6 and 1.1 +/- 0.4%, respectively. Cotransfection by pSG5dys and pRc-CMV-10.1 plasmids (10 and 10 mkg of each plasmids DNA per mouse) reduced the share of D(+) SMF to 1.1 +/- 0.5% and SMF destruction to 0.9 +/- 0.3%. pSG5dys transfection considerably reduced the share of SMF having peripherally located nuclei, thus indicating a decrease in SMF differentiation level after transfection. Cotransfection of ACR-1 gene and a dystrophin minigene did not suppress further cytodifferentiation of mdx muscle fibers. A conclusion is made that ballistic transfection by hACR-1 gene reduces the level of apoptosis in mdx mice SMF without changing the level of SMF differentiation. The cotransfection of mdx mice muscle by hACR-1 and human minidystrophin gene reduces SMF destruction and supports SMF differentiation, too.

[Seasonal features of the effect of adaptogens on sex behavior of experimental animals]. / Sezonnye osobennosti vliianiia adaptogenov na polovoe povedenie éksperimental'nykh zhivotnykh.

Seasonal variation of the aphrodiatical activity of pantocrin (deer antler extract), ginseng root tincture, and eleuterococcus extract was studied. It was found that a one-month treatment with these adaptogen preparations in a daily dose of 100 mg/kg (for dry parent substance) stimulate the sexual behavior of puberal mice and rat males in winter. The maximum effect was observed for pantocrin. In summer months, the stimulating effect of adaptogens was not manifested. The reference drug (tentex forte) increased the sexual behavior irrespective of the season, the effect being more pronounced in summer months. These results are explained by assuming that the adaptogens studied possess a weak gonadotropic activity, which favors normalization of the testosterone level (reduced in winter season) in experimental animals.

Effect of knotweed extract on alkeran-induced changes in rat ejaculate.

We studied the effects of aqueous knotweed extracts in alkeran-induced experimental pathozoospermia. Therapeutic effect of knotweed extract in experimental cytostatic hypogonadism was demonstrated (the preparation improved spermatozoon motility).

[Nonhistone chromosome proteins HMG1 and HMG2 stabilize one of the sequence-specific complexes, formed on the promotor of human retroposons of the ALU-family of other nuclear proteins]. / Negistonovye khromatinovye belki HMG1 i HMG2 stabiliziruiut odin iz sikvensspetsificheskikh kompleksov, obrazuiushchiisia na promotore chelovecheskikh retropozonov ALU-semeistva drugim iadernym belkom.

Human retroposons of the Alu family have an internal promoter for RNA polymerase III also present in tRNA genes, but Alu are poorly transcribed by this polymerase in somatic cells in vivo, which is probably due to an efficient system of repression of the Alu transcription. The key control promoter element of the tRNA genes, known as B-box, binds basal transcription factor TFIIIC2, which initiates assembly of the full transcription complex. Previously we identified several human nuclear factors which bind to Alu Subregion covering B-box and the adjacent sequences, which can be involved in repression of Alu transcription. In this study we identified one factor, F1, as HMF1/2 and another factor, F2, as TFIIIC2-like protein reacting with B-box. Both the factors are not alu-specific, whereas the third identified Alu-binding factor, F3, seems to be specific for the Alu-specific subsequence located just downstream from B-box and can discriminate evolutionary "young" and "old" Alu subfamilies producing different numbers of transcripts in vivo. Although HMG1/2 binds to Alu in a sequence-unspecific manner, the proteins are capable of stabilizing the sequence-specific complex F3 which can be functionally significant. We believe that the factors binding to Alu B-box subregion interfere somehow with the functions of the basal factor TFIIIC2 and, hence, can be co-repressors of Alu transcription by RNA polymerase III.

A human B-box-binding protein downregulated in adenovirus 5-transformed human cells.

Internal promoters of some genes transcribed by RNA polymerase III (e.g. tRNA genes, adenovirus VA1 RNA gene, human retroposons of the Alu family) contain a conserved sequence element, B-box, interacting with basal transcription factor TFIIIC2 which initiates assembly of the full transcription complex on the genes, and which represents the major determinant of the efficiency of their expression. In this study we have identified in human nuclear extracts a protein which interacts with VA1 B-box DNA and forms a high-affinity complex which is very stable after the addition of a large excess of competitor DNA. Unlike TFIIIC2, the B-box-binding activity of the B-box-binding protein is found to be decreased in adenovirus 5-transformed human cells. In these cells (line 293) increased transcription of VA1 and tRNA genes in vivo and in vitro was previously detected by other workers. Our results suggest that besides TFIIIC2, an additional B-box-binding protein factor may be involved in the regulation of expression of the RNA polymerase III-transcribed genes.

[The DNA-binding activity of the membrane protein annexin II and its interaction with antibodies to the chromatin nuclear ribonucleoprotein (alpha-RNP)]. / DNK-sviazyvaiushchaia aktivnost' primembrannogo belka anneksina II i ego vzaimodeistvie s antitelami k iadernomu ribonukleoteinu khromatina (al'fa-RNP).

By screening with labeled Alu DNA, a clone was isolated from cDNA expression library, which appeared identical in sequence to the well-known Ca-phospholipid-binding protein annexin II. To evidence the DNA-binding activity of recombinant annexin II and its presence in the cell nucleus, we have expressed full-length mouse annexin II cDNA in bacteria with pGEMEX vector. The expressed protein was studied with electrophoretic mobility shift assay and for its reaction with polyclonal antibody to chromatin-associated ribonucleoprotein (alpha-RNP), which is one of the major acid-dissolvent components of the nucleus. The obtained results confirm the DNA-binding activity of recombinant annexin II. Annexin II reacts with polyclonal antibody to rat alpha-RNP. So, annexin II is a major nuclear DNA-binding protein in mammalian cells.

Evidence for a regulatory protein complex on RNA polymerase III promoter of human retroposons of Alu family.

Abundant human retroposons of the Alu family produce few RNA polymerase III (RPIII)-dependent transcripts in vivo. This suggests that either the bulk of the repeats has no proper promoter elements or that transcription of Alu by RPIII is repressed. In this study, we analyzed complexes formed by human nuclear proteins with the Alu B-box and with an adjacent downstream sequence (DB-sequence). Four complexes (C1-C4) were detected and two of them (C2 and C3) were found to be induced by different proteins. C3 formation was found to be sensitive to minor sequence variation within the Alu DB-sequence. The C2 complex is specifically repressed by the competing VA1 B-box oligonucleotide and was found to be very stable. In addition, it is downregulated in human cells transformed by adenovirus 5. This is consistent with a view that the C2 complex is formed by a protein (designated as ACR1) that is different from TFIIIC2. The ACR1 protein may be involved in the modulation of Alu transcription in vivo by interfering or cooperating with TFIIIC2. A similar complex is detected with the efficiently transcribed adenovirus VA1 RNA gene B-box. We compared the affinity of complexes formed by ACR1 with Alu and VA1 B-boxes. It was found that both B-boxes bind ACR1 with equal affinity with a dissociation constant of about 2 nM. However, DB-sequences in Alu and VA1 promoters are non-homologous, and C3/C4 complexes are found to be formed with Alu DB, but not formed with VA1 DB sequences. The Alu-specific protein forming C3 (named as ACR2) may cooperate with ACR1 in selective repression of RPIII-dependent Alu transcription in vivo.