Asymmetric DNA methylation between sister chromatids of metaphase chromosomes in mouse embryos upon bisphenol A action.

Earlier we showed that asymmetric methylation of sister chromatids (AMSC) was a specific characteristic of differentiation potency, and supposed that AMSC could be a useful marker of environmental impact connected with differentiation and/or dedifferentiation. Here we investigated the level of AMSC in chromosomes and the nuclei methylation in mouse preimplantation and postimplantation embryos, in comparison with the undifferentiated cells of mouse embryonal carcinoma cell line F9, and human differentiated HEK293 cells upon BPA influence. We found that exposure of mouse preimplantation embryos to BPA caused a significant decrease in the level of AMSC in chromosomes and the nuclei methylation. The BPA exposure of potentially differentiating F9 cells had no any influence on DNA methylation in nuclei but significantly decreased the number of AMSC. The level of DNA methylation and AMSC in HEK293 cells were not also changed. These data indicate that BPA exerts significant influence on differentiating and potentially differentiable cells. The most sensitive BPA targets are preimplantation embryos and stem cells.

[THE INFLUENCE OF THE PREPARATION PRETREATMENT ON IN SITU DETECTION OF 5-METHYLCYTOSINE IN METAPHASE CHROMOSOMES AND IN INTERPHASE NUCLEI].

Qualitative and quantitate analysis of DNA methylation in situ at the level of cells, chromosomes and chromosomal domains is extremely important for the diagnosis and treatment of various diseases, the study of ageing and the consequences of environmental impacts. An important question arises, whether the revealed in situ methylation pattern reflects DNA methylation per se and (or) availability of the DNA for antibodies, which in turn depends on the peculiarities of chromatin structure and chromosome condensation. These events can lead to an incorrect evaluation of the actual pattern of DNA methylation. To avoid this shortcoming as far as possible, we have modified the most widely used method of revealing 5-methylcytosine in situ with monoclonal antibodies. Here we have shown that the detection of DNA methylation staining of chromosomes including C-heterochromatin, chromosomal arms and sister chromatids is drastically dependent on pretreatment of chromosomal preparations for immunocytochemical study using fluorescent antibodies. Using undifferentiated stem cells of mouse embryonal carcinoma line F9, it has been found that change in preparations storage results in a sharp fluorescence decrease up to complete disappearance of the signal in centromeric heterochromatin. With the help of the method described in the work, we have first revealed the asymmetry of sister chromatids methylation in metaphase chromosomes of F9 cell and lymphocytes of human periphery blood. This may lead to asymmetry of transcriptional signature of daughter cells after division. The proposed here modification of 5-methylcytosine detection in situ provides a more complete characterization of methylation of chromosomes and chromosomal domains, compared to previously published methods.

[[Length polymorphism of minisatellite repeat B2-VNTR of the bradykinin B2 receptor gene in healthy Russians and in patients with coronary heart disease].

Bradykinin B2 receptor is involved in many processes, including the regulation of blood pressure and smooth muscle contraction, vasodilation, inflammation, edema, cell proliferation, pain. It is suggested that this receptor may be one of the factors that have cardioprotective and infarct-limiting effects. It is assumed that certain genetic variants in both coding and non-coding regions ofBDKRB2 gene may influence its expression. In the 3'-untranslated region of BDKRB2 exon 3 the minisatellite repeat B2-VNTR is located. B2-VNTR has previously been shown to affect the BDKRB2 mRNA stability. Therefore, it is important to perform the molecular genetic analysis of this minisatellite in patients with different forms of coronary heart disease in order to reveal possible associations between specific B2-VNTR alleles and certain clinical forms of coronary heart disease. In the present study, a comparative analysis of the allele and genotype frequencies of B2-VNTR was carried out in groups of healthy individuals and patients with two clinical forms of coronary heart disease (angina pectoris and myocardial infarction), ethnically Russian. The results of the B2-VNTR length polymorphism analysis indicate that this tandem repeat may be attributed to a class of low polymorphic and non-hypervariable minisatellite. In all analyzed groups we revealed three B2-VNTR alleles, consisting of 43, 38 and 33 repeat units. Alleles of 43 and 33 repeats were major in all investigated groups. No statistically significant differences were found in the B2-VNTR allele and genotype frequencies between men and women in control group, and also between healthy men and men with angina pectoris and myocardial infarction. Thus, B2-VNTR length polymorphism was not associated with these clinical forms of coronary heart disease in Russian men. However, we do not exclude the possibility of association between the B2-VNTR short alleles (38 and 33 repeats) and cardioprotective effects of bradykinin B2 receptor in women with coronary heart disease. This hypothesis requires further investigation.

[Human intra-intronic minisatellite UPS29 associated with neurological diseases regulates reporter gene EGFP expression depending on cell type].

Earlier, it was established that polymorphism of minisatellite UPS29 located in one of introns of human gene CENTB5 (ACAP3) was associated with Parkinson's disease and epilepsy. The main aim of this work was to elucidate if that minisatellite could regulate reporter gene activity, and if such activity was tissue (cell)-specific. To this end there was used transient transfection of HeLa cells, mouse embryonal carcinoma line F9, and rat astrocytes cultures with plasmides which contained reporter gene EGFP under eukaryotic promoter ROSA26 and different allelles of minisatellite UPS29. It was found that UPS29 possessed enhancer-like activity in neuronal type cells.

[The study of preimplantation development of mice embryos labelled with green fluorescent protein gene (EGFp)].

One of the crucial problems of developmental biology is the study of mechanisms of regulation of gene expression in early embryogenesis. Here we studied dynamics of mosaic appearance of a marker fluorescent protein in in vitro developing mice embryo derived from zygotes after microinjections to male pronuclei of cloned DNA fragment carrying EGFP under control of different promoters. Main attention was paid to initial stages of development, when structural rearrangements and reprogramming of both parental genomes, activation of zygotic genes, and control of development by embryo genome take place.

[The role of the yolk sac in copper metabolism during rat embryogenesis]. / Rol' zheltochhogo meshka v metabolisme medi v c'mbriogeneze krys.

Using the immunoblotting method, the synthesis of two copper-transporting P1-type ATPases, ATP7A (a candidate for the product of the Menkes disease gene) and ATP7B (presumed product of the Wilson disease gene), in the yolk sac cells of rat embryos at days 11 and 20 of embryogenesis was demonstrated. Concomitantly, yolk sac cells produce ceruloplasmin, a soluble copper-transporting glycoprotein, a proportion of which in secreted proteins progressively diminishes, attaining 5.2% at day 11 and 3.1% at day 20 of development. At different stages of embryogenesis, yolk sac cells synthesize two molecular forms of [14]C-ceruloplasmin, one of which is secreted towards the embryo, whereas the other, towards the decidual membrane. Two forms of ceruloplasmin secreted in polar directions differ in the rate of secretion. The role of the yolk sac as a key organ controlling the delivery and secretion of copper in the embryo during the postimplantation period is discussed.

[Identification of a fragment of ceruloplasmin, interacting with copper-transporting Menkes ATPase]. / Identifikatsiia uchastka tseruloplazmina, vzaimodeistvuiushchego s med'transportnoi ATP-azoi Menkesa.

The interaction was studied of ceruloplasmin (Cp, EC 1.16.3.1), a copper-containing plasma protein, with two synthetic peptides P15 and P16 whose structures correlate with those of the noncytosolic regions of the copper transfer P1 type ATPase (ATP7A), apparently encoded by the Menkes disease gene (Atp7a). Pentadecapeptide P15 and hexadecapeptide P16 were synthesized using the solid phase method. They correspond to fragments of two extracellular loops ATP7A, of which one loop is apparently involved in the copper ion transfer (P16) whereas the other is not (P15). The protein footprinting showed that P16 binds to a fragment of the ceruloplasmin domain 6. Kinetics of the ceruloplasmin-P16 binding was studied by affinity chromatography on P16 immobilized on a macroporous disk, and the Kd value (1.5 x 10(-6) M) of this interaction was determined. The ATP7A involvement in the copper ion transfer to nonhepatocyte cells is discussed.

Isolation and partial characterization of cDNA clone of human ceruloplasmin receptor.

An individual clone, presumably carrying a 3 bp fragment of ceruloplasmin receptor cDNA was isolated from the expression library of human placenta cDNA using polyclonal specific antibodies to ceruloplasmin receptors. EcoR1-hydrolysate of isolated DNA was cloned in a pTZ19 bacterial vector and sequenced in the forward and reverse direction. The comparison of the revealed sequence with known sequences of human genome revealed its high similarity to ceruloplasmin cDNA.

[Effects of infant formula feeding on the distribution of copper in the body of 8-day-old rats]. / Vliianie iskusstvennogo vskarmlivaniia na raspredelenie medi v organizme 8-dnevnykh krysiat.

The oxidase activity of ceruloplasmin (Cp, EC 1.16.3.1), the content of immunoreactive Cp and copper ion concentration were measured in the serum of eight day-old rats receiving either breast feeding (control group) or commercial nutritive mixture which has been recommended for the newborn children beginning from zero age (experimental group). It was shown that the artificial feeding caused almost 3-fold increase of Cp oxidase activity and copper content in the serum when compared to age-matched controls. No changes in the copper content per Cp molecule were observed. Dot-hybridization of the total liver polyribosomal RNA with Cp [32P]cDNA showed that the increased Cp level in the blood of the rats of experimental group correlated well with the level of expression of Cp gene. The copper content in the liver of experimental rats was two times lower that in control animals while no differences was found in the brain copper content between two groups of rats. The role of the regulation of Cp gene expression in the lactating mammary gland and of milk Cp in the copper homeostasis in the newborn body is discussed.

[A comparative study of the transport dynamics of the peptide moiety of the milk ceruloplasmin molecule in the body of rats with embryonic and adult types of copper metabolism]. / Sravnitel'noe izuchenie dinamiki peremeshcheniia peptidnoi chasti molekuly tseruloplazmina moloka v organizme krys pri émbrional'nom i vzroslom tipakh metabolizma medi.

In chase experiments, we followed the distribution of [125I]-ceruloplasmin prepared from human breast milk orally administered to young rats. Experiments were conducted using six-day-old rat pups (the embryonic type of copper metabolism) or 35-day-old ones (the adult type of copper metabolism). Using the technique of rocket immunoelectrophoresis, we have demonstrated that in six-day-old rats [125I]-ceruloplasmin was transferred from the gastrointestinal tract to the bloodstream and could be detected there over a period of 4 h. In 35-day-old rats, milk ceruloplasmin was digested in the upper part of the intestinal tract. The dynamic aspects of the distribution of labeled milk ceruloplasmin in the body of six-day old rats over a period of 4 h point out that, under the conditions of embryonic copper metabolism, it can serve as a transporter of copper ions to extrahepatic organs. We discuss the role of milk ceruloplasmin in copper metabolism in mammals during the neonatal period.

Study of intracellular localization and traffic of newly synthesized ceruloplasmin receptor in cultured human fibroblasts.

Ceruloplasmin (Cp) receptor in cells of non-hepatocyte lineage (human HT-1080 fibroblasts) is synthesized by membrane-bound polyribosomes and then becomes a resident of the plasma membrane. The intracellular traffic of [14C]Cp receptor was followed in pulse-chase experiments using specific antibodies. It was shown that pulse-labeled Cp receptor, after reaching the place of its residence in the plasma membrane, is retained there for 90 min and then appears in the cytosol. Immunoactive 20-kD fragments of Cp receptor were found in the culture medium 1.5 h later. The intracellular traffic of 125I-labeled Cp bound to the fibroblast cell surface was traced in parallel chase experiments. It was shown that the internalized Cp receptor was recovered from the floating fraction of the cytosol. Comparison of the dynamics of the retention of internalized [14C]Cp receptor and 125I-labeled Cp in the subcellular compartments demonstrated that the traffic of both proteins within the fibroblasts is coordinated in time and proceeds via a common route. The role of Cp receptor in copper uptake by non-hepatocyte cells is discussed.

Comparative analysis of the molecular heterogeneity of ceruloplasmin from human blood and breast milk.

The content of ceruloplasmin (Cp) was determined in 96 samples of human breast milk using rocket immunoelectrophoresis and measurement of Cp oxidase activity. The concentration of immunoreactive Cp in milk decreased about 9 times during the first 20 days of lactation while the specific oxidase activity decreased only 4 times. Two-dimensional electrophoresis of purified milk Cp before and after its treatment with chelating agents showed that copper atoms in milk Cp are more sensitive to EDTA treatment that those in blood Cp. The comparison of the different lectin-binding ability of blood and milk Cp's revealed a difference in the composition of their carbohydrate chains. The mechanisms controlling the uptake of copper ions by newborns at the level of the expression of the Cp-encoding gene in the mammary gland of the mother are discussed.

Reconstitution of the intercellular transfer pathway of the peptide moiety of ceruloplasmin in mammals.

According to rocket immunoelectrophoresis, the antigenic properties of the ceruloplasmin (Cp) receptor present on the surface of human fibroblasts are similar to those of the Cp receptor of the erythrocyte plasma membrane. Using antibodies to Cp receptor, it was demonstrated that Cp binds to the surface of fibroblasts only via the Cp receptor. Ceruloplasmin was labelled with radioactive iodine and its binding to cultured human HT-1080 fibroblasts was studied; at saturating concentrations [125I]Cp interacts with cell surface of fibroblasts (but not hepatocytes) with high affinity (Kd = 80 nM). Subsequent to specific binding fibroblasts absorb [125I]Cp and in about 120-150 min start to release it into the incubation medium. Two fractions of released Cp are clearly detected by non-denaturing polyacrylamide gel electrophoresis. The relative mobility of one of these fractions corresponds to apo-Cp and the other has lower mobility than native Cp. The molecular weight of released [125I]Cp is changed insignificantly. Released Cp does not bind again to the fibroblast surface but is bound, absorbed, degraded, and secreted by hepatocytes. The molecular mechanism of cell-specific transfer of Cp in the human body is discussed and possible functions of this mechanism in copper absorption, metabolism, and excretion in mammals are considered.