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.

Effects of heparin on synaptic activity in the hemorrhagic stroke model in vitro.

We studied the ability of heparin to modify synaptic activity on the original stroke model in vitro. Cultured slices of the brain slices from hypertensive rats were treated with the autoblood clot. Administration of heparin (2 mg/ml) before autoblood treatment had a protective effect on ionotropic glutamatergic and GABAergic receptors, whose activity was inhibited by the blood.

[Application of alpha-tocopherol acetate prevents swelling of brain slices during the autoblood clot action].

The swelling of olfactory cortex slices of the hypertensive SHR rats under the long autoblood action have been studied. The influence of a preincubation of slices with vitamins E, C and D on a degree of swelling have been detected by their weighing before and after exposure to autoblood. The water-soluble form of vitamin E have exerted a substantial antiswelling action exceeding the same of vitamin D, whereas vitamin C had no any effect.

Neuroprotective properties of l-carnosine in the brain slices exposed to autoblood in the hemorrhagic stroke model in vitro.

Neuroprotective properties of L-carnosine have been studied in our in vitro model on olfactory cortex slices of hypertensive rats under a long autoblood (blood clot) influence. Application of L-carnosine (5 mg/ml) on slices before autoblood influence leads to restoration of the activity of glutamatergic and GABA-ergic receptors inhibited in the presence of autoblood and interferes with swelling of slices. L-carnosine protects a bioelectric activity of nervous cells in case of long influence of autoblood and also renders an anti edema effect. This model of hemorrhagic stroke may provide a perspective for investigating the mechanisms of neuroprotection.

[Development of edema in nervous tissue in the model of hemorrhagic stroke in vitro].

The development of edema in the surviving olfactory cortex slices by the control of bioelectric activity of neurons under the long-term influence of autoblood has been studied. The level of disturbance of neuronal activity was revealed by comparing focal potentials with their control values; the degree of tissue swelling was detected by their weighting before and at various time-points after exposure to autoblood. The use of this model of hemorrhagic stroke and pharmacological blockade of glutamate receptors allowed to find the correlation between the degree of edema in nervous tissue and the level of activity of ionotropic and metabotropic glutamate receptors.

[Hemorrhagic stroke: molecular mechanisms of pathogenesis and perspective therapeutic targets].

Key mechanisms of an induction and development of a hemorrhage insult are considered in the review. Action of the whole hemoglobin, products of blood destruction, and also the effects caused by nitric oxide are analyzed. The special attention is given to processes of a blood-brain barrier disruption, a water homeostasis and to development of a brain edema. The role of serum proteins and thrombin is considered. Data about involving of the heat shock proteins in development of a cerebral insult are resulted in a final part of the review.

[Modeling of hemorrhage stroke in vitro].

A method of modeling of hemorrhage stroke suggested in the study includes the application of the autoblood on the surviving brain slices for 25-40 min followed by their washing. The parameters of evoked bioelectrical activity (focal potentials) of the slices are registered. The extent of nervous cells injury is established by comparing the focal potentials parameters in the control and during the blood application. The potential to cell recovery after the blood exposure is defined by comparing of these parameters in the control and after washing. The method permits to increase the model repeatability to 100% and reveals the cell and molecular mechanisms responsible for the development of hemorrhage stroke.

Effects of long-term exposure of nerve cells to autoblood in in vitro model of hemorrhagic stroke.

Changes in bioelectrical activity of nerve cells after their long-term exposure to autoblood were studied in vitro on cultured brain slices. This model simulated the events characteristic of a hemorrhagic stroke. Brain slice was placed into a glass vial with autoblood for 60-420 min, after which the slice was transferred into a perfusion chamber and after washing from autoblood their focal potentials were recorded. The level and reversibility of disorders in nerve cell activity were detected by comparing the parameters of focal potentials with the control values. Delayed effects of autoblood were detected, manifesting in the progress in disorders of nerve cell activity with prolongation of exposure in the blood, and the period after which they could be restored was determined.

Neurotropic effect of exogenous L-carnosine in cultured slices of the olfactory cortex from rat brain.

Incubation of cultured slices of the olfactory cortex from rat brain with L-carnosine in concentrations of 50, 250, and 500 M induced activation of glutamatergic and GABAB-ergic mechanisms and facilitated long-term posttetanic potentiation. The effect of L-carnosine is mediated by its effect on AMPA- and NMDA-related glutamatergic receptors and on inhibitory GABAB receptors.

Heat shock protein HSP70 increases the resistance of cortical cells to glutamate excitotoxicity.

Preincubation of cultured slices of the olfactory cortex of rat brain with heat shock protein in a concentration of 1 microg/ml protected the pre- and postsynaptic mechanisms of glutamatergic synaptic transmission from glutamate excitotoxicity (50 mM) inducing blockade of excitatory postsynaptic function and reducing presynaptic processes. It was hypothesized that heat shock protein protects AMPA and NMDA receptor-mediated processes.

[Modification of rat behavioral reactions by peptides secreted by olfactory cortex cells upon their tetanization]. / Modifikatsiia povedencheskikh reaktsii krys peptidami, sekretiruemymi kletkami oboniatel'noi kory pri ikh tetanizatsii.

We studied neurotrophic properties of neuropeptide extracts from tetanized slices of the rat olfactory cortex. Two types of the extracts were studied, EP and ED, obtained from perfusates of the tetanized slices after long-term potentiation and from the slices after long-term depression, respectively. The effects of these two extracts were tested on rats using the open field and passive avoidance tests upon intranasal administration of the extracts (ca. 1 ng total protein). EP showed a neurotropic effect combined with a moderate sedative-hypnotic effect, while EP had obvious neurotropic and nootropic properties, affecting mainly the mechanisms of memory consolidation, and had an activating effect combined with a minor, delayed sedative effect. The functional significance of the secreted endogenous neuropeptides and the mechanisms of their effects are discussed.

[Endogenous neuropeptides and regulation of the brain functional plasticity]. / Uchastie éndogennykh neiropeptidov v reguliatsii funktsional'noi plastichnosti mozga.

This review analysed the data about the neuropeptides secreted from the different brain structures. Involvement of the endogenous neuropeptides in synaptic plasticity was tested at the model of long-term potentiation (LTP) as a form of learning and the memory formation. The most of these neuropeptides or their fragments involves in the induction and maintenance of the LTP and provides the transformation of short-term excitability of the neurones into the long-term one. They may be considered as nootropic compounds. It is proposed that the system of peptidergic regulation of neuronal plasticity functionates in the brain and there are a possibility to correct the activity of this system during the different pathology.

Role of the phosphoinositide signal system and methylation of phosphatidylethanolamine in the development of long-term post-tetanic potentiation in rats.

High-frequency stimulation eliciting long-term post-tetanic potentiation of neuronal excitation in slices of the rat olfactory cortex was accompanied by changes in the metabolism of phospholipid components of cell membranes. At the first stage of the development of long-term potentiation (10 min after tetanization), there was a reduction in phosphoinositide metabolism. The maintenance phase of the potentiated state (30 min after tetanization) was associated with a three-fold increase in the incorporation of 14C-labeled groups from adenosylmethionine into phosphatidylethanolamine methylation products and with normalization of phosphoinositide metabolism. Sixty minutes after tetanization, when potentiation had decayed, there was activation of phosphoinositide metabolism and the intensity of phosphatidylethanolamine methylation returned to the control level. It is suggested that the phosphoinositide system plays an important role in the induction of long-term potentiation, as well as at the stage of recovery of normal neuronal excitability, while the long-term maintenance phase of elevated neuronal excitability was associated with long-lasting changes in the level of phosphatidylethanolamine methylation. The effect of glutamate receptor agonists on the carbachol-stimulated phosphoinositide response in potentiated slices was found to differ from that in nonpotentiated slices. The development of the long-term potentiated state is thus accompanied by a modulatory action of glutamate on the phosphoinositide response.

[The role of the phosphoinositide signal system and phosphatidylethanolamine methylation in the development of long-term posttetanic potentiation in rats]. / Rol' fosfoinozitidnoi signal'noi sistemy i metilirovaniia fosfatidilétanolamina v razvitii dlogovremennoi posttetanicheskoi potentsiatsii u krys.

A long-term potentiation of the rat olfactory cortex slices induced by a high-frequency stimulation is followed by membrane phospholipid changes. Activation of the phosphoinositide metabolism and normalising of phosphatidylethanolamine methylation occurs within 60 min after tetanisation when the potentiation fades. The findings reveal a regulating role of the phospholipid signal system at different stages of the long-term potentiation.