[Use of the most recent reagent (CuFL) for stimulation of NO synthesis by the medicinal leech salivary cell secretion in the cultures of human endothelium cells (HUVEC) and in rat cardiomiocytes].

The medicinal leech salivary cell secretion (SCS) may stimulate NO-production in cultures of human endothelium cells (HUVEC) and rat cardiomiocytes (RCM). This effect was detected using a NO specific reagent, - the complex Cu2+ with a fluorescein derivative (Cu-Fl). NO had also been detected in the cells by fluorescent electronic microscopy and determined quantitatively in the cells and in culture fluid by the fluorescence method. SCS stimulated NO synthesis in HUVEC cells (but not in RCM) is accompanied by NO release into intercellular space. Localization of NO synthesis centers is presented and it is shown that the increase in NO levels during the SCS action on HUVEC and RCM is associated with the increase in the activity of eNOS/nNOS, but not iNOS. In endothelial cells SCS activates nitrosylation processes, assessed by the increase of nitrite-ions in the culture medium. It is therefore important to use Cu-Fl, other than Griss-reagent, during the first hour of analysis of NO synthesis. The NO-depended mechanism of SCS action on endothelial cells might be a factor in providing of its positive action in hirudotheraphy.

[Delayed appearance of hypertension in spontaneously hypertensive rat (SHR) injected with CpG-rich DNA early in ontogenesis].

In this study we have investigated properties of blood serum extracellular DNA (cell-free DNA) from patients with essential arterial hypertension (AH). Cell-free DNA concentration was not changed in the control AH group compared to norma (healthy donors) but fragments of CpG-rich cell-free DNA marker content were increased at transcribed area of ribosomal repeat (TArDNA, CpG-DNA). To evaluate effect of CpG-DNA on AH development in 2-day SHR line and in control normotensive line (WKY), 700 ng of human TArDNA single subcutaneous injection were inoculated to obtain anti-CpG-DNA polyclonal antibodies. These antibodies could change CpG-DNA contents in total cell-free DNA. Blood pressure (BP) in 9-week SHR line rats immunized with CpG-DNA was equal to BP of WKY rats. Then BP of immunized SHR steadily increased with age and reached high value 8 weeks later compared to control SHR rats. Cell-free DNA analysis in 17-week SHR line rats showed significantly reduced concentrations of cell-free DNA and also showed decrease in small DNA fragments content, but increased content of CpG-DNA (rat TArDNA). These changes were accompanied with 3.5-fold blood endonuclease activity increase and decrease of free (unbound to cell-free DNA) anti-CpG-DNA antibodies quantity. Total anti-CpG-DNA antibodies quantity in immunized rats wasn't changed compared to control animals. Thus, observed effect of increase in stable BP elevation age in immunized SHR line rats doesn't relate to increase of anti-CpG-DNA antibody production. Possible reason of this effect is further discussed.

[The study of therapeutic effect of synthetic bradykinin "gene" contained in retrovirus vector on spontaneously hypertensive rats].

Administration of DNA plasmid pPS-3-neo (brd) with synthetic bradykinin " gene " to 2-days old male spontaneously hypertensive rats (SHR) leads to 2 weeks delay in development of arterial hypertension. Lowering of SBP and positive results of PCR DNA of various organs observed in synthetic bradykinin " gene " transgenic SHR but not in control SHR confirm therapeutic effect of synthetic bradykinin " gene " . This data indicate one of possible ways of gene therapy of arterial hypertension as well as other pathological states by introduction of transgene directly into genome of the organism.

[Cardiomyocyte culture as a highly sensitive system for testing pharmacological activity of specific bradycardic agents].

Two antiarrhythmic agents were studied: verapamil and bradizole, a new bradycardic drug. It was found that both drugs exhibit a dose-dependent negative chronotropic effect on the culture of contractile cardiomyocytes of newborn rats. Bradizole showed more pronounced bradycardic properties than verapamil.

Effect of hypoxia during early organogenesis on cardiac activity and noradrenergic regulation in the postnatal period.

Cardiac activity in rats during the postnatal period was studied in vitro and in vivo after exposure of rat pups to antenatal acute hypobaric hypoxia at the stage of organogenesis (day 9-10 of gestation). Cultured cardiomyocytes from rat pups exposed to antenatal hypoxia were characterized by increased rate of contractions and decreased reactivity to norepinephrine. Heart rate elevation, predominance of sympathetic influences on cardiac activity, and significant increase in norepinephrine concentration in the cerebral cortex were found in freely moving animals exposed to antenatal hypoxia. Our results indicate that hypoxia at the stage of organogenesis modulated cardiac activity during the postnatal period, which manifested at the level of effector structures in the heart and activity of regulatory systems.

[Different effect of the ''recombinant'' bradykinin on the contractile activity of cultured atrial and ventricular cardiomyocytes]. / Razlichnoe vliianie "rekombinantnogo" bradikinina na sokrashchenie kul'tiviruemykh kardiomiotsitov predserdii i zheludochkov.

The physiological activity of the "recombinant" bradykinin expressed by retrovirus recombinant pPS-3-neo (brd) was tested on cultural atrial (aCMC) and ventricular (vCMC) cardiomyocytes in newborn rats. The "recombinant" bradykinin was shown to have a chronotropic effect on aCMC and an inotropic effect on vCMC. The effects are in line with the action of the synthetic bradykinin preparation at a concentration of around 10(-15) M. A pretreatment of CMC by parmidine, i.e. a bradykinin antagonist, blocked the effect of bradykinin. The contractive CMC activity in the cultural cell medium, transferred by pPS-3-neo without the bradykinin gene, was not different from the control value.

[Construction by using retrovirus vector of recombinant with synthetic bradykinin "gene" for investigations of human gene expression in mammalian cells]. / Konstruirovanie na osnove retrovirusnogo vektora rekombinanta s sinteticheskim "genom" bradikinina s tsel'iu izucheniia ékspressii genov cheloveka v kletkakh mlekopitaiushchikh.

The synthetic gene of bradykinin was built into the retrovirus vector pPS-3-neo under the guidance of LTR promotor, followed by pPS-3-neo (brd) vector transfection of strain 293 cells. The physiological activity of the expressed bradykinin was tested on cultured neonatal rat cardiomyocytes. The culture medium of strain 293 cells transferred by pPS-3-neo (brd) produces a positive chronotropic effect that is directly related to the time parameters of preparation of recombinant bradykinin, which are comparable with the curve of chronotropic effect of synthetic bradykinin at concentrations of 10(-17) to 10(-16) M. The control of bradykinin "gene" expression was due to the lack of chronotropic responses of cardiomyocytes to the kinin receptor blocker parmidine and the transfection of strain 293 cells with the retrovirus vector without bradykinin "gene".

[The electron microscopic study of the cytoplast mitochondria of an ESK cell culture after the action of actinomycin D. Embryonic swine kidney]. / Elektronno-mikroskopicheskoe issledovanie mitokhondrii tsitoplastov kletok kul'tury SPEV posle deistviia aktinomitsina D.

The inhibitor of RNA synthesis actinomycin D substantially changed mitochondrial morphology and ultrastructure of cytoplasts in comparison with mitochondria of intact cells. These changes included an increase of matrix condensation, the number of cristae, and extension of intercristal and intracristal spaces. We suggest that mitochondria are characterized by significant independence from the nucleus that possibly controls a considerable part of mitochondrial RNA.

[The creation of an SV40 virus-based vector as an experimental model for the study of gene expression]. / Sozdanie na osnove virusa SV40 vektora kak éksperimental'noi modeli dlia izucheniia ékspressii genov.

The purpose of the studies is to design the recombinant virus SV 40 where the C-end of the basic structural protein of virus P-1 was replaced by a synthetic sequence of the neuropeptide bradykinin. The recombinant virus SV (SV 40/Brd) was obtained. In this virus 60 n.p. with 3'-end of VP-1 gene was substituted for 36 n.p. synthetic gene of bradykinin without impairing the frame of translation. The biological activity of SV 40 (Brd) was tested on the cultured cells CV-1 permissive for this virus. An immunofluorescence method was used to detect T antigen and to examine the cytopathic action of this recombinant. The gene engineering design does not make the recombinant loose its biological properties typical of wild virus.

[Enucleation and reconstruction of murine L-cell fibroblasts]. / Enukleatsiia i rekonstruktsiia myshinykh fibroblastov linii L.

Two types of teflon chambers are suggested for the procedures of enucleation and reconstruction of cells cultivated in the monolayer. The first chamber simplifies the enucleation and permits performing it on the standard cover glasses with a small volume of medium with cytochalasin B. The second chamber permits purifying the karyoplast fraction from intact cells by their adhesion on the cover glasses. Then the karyoplasts are sedimented by centrifugation on cytoplasts located on the cover glasses. This procedure promotes sticking of fragments and their subsequent fusion in the presence of polyethylene glycol. In total the number of reconstructed cells increases many times.

[Assessment of mitochondrial function in the surviving cytoplasts of cultured cells of pig embryonic kidney by using ethylrhodamine]. / Otsenka funktsional'nogo sostoianiia mitokhondrii v perzhivaiushchikh tsitoplastakh kul'tivirovannykh kletok pochki émbriona svin'i s pomoshch'iu étilrodamina.

A study was made of the functional state of chondriome in cytoplasts previously cultivated for a long time under vital staining with fluorescent dye ethylrhodamine (10 micrograms/ml) which is known to accumulate preferentially in mitochondria. The energization was estimated by the intensity of mitochondrial fluorescence. It was realized that 30 minutes after enucleation cytoplasts retained the same fluorescence as did the untreated cells, and that the mitochondrial distribution within the cell was very similar. Such a high intensity of fluorescence seen within one day after enucleation gives a strong evidence on the high degree of independence on the cell nucleus of organelles that provide the energy to metabolic processes. In the course of survival in the cultivation medium for 1-4 days the intensity of fluorescence is shown to fall, especially on the second day after enucleation. All these changes coincide with the changes in cytoplast shape: originally well spread bodies transform into squeezed, ball-shaped or strongly deformed ones. The adhesive ability is going down, and in result only single units of cytoplast can hardly be found on the cover slips by the 4th day after enucleation. These changes give evidence on the enhanced degeneration of cultured cytoplasts.

[Modification of a method of cell enucleation using cytochalasin B and the study of cytoplast viability]. / Modifikatsiia metoda énukleatsii kletok s pomoshch'iu tsitokhalazina B i izuchenie zhiznesposobnosti tsitoplastov.

The modification of Prescott's (Prescott et al., 1972) method of enucleation in vitro was described. A special teflon chamber faciliatating the enucleation of monolayer cultured cells to produce cytoplasts and karyoplasts was constructed. Mouse L-cells were enucleated by exposing to cytochalasine B (10 gamma/ml) followed by centrifugation. The fraction of cells enucleated in the chamber was about 98%. The life time of cytoplasts in cultural medium after their enucleation was 48 hours (sometimes 56-72 hours) as tested by vital neutral red staining. The cytoplasts that survived were shown to accumulate large lysosomes, and the evidence of appearing ring-like fibrillar structures was provided using a simple technique of cytoskeleton observation under light microscope.

Reactivation of rat peritoneal leukocyte and mast cell nuclei in heterokaryons with actively growing mouse cells.

Leukocytes and mast cells of rat peritoneal exudate (PE) were fused in vitro with actively growing mouse cells. Segmented ring-shaped nuclei of granulocytes undergo drastic changes which result in dispersion of tightly condensed chromatin and gradual disappearance of the opening in the centre of the nucleus. These changes are paralleled by a resumption of RNA and DNA synthesis, as shown by autoradiography with [3H]uridine and [3H]thymidine. Solid inactive nuclei of mast cells, lymphocytes, monocytes and macrophages also resume DNA replication and high level of RNA synthesis. Fusion of thymidine kinase-deficient 3T3-4E cells with PE cells results in the incorporation of [3H]thymidine into the nuclei of heterokaryons. This may be considered evidence of the phenotypic expression of rat thymidine kinase gene in heterokaryons. A similar way in which segmented and non-segmented dormant nuclei undergo reactivation suggests that the reversibility of nuclear inactivation is a common feature of differentiated somatic cells.

[Morphologic and radioautographic study of reactivation of peritoneal exudate cell nuclei in heterokarya]. / Morfologicheskoe i radioavtograficheskoe issledovanie reaktivatsii iader kletok peritoneal'nogo ékssudata v geterokarionakh

The heterokaryons of undifferentiated mouse fibroblasts (L and 3T3-4E/TK-) and various cell elements of the rat peritoneal exudate were obtained under the treatment with inactivated Sendai virus. The reactivation of RNA and DNA synthesis in the nuclei of highly differentiated periotoneal exudate cells and the synthesis of thymidine kinase controlled by the nuclei of peritoneal exudate cells were shown to occur in the heterokaryons. During the process of reactivation, the ring-like nuclei of polymorphonuclear leucocytes acquired the form characteristic of the reactivated nuclei of mononuclears. The morphological changes of heparin-containing granules in the cytoplasm of the heterokaryons of mast cells and undifferentiated fibroblasts suggest the degeneration and breakdown of granules.