The effect of transforming growth factor alpha (TGFt) on the expression of imprinted Igf2 and Peg1/Mest genes was studied in diploid parthenogenetic embryos (PEs) of (CBA x C57BL/6)F1 mice during the postimplantation period of embryogenesis. The PEs were treated with TGFalpha in vitro at the morula stage and, after they developed to the blastocyst stage, were implanted into the uterus of false-pregnant females. On the tenth day of pregnancy, the PEs were explanted for subsequent in vitro culturing for 24 or 48 h. The expression of the imprinted Igf2 and Peg1/Mest genes was studied by means of whole mount in situ hybridization using digoxigenin-labeled antisense RNAs. The expression of the imprinted Igf2 and Peg1/Mest genes was studied in embryos on the tenth day of in utero development before culturing and after 24 and 48 h of culturing in vitro. The expression of Igf2 before culturing was detected only in the brain of 60% of PEs on the tents day of pregnancy (the 21-to 25-somite stages); while the Peg1/Mest expression was not detected at all. In control (not treated with TGFalpha) PEs, neither gene was expressed at the same 21- to 25-somite stages. After 24 h of culturing, the Igf2 expression was detected in the brain of 71% of PEs at the 30- to 35-somite stages, while the Peg1/Mest expression was not detected. In control (untreated) PEs, neither imprinted gene was expressed at the 30- to 35-somite stage. After 48 h of culturing, Igf2 was expressed in the regions of the brain, developing jaws, heart, liver, and somites of all TGFalpha-treated PEs at the 40- to 45-somite stages; and Peg1/Mest was expressed in the brain, heart, and liver of these embryos. In control (untreated) PEs, neither Igf2 nor Peg1/Mest was expressed at these stages The expression patterns of the imprinted Igf2 and Peg1/Mest genes in PEs at the most advanced developmental stages (40-45 somites) and in normal (fertilized) embryos at the same stages were similar; however, their expression rate in PEs was substantially lower than in normal embryos. These data indicate that exogenous TGFalpha can reactivate the expression of the two imprinted genes, modulating the effects of genomic imprinting in such a way that the PE development is improved and substantially prolonged.

The study addresses the control of plant cell division and differentiation using the model of tumor-forming lines of radish. Expression of the genes involved in control of the cell cycle (CycD3), maintenance of meristematic cell activity (STM, WUS, and KNAT1), and primary response to cytokinin (ARR) was studied in inbred radish lines characterized by tumor growth at different stages of development. The influence of exogenic cytokinin on the expression of the genes of interest is analyzed. The possible role of the CycD3, KNAT1, STM, WUS, and ARR5 in tumor formation in radish is discussed.

Prolactin (PRL) is one of the pituitary hormones participating in the control of mammalian folliculo- and oogenesis. In the present study, the joint effect of PRL (50 ng/ml) and dibutyryl cAMP (dbcAMP, 1 mM) on oocyte maturation and the morphologic-functional state of surrounding cumulus cells was investigated in vitro. It has been shown that PRL totally suppresses the braking impact of dbcAMP on meiosis reinitiation and the completion of oocyte nuclear maturation. Furthermore, PRL partly inhibited cumulus expansion induced by dbcAMP, although it exerted the opposite effect in the control medium. In the presence of PRL, the inhibitory impact of dbcAMP on the proliferative activity of cumulus cells and on the PRL-elicited braking of destructive processes in the cells has been found. In cumulus cells, mRNA expression of PRL receptor long isoform was revealed by the RT-PCR method. The data obtained suggest an interaction of signal cascades induced by PRL and cAMP in bovine oocyte-cumulus complexes, with the coupling site of these cascades in oocytes being apparently different from that in cumulus cells.

HSV-1 ICP27, an immediate early protein of herpes simplex virus type 1, is involved in viral replication, transcriptional activation, RNA stability, apoptosis, and reactivation from viral latency. The reactivation from viral latency is closely related to apoptosis and oxidative stress. To understand the effect of ICP27 upon apoptosis, we tested for cell death of ICP27-expressing cells (3-3) in the presence of hydrogen peroxide. Under oxidative stress, 3-3 cells were sensitive to death, displaying typical apoptosis features such as oligonucleosomal DNA fragmentation, chromatin condensation, and G0/G1 accumulation. To dissect the sensitizing mechanism of ICP27 in apoptosis, we analyzed the level of intracellular reactive oxygen species (ROS) in 3-3 cells. We found that 3-3 cells exhibited increased ROS levels compared to Vero cells devoid of ICP27. We also observed that the increased levels of intracellular ROS in 3-3 cells were caused by disturbances in antioxidant enzymes. In 3-3 cells, the elevated ROS increased the AP-1 activity, subsequently the expression of Bax increased, and the expression of Bcl2 was reduced. In addition, 3-3 cells were sensitive to various apoptotic agents. Taken together, these results indicate that HSV-I ICP27 sensitizes the cells to apoptosis by elevating the intracellular levels of ROS.

Hypoxia is a common environmental stress that influences signaling pathways and cells function, which through initiating intracellular signaling pathways and hence leading to the activation of the transcription factor hypoxia-inducible factor-1 (HIF-1). In this study, we initially confirm that hypoxia activates HIF-1alpha protein expression in a time-dependent manner with a maximum reached at 60 min in vitro and 4h in vivo in gastric mucosa epithelial cells. The expression of HIF-1alpha is correlated with the activation of HIF-1 DNA binding and transcriptional activity. Hypoxia dose not affect HIF-1alpha mRNA transcription but regulates HIF-1alpha protein expression through a translation-dependent pathway to regulate protein synthesis. Hypoxia could induce phosphorylation of Akt, MAPK (ERK), and target of p70S6K1. PI3K and MAPK inhibitor, LY294002 and U0126 could inhibit hypoxia-induced HIF-1 and VEGF expression. We also investigated the role of reactive oxygen species (ROS) involved in HIF-1 and VEGF expression Exogenous addition of H2O2 was sufficient to activate Akt and ERK, scavengers of H2O2 significantly inhibited hypoxia-induced Akt and ERK, and subsequent HIF-lax expression and transcriptional activity. In conclusion, our data suggested that hypoxia- PI3K signaling through Akt and ERK kinases regulated ROS-dependent, hypoxia- induced HIF-1 activation and VEGF expression in gastric mucosa epithelial cells.

We studied the effect of hydrogen peroxide on morphological characteristics and resistance of common wheat calluses (Triticum aestivum L.) to Tilletia caries Till. The induction of the defense response and morphogenesis in calluses depended on H2O2 concentration. A correlation was revealed between the elevated concentration of hydrogen peroxide in wheat calluses and high activity of oxalate oxidase in the cell wall. Administration of H2O2 into the callus culture medium was followed by rhizogenesis, induced the formation of dense regions, and inhibited fungal growth on calluses. Hydrogen peroxide at high concentrations was less potent in inhibiting the growth of fungi. A relationship was found between oxalate oxidase activity, H2O2 concentration, and morphogenetic and defense responses of calluses induced by exogenous hydrogen peroxide. These data suggest that the induction of H2O2 generation is one of the approaches to increase callus resistance.

It was shown earlier, that ginseng embryogenic cell culture 2c3 was obtained as a result of callus cells transformation with the Agrobacterium rhizogenes rolC oncogene. In the present report we determine that inhibitors of Ca2+-channels (LaCl3, verapamil, niflumic acid) certainly lowered the quantity of somatic embryos in the 2c3 cell culture. This is the evidence of the influence of calcium-dependent signal system on plant embryogenesis. Protein kinases inhibitors W7 and H7 also caused the lowering of somatic embryos quantity in the 2c3 cell culture. We analysed changes of CDPK genes expression in embryogenic 2c3 cell culture. Total expression decreased 1.2-1.5 times comparing with the control callus culture. CDPK expression in the 2c3 embryogenic culture lowered by the inhibition of expression of the gene subfamilies PgCDPK1 (PgCDPK1a and PgCDPK1b) and PgCDPK3 (PgCDPK3a). At the same time, expression of PgCDPK2 gene subfamily (PgCDPK2b and PgCDPK2d) was increased. We suppose that genes of PgCDPK2 subfamily might be responsible for the embryogenesis initiation in the 2c3 ginseng cell culture. It was shown for the first time that the rolC gene and the process of embryogenesis could change expression of particular forms of CDPK genes.

To investigate the effect of E-64d, a selective inhibitor of calpain, on the expression of calpain and calpastatin in rat retina subject to ischemia/reperfusion injury (IRI). An animal model of retinal IRI was set up by increasing the intraocular pressure (110 mmHg) of a rat eye for 1 h. The retinal thickness and morphologic changes were detected by histology. The protein expression of m-calpain (a calpain isoform) in the retina was assessed by immunohistochemistry and Western blot assay. The mRNA of m-calpain as well as calpastatin (an endogenous protein inhibitor of calpain) in the retina was assessed by RT-PCR, and the ratio of m-calpain/calpastatin was then calculated. To evaluate the effect of E-64d on the expression of calpain, the drug (5 microl of 100 microM) was injected intravitreously immediately after IRI. There were retinal edematous changes, particularly in the inner plexiform layer after IRI. The protein expression of m-calpain in the retina was increased 24h after IRI, an effect that was inhibited by E-64d (P < 0.05). The mRNA expression of m-calpain and calpastatin was also increased 24 h and 3 h after IRI, respectively. Neither m-calpain nor calpastatin mRNA expression was influenced by E-64d (P > 0.05). The mRNA ratio of m-calpain to calpastatin was increased at the 6 h, 24 h and 72 h after IRI, and only at 24 h the increase of the ratio of m-calpain to calpastatin was inhibited by E-64d (P < 0.05). In the rat retina of IRI, E-64d inhibits the increase of m-calpain protein expression, as well as the mRNA ratio increase of m-calpain to calpastatin. E-64d also inhibited the retinal damage induced by IRI, suggesting a role for E-64d in the protection of the retinal apoptosis induced by IRI.

In 2005 we have described in exponentially growing E. coli cells a new fundamental genetic phenomenon,--quasi-adaptive response to alkylating compounds (quasi-Ada). Phenotypic expression of quasi-Ada is similar to the true Ada response. However, in contrast to the letter, it develops in the course of pretreatment of the cells by a sublethal dose of nonalkylating agent, an NO-containing dinitrosyl iron complex with glutathione (DNICglu). To reveal the mechanisms of quasi-adaptation and its association with the function of the Ada regulatory protein, here we used a unique property of dual gene expression regulation of aidB1 gene, a part of the Ada-regulon, namely its relative independence from Ada protein in anaerobic conditions. Based on the results of aidB1 gene expression analysis an EPR spectra of E. coli MV2176 cells (aidB1::lacZ) in aerobic and anaerobic conditions after the corresponding treatments, we conclude that the function and the spatial structure of meAda and [(Cys-)2Fe+(NO+)2]Ada are identical and thus the nitrosylated protein represents a regulator of the Ada regulon gene expression during quasi-adaptation development.

The overexpression of a new cytokine-induced apoptosis inhibitor 1 (CIAPIN1) gene has been shown previously to promote a multidrug resistant phenotype in gastric cancer cells through the upregulation of MDR1 and MRP1. In the present study, we constructed the siRNA eukaryotic expression vectors of CIAPIN1 and transfected them into SGC7901/VCR cells to examine whether the down regulation of CIAPIN1 increased cell sensitivity towards chemotherapeutic drugs. After transfection, the expression of CIAPIN1 was dramatically decreased in CIAPIN1 siRNA transfectants compared with that in parental cells and empty vector control cells. The down-regulation of CIAPIN1 significantly enhanced the sensitivity of SGC7901/VCR cells to vincristine (VCR), adriamycin (ADR) and etoposide (VP-16), but not to 5-fluorouracil (5-FU) and cisplatin (CDDP). Cell capacity to efflux adriamycin decreased markedly in CIAPIN1 siRNA transfectants, and correlation between CIAPIN1 down regulation and decreased MDR1 transcriptional activity were observed. CIAPIN1 siRNA could significantly down regulate the expression of Bcl-2, and up-regulate the expression of Bax, but not alter the expression of PTEN in gastric cancer cells. These observations suggested that the siRNA constructs of CIAPIN1 we obtained could effectively down-regulate the expression of CIAPIN1 and reverse the resistant phenotype of gastric cancer cells. The further study of the biological functions of CIAPIN1 may be helpful for understanding the mechanisms of multidrug resistance of gastric cancer and developing possible strategies to treat gastric cancer.

The effects of the preparation Furolan, (2-furyl-2)-1,3-dioxolane, on the degree of mRNA polyadenylation and the pattern of protein synthesis in the ripening grain of several soft winter wheat (Triticum aestivum L.) cultivars were studied. It was demonstrated that Furolan stabilized mRNA in a cultivar-specific manner, thereby accelerating to various degrees the biochemical processes taking place in the ripening grain. Of the wheat cultivars studied, Krasnodarskaya 99 was the most responsive cultivar with respect to a set of changes in nucleic-protein metabolism; the cultivar Deya was next followed by the cultivar Bat'ko. The cultivar Kroshka did not respond to the treatment with Furolan. The cultivar specificity of this preparation allows its practical application to be optimized.

The effect of salicylic acid (SA) on oxalate oxidase and peroxidase activities and hydrogen peroxide (H2O2) production in leaf cells has been studied in wheat of the susceptible cultivar Zhnitsa infected by the fungus Septoria nodorum, a pathogen of wheat leaf blotch. The results show that fungal hyphae spread into interstices between mesophyll cells and that infected tissues contain H2O2. Treatment with SA results in enhanced H2O2 production in mesophyll cells, which is due to activation of oxalate oxidase and peroxidase in the cell wall. It is proposed that the modulating effect of SA on oxide reductase activities is involved in the induction of protective response to fungal infection in wheat plants.

The effect of cationic microbial ribonuclease from Bacillus intermedius (binase) on normal precursors of myeloid cells of FDC-P1 mice and kit-transformed precursors expressing the receptor of the growth factor of stem cells has been studied by flow-through cytometry. Selective apoptogenic properties of binase toward kit-transformed cells were revealed. Viable kit-transformed cells responded to binase by an increase in the concentration of cytosolic calcium. The content of calcium in the cytosol of both cell types in which apoptosis was induced by binase decreased in a dose-dependent manner. The death of cells was not accompanied by a substantial decrease in the content of intracellular RNA. A possible mechanism of binase-induced effects, which involves changes in the expression of genes due to the interference of exogenous RNAse into the RNA interference, was considered.

Effects of chronic intranasal administration of ACTH(4-10) analog Semax (MEHFPGP) on exploratory activity, anxiety level, and depression-like behaviour were studied in white rats. The peptide was injected daily in dose 0.05 mg/kg during 10 or 14 days. It was shown that chronic Semax administration at 1-2 weeks induced anxiolytic and antidepressant effects but did not influenced the exploratory activity in non-stressogenic environment. The Semax effects may be the results of activation of the brain serotoninergic system as well as increased BDNF expression in the rat hippocampus.

The studies, conducted on mice and rats with experimentally obtained hypercholesterolemia. Revealed that hepatotropic means of animal and plant origin--hepathosan, entherosan, karsil and alcohol, rich in biologically active substances, make for acceleration of cholesteroles hydroxylation in liver and increase of Hmger genes expression level up to planned indices. It was determined that hepathosan has the strongest modulating effect among hepatotropic means.

Effect of vasopressin on the expression of Hyal-1 and Hyal-2 genes in different functional zones of Wistar and homozygous vasopressin-defficient Brattlboro rat kidneys was analysed using RT-PCR mehod. It was found that, in Wistar rats the content of Hyal-1 mRNA was higher in the medulla than in other kidney zones at the normal water and food regimen. The level of Hyal-1 mRNA in the cortex and the medulla of Brattlboro rat kidney exceeded that of papilla. There were no significant differences in the Hyal-2 mRNA content detected between functional zones of Wistar and Brattlboro rat kidneys. The treatment by dDAVP, the agonist of V2 vasopressin receptor (Desmopressin, 10 microg/100 g b.w.i.p. twice a day for two days) induced an increase in urine osmolality and significant increase in the Hyal-1 and Hyal-2 mRNA content in the medulla without changes in the cortex and papilla. The effect was more pronounced in Brattlboro rat kidney. These results demonstrate that, in control conditions, genes encoding Hyal-1 and Hyal-2 were expressed independently in all functional kidney zones in the both in normal Wistar and in vasopressin-defficient Brattlboro rats. Desmopressin (dDAVP) exerts a stimulating effect on Hyal-1 and Hyal-2 gene expression in the medulla.

To quantitatively determine minimal residual disease (MRD) by real-time polymerase chain reaction (PCR) in patients with a chronic phase (CP) of chronic myeloid leukemia (CML).

The objective of this work was to perform a parallel analysis of activation of the rat anterior hypothalamus cells as judged by c-Fos protein expression, and of the expression of interleukin-2 (IL-2) under different influences, i. e., mild stress (handling) and adaptation to it, and intranasal administration of saline and the peptides Vilon (Lys-Glu) and Epithalon (Ala-Glu-Asp-Gly). Changes in the counts of cells positive for c-Fos- and IL-2 proteins were studied in structures of the lateral (LHA) area, anterior (AHN), supraoptic (SO) and paraventricular (PVH) nuclei of Wistar rat hypothalamus. Quantity of the interleukin-2-positive and c-Fos-positive cells was calculated. The findings were: a negative correlation between the activation of cells and the amount of IL-2 in the cells in the hypothalamic structures under study, and the specific patterns of changes in the counts of cells positive for c-Fos and IL-2 under stress and adaptation to stress.

A new approach for the evaluation of oxidizability of proteins and lipids in the same sample of blood plasma was proposed. We tested a method for evaluation of metal-catalyzed oxidation of fibrinogen by the formation of bityrosine cross-links during oxidation detected by the increase in fluorescence at 415 nm. A correlation was revealed between parameters of oxidizability estimated by this marker and carbonyl derivatives (dinitrophenylhydrazine method). Oxidizability of total proteins from whole plasma was compared with oxidizability of plasma lipids (marker malonic dialdehyde). Study of these parameters in patients with coronary heart disease showed that the proposed experimental approach allows us to divide the sample into several subgroups differing in the resistance to oxidative stress. These data can be used for diagnostic and prognostic purposes.

Stress stimuli are known to influence the intensity if immune response. To elucidate the role of central regulating structures in this changes, analysis of activation level of hypothalamic neurons (revealed by quantity of c-Fos-positive cells) was carried out in rats within 2 hours after intravenous LPS injection and after this--impact associated with electric pain stimulation (EPS). The investigation was carried out in 52 male Wistar rats, 200-250 g. The c-Fos protein expression was analyzed with immunohistochemical method. The increase of c-Fos-positive cells number in 2 hours after LPS injection was observed in AFTN, PVH, LHA, VMH, DMH and PH. After electrical pain stimulation, the quantity of c-Fos-positive cells increased in the same structures. Combined application of electric pain stimulation and LPS injection results in diminished activation level in AHN, PVH, LHA and VMH as compared with typical response to single LPS injection without EPS. The EPS suppresses intensity of the immune response induced by injection of LPS (revealed by local hemolysis method with calculation of antibody-forming cells quantity (%) in the rat spleen). Thus the activation level changes of hypothalamic structures (AHN, PVH, LHA, PH) correlate with development of stress-induced immunosuppression, i. e. morphofunctional description of hypothalamic structures activation as revealed by pattern of activated cell alterations in hypothalamic structures during realization of stress-induced changes of immune system responses to antigen injection.