Chronic Alcohol Intoxication Is Not Accompanied by an Increase in Calpain Proteolytic Activity in Cardiac Muscle of Rats.

Enzymatic activity of Ca2+-dependent calpain proteases as well as the content and gene expression of µ-calpain (activated by micromolar calcium ion concentrations), calpastatin (inhibitor of calpains), and titin (substrate for calpains) were investigated in cardiac muscles of rats subjected to chronic alcoholization for 3 and 6 months. There was no increase in the "heart weight/body weight" parameter indicating development of heart hypertrophy in the alcoholized rats, while a decreasing trend was observed for this parameter in the rats after 6-month modeling of alcoholic cardiomyopathy, which indicated development of atrophic changes in the myocardium. Fluorometric measurements conducted using the Calpain Activity Assay Kit did not reveal any changes in total calpain activity in protein extracts of cardiac muscles of the rats alcoholized for 3 and 6 months. Western blot analysis did not show reliable changes in the contents of µ-calpain and calpastatin, and SDS-PAGE did not reveal any decrease in the titin content in the myocardium of rats after the chronic alcohol intoxication. Autolysis of µ-calpain was also not verified, which could indicate that proteolytic activity of this enzyme in myocardium of chronically alcoholized rats is not enhanced. Using Pro-Q Diamond staining, changes in phosphorylation level of titin were not detected in cardiac muscle of rats after chronic alcoholization during three and six months. A decrease in µ-calpain and calpastatin mRNA content (~1.3-fold, p ≤ 0.01 and ~1.9-fold, p ≤ 0.01, respectively) in the myocardium of rats alcoholized for 3 months and decrease in calpastatin mRNA (~1.4-fold, p ≤ 0.01) in animals alcoholized for 6 months was demonstrated using real-time PCR. These results indicate negative effect of chronic alcohol intoxication on expression of the abovementioned genes.

[Mechanisms of human plasma proteins adsorption on the surface of perfluorocarbon emulsion stabilized with proxanol 268].

It has been shown that sorption of most proteins with the molecular weight lower than 200 kDa from human blood plasma on the surface of perfluorocarbon emulsion, stabilized with proxanol 268, is mainly based on hydrophobic interaction, whereas sorption of immunoglobulin G is mainly the result of electrostatic interaction. The removal of lipidic components from plasma leads to the increase of a total amount of adsorbed proteins by 35%. Particularly, when lipidic components are removed, sorption of apolipoprotein AI and immunoglobulin G is considerably bettered as well as sorption of other proteins with the molecular weight of about 50 and 60 kDa occurs. It has been out that apolipoprotein AI in the adsorbed condition loses its capability of tryptophan fluorescence, which might be probably determined by the quenching influence of the perfluorocarbon core of nanoparticle. We think that the findings obtained also indicates considerable conformational rearrangements of this protein during adsorption. It was shown, that the fluorescence of proteins with sorption on nanoparticles in emulsion based on the hydrophobic interaction, is completely or partially quenched.

[Blood plasma protein adsorption capacity of perfluorocarbon emulsion stabilized by proxanol 268 (in vitro and in vivo studies)].

The adsorption abilities of the perfluorocarbon emulsion stabilized by Proxanol 268 were investigated in vitro and in vivo. In vitro, the saturation point for the blood plasma proteins was nearly reached after five minutes of incubation of the emulsion with human/rabbit blood plasma and was stable for all incubation periods studied. The decrease in volume ratio (emulsion/plasma) was accompanied by the increase in the adsorptive capacity of the emulsion with maximal values at 1/10 (3.2 and 1.5 mg of proteins per 1 ml of the emulsion, for human and rabbit blood plasma, respectively) that was unchanged at lower ratios. In vivo, in rabbits, intravenously injected with the emulsion, the proteins with molecular masses of 12, 25, 32, 44, 55, 70, and 200 kDa were adsorbed by the emulsion (as in vitro) if it was used 6 hours or less before testing. More delayed testing (6 h) revealed elimination of proteins with molecular masses of 25 and 44 kDa and an additional pool of adsorpted new ones of 27, 50, and 150 kDa. Specific adsorptive capacity of the emulsion enhanced gradually after emulsion injection and reached its maximum (3.5-5 mg of proteins per 1 ml of the emulsion) after 24 hours.

[Possible reasons for the variability of the inotropic insulin effect in papillary muscles of ground squirrel myocardium].

The effects of insulin (0.1-50 nM) on isometric twitch force (0.1 to 1.0 Hz; 30 +/- 1 degree C; 1.8 mM Ca(2+)) were studied in right ventricular papillary muscles from active ground squirrels of different seasons (summer, n = 14; autumn, n = 16 and winter interbout, n = 16) in control conditions and after one-hour pretreatment of PM with 2 mkM nifedipine (an L-type Ca(2+)-channel inhibitor) and 1.0 mM orthovanadate (a tyrosine phosphatase inhibitor). In active animals of different seasonal periods insulin causes both positive and negative inotropic effects. At low frequencies (0.1-0.5 Hz), insulin of low concentrations (0.1-1.0 nM) induces a transient (within the first 20 min after application) positive effect (about 15-25%). Application of high hormone concentration (10 nM) in a low range of stimulation frequencies causes a biphasic effect (a small initial positive inotropic effect followed by a marked negative one). At frequencies above 0.5-Hz stimulation, insulin of 10 nM concentration causes presumably a negative inotropic effect. It was proposed that ICaL is possibly involved in the insulin-induced negative inotropy in ground squirrels hearts. Alteration of protein phosphorylation in tyrosine residues is known to be a major link in the mechanism of insulin action. We performed a study on orthovanadate action (a known inhibitor of tyrosine phosphatase) on the inotropic insulin effect. In the group of summer animals the pretreatment of papillary muscles with orthovanadate (100 mkM) does not change the negative inotropic effect of insulin in a low range of stimulation frequencies but almost completely removes this effect at stimulation frequencies above 0.3 Hz (n = 4). Nifedipine (1-1.5 hr pretreatment), a blocker of L-type calcium channel, reduces the inhibitory effect of insulin in autumn and winter animals, and on the contrary intensifies it in summer animals. This fact indicates that different mechanisms must be involved in insulin actions in animals of summer and winter periods. The main findings of the present study are that insulin induces positive, negative or no inotropic effects in papillary muscles of ground squirrels myocardium. The character of the effects of insulin depends on the physiological state of animals; time and concentrations of the hormone applied; affected by conditions that alter cellular Ca(2+) loading and the ratio of protein-tyrosine kinases/phosphatases activity.

[Modified peroxiredoxins as prototypes of drugs a powerful antioxidant].

The possibility of using modified peroxyredoxins as powerful antioxidant agents has been considered. Peroxyredoxins immobilized on perfluorocarbon emulsions and PTD-modified peroxyredoxins have been studied. It has been shown that peroxyredoxins efficiently bind to particles of perfluorocarbon emulsions, while maintaining their antioxidant properties. A panel of PTD-modified peroxyredoxins has been created and peroxyredoxins most effective both in antioxidant properties and the ability to penetrate cells have been selected. The modified peroxyredoxins obtained may serve as the basis for the design of drug with powerful antioxidant action.

[Use of perfluorocarbon emulsions for administration of photosensitizing preparations into bone marrow stem cells].

It has been shown that, upon incubation of mouse bone marrow stem cells (BMSC) in vitro with the nanoparticles of perfluorocarbon (PFC) emulsion stabilized by proxanol 268, these nanoparticles penetrate into cells and stay there for a long time (up to 20 days of observation). It has been found that, under in vitro conditions, mouse BMSC loaded with the nanoparticles of both the original emulsion and the emulsion preliminarily incubated with radachlorine do not differ from control stem cells in the rate of division, stretching on a plastic support, and the formation of a monolayer. It has been shown that the exposure to laser radiation of BMSC incubated with the nanoparticles of a PFC emulsion preliminarily incubated with radachlorine under in vitro conditions leads to the death of these cells due to the destruction of the cell membrane. The treatment with laser radiation of BMSC incubated with the nanoparticles of the starting PFC emulsion (without preliminarily incubation with radachlorine) causes no death of these cells. It has been shown in in vivo experiments that, when transplanted to the organism of a recipient mouse, BMSC of a donor mouse incubated with the nanoparticles of a PFC emulsion preliminarily incubated with radachlorine retain their functional activity, in particular the ability to migrate in the animal body. In this case, radachlorine contained in these stem cells retains its major function, to induce the death of stem cells by the action of laser radiation due to the destruction of the cell membrane. The observation period after the transplantation was 5-7 days.

[Dynamics of a vortex with the U-shaped filament in the heart of a ground squirrel].

The dynamics of an electrical scroll wave with the U-shaped filament with both ends of the filament being "anchored" on the endocardial surface and the dependence of the structure of pseudoECG on the dynamics of the vortex during the development of polymorphic tachysystolia have been studied by applying premature stimuli to the "target phase" with subsequent registration of the spatial and temporal distribution of electrical potential throughout the surface (endocardial and epicardial) of a thin (approximately 1 mm) preparation. It was found that (1) the psedoECG of the polymorphic form during the tachysystolia attack can be observed in the case that the position of the filament ends on the surfaces of the preparation does not practically change from turn to turn (filament ends are "anchored"); (2) the thread of a scroll wave during this attack can twist and untwin (twisted filament), just as it was the case for scroll waves with a straight filament; (3) in the case of pseudoECG of polymorphic form, the twisting and untwining of the filament were stronger (the angle of maximal twisting was 120 degrees and more), and the angle of twisting changed by a substantially greater value from turn to turn as compared with the pseudoECG of monomorphic form; (4) in the case of pseudoECG of polymorphic form, the time interval between the appearance of waves on the surfaces of the preparation (Tepi-endo) was substantially greater and changed to a greater extent from turn to turn of the vortex; and (5) simultaneously with the appearance of pseudoECG of polymorphic form and the onset of changes in the twisting of the scroll and the Tepi-endo interval indicated in (2-4), significant changes in the patterns of coverage of the surface by excitation occurred. Based on the results obtained, an explanation of the reasons for the appearance of excitation breakdown patterns on the surface of the myocardium was proposed, which differs from the traditional viewpoint. These patterns may be the result of reflection on myocardial surfaces of the activity of not different simultaneously occurring sources of initiation of excitation but of a single three-dimensional vortex whose filament twists when passing through the thickness of the myocardium and can closely approach one or the other surface.

[Sorption of plasma proteins by fluorocarbon emulsions stabilized by various surfactants].

It has been found in in vivo and in vitro experiments that, as a perfluorocarbon emulsion stabilized by Proxanol 268 comes in contact with blood plasma proteins, plasma proteins with molecular masses from 25 to 170 kDa and above are adsorbed on the surface of emulsion particles. Among the adsorbed proteins, fibronectin and fibrinogen were identified by immunoblotting. In in vivo experiments, during circulation in the blood flow, considerable amounts of plasma proteins are adsorbed on Proxanol-stabilized emulsion particles; the amount of adsorbed proteins increases with the time the particles are in the blood flow. Considerably lesser amounts of proteins are adsorbed during circulation in the blood flow on emulsion particles stabilized by egg yolk phospholipids, and their qualitative composition differs from the composition of proteins adsorbed on Proxanol-stabilized emulsion particles. A preliminary incubation of the Proxanol-stabilized emulsion with heparin decreases the amount of the adsorbed proteins and changes their qualitative composition.

[An emulsion based on 1-bromoperfluorooctane: X-ray contrast properties and mechanisms of X-ray contrasting].

It has been shown that the emulsion of a mixture of the perfluorocarbons 1-bromoperfluorooctane and perfluoro[1-(4-methylcyclohexyl)piperidine], stabilized by egg yolk phospholipids, makes it possible to contrast rapidly (beginning with the 10th minute) and for a long time (up to 5 days) the tissues of various organs, such as liver, spleen, adrenal glands, heart, and the peritoneal part of the aorta. The roentgenograms of rat organs in in vivo experiments were evaluated by computer-assisted morphodensitometry. The contrasting of organs at early terms of the circulation of emulsion in the body is related to a high concentration of 1-bromoperfluorooctane in the blood flow, and the contrasting at later terms is related to the accumulation of emulsion particles by cells of the reticuloendothelial system.

[Dynamics of the electrical activity in the form of a vortex with a straight filament in ground squirrel heart].

Attacks of tachysystolia have been studied, which were induced by premature stimuli (amplitude up to 4-5 diastolic thresholds, duration 4 ms) applied after a set of rectangular impulses (amplitude 2 diastolic thresholds, duration 4 ms, frequency 0.5 or 2 Hz). The spatial and temporal distribution of electrical potential throughout the surface of a thin (approximately 1 mm) preparation was registered by two multi-electrode arrays (32 unipolar electrodes each). One array recorded the distribution of electrical potential on the endocardial surface and the other, on the epicardial one. Wave isochronous pictures (maps) corresponding to spatial and temporal propagation of excitation on the surfaces of the preparation were reconstructed on the basis of electrograms registered on each of the surfaces. On the basis of these maps, the three-dimensional structure of scroll waves, including the location, direction and velocity of the shift of filament ends as well as the shape of the thread was analyzed. The analysis of the data obtained in our experiments allow one to conclude that, under tachysystolias caused by three-dimensional scroll wave with a straight filament, there occur the following kinds of wave thread movements: (1) the wave thread may change its location from turn to turn and on the whole be located at different angles to the preparation surfaces; (2) the wave thread may precess, when one of the filament end is "secured" on the surface and the other constantly changes its location on the opposite surface; (3) the wave thread may periodically intertwine (twisted filament) and untwine; (4) dimensions of the scroll wave kernels (sections of the filament on the surfaces) may change from turn to turn both simultaneously on both surfaces (endocardial and epicardial) and on one of them only; (5) the wave thread may curve when it goes within the wall from endocardial to epicardial surfaces; the curve may come rather close the surfaces of the myocardial tissue.

[Effect of perfluorocarbon emulsions on function of enzyme systems responsible for generating active forms of oxygen in neutrophils]. / Vliianie émul'sii perftoruglerodov na funktsionirovanie fermentnykh sistem, otvetstvennykh za generatsiiu aktivnykh form kisloroda v neitrofilakh.

The ability of the emulsion of perfluoroorganic compounds stabilized with proxanol 268 to affect the functions of peritoneal neutrophils was evaluated. The functional activity of neutrophils was estimated from the intensity of generation of reactive oxygen species using the method of chemiluminescent analysis. The emulsion was shown to suppress the neutrophil responses to phorbol-12-myristate-13-acetate in a dose-dependent manner. No inhibition of the activity of neutrophils in the presence of the emulsion was observed in N-formylmethionylleucylphenylalanine stimulated cells. The data obtained indirectly confirm the suggestion that the perfluoride emulsion inhibits neutrophil NADPH oxidase activity. In the presence of the perfluoride emulsion, myeloperoxidase plays a more important role in the generation of luminescent responses in both N-formylmethionylleucylphenylalanine- and phorbol-12-myristate-13-acetate-stimulated neutrophils. The effect of perfluoride emulsion results in the preferential myeloperoxidase-produced generation of reactive oxygen species in the neutrophil respiratory burst.

[Radiocontrast properties of the emulsion of the perfluoroorganic compound lipobrom]. / Rentgenokontrastnye svoistva émul'sii perftotorganicheskogo soedineniia lipobroma.

X-ray contrast properties of the new perfluoroorganic emulsion lipobrom were studied in comparison to those of a traditional clinical Roentgen diagnostic agents omnnipaque and ultravist and the well-known blood substitute perftoran. The x-ray patterns were evaluated by a computer morphodensitometry technique.

[Sorption of plasma components on the surface of particles of fluorocarbon emulsions stabilized by proxanol 268]. / Sorbtsiia komponentov plazmy krovi na poverkhnosti chastits ftoruglerodnykh émul'sii, stabilizirovannykh proksanolom 268.

It was found in the experiments in vivo and in vitro that the contact of perfluorocarbon emulsion stabilized with proxanol 268 with blood plasma leads to the sorption of various plasma proteins on the surface of emulsion particles. The profile of the proteins sorbed is complex and includes proteins with molecular weights ranging from 14 to 94 kDa. Among proteins sorbed on the emulsion particles circulating in blood, IgG was identified. Incubation of the emulsion stabilized with proxanol 268 with human blood plasma in vitro was shown to result in the sorption of IgG and IgA the perfluorocarbon particles. The sorbtion of serum proteins and immune complexes circulating in blood on the surface of perfluorocarbon particles stabilized with proxanol 268 was revealed to activate the complement system.

[Characteristics of force-frequency relations in the myocardium of the squirrel Citellus undulatus]. / Osobennosti ritmoinotropnykh otnoshenii v miokarde suslika Citellus undulatus.

The force-frequency relationship (FFR) in papillary muscles of the heart of active ground squirrel in different seasons was studied. For comparison, similar preparations from rat and rabbit were used. It was shown that the FFR of papillary muscles of active ground squirrel undergo significant seasonal changes. In summer and a part of autumn squirrels, a negative staircase (a decrease in the isometric force with increasing stimulation frequency) similar to that in adult rat was revealed. The FFR of the majority of autumn, winter and spring squirrels were polyphasic and contained both positive and negative components. Changes in the force in response to the introduction of pauses at a constant stimulation frequency were recorded. Two types of the post-rest recovery pattern were revealed in the myocardium of ground squirrels. For frequencies range with the negative direction of FFR, a typical pattern of rest-potentiation similar to that in rat papillary muscles was observed. The amplitude of the first post-rest contraction (F1) was usually higher than that of the preceding steady-state contraction. In papillary muscles of autumn animals the F1 value was greater that in summer, which suggests an enhanced release of Ca2+ from the sarcoplasmic reticulum. There was no post-rest potentiation in the range of frequencies with positive direction of FFR, and the post-rest recovery pattern in these cases was principally different from those of rat and rabbit preparations. It was proposed that seasonal differences of the FFR of active ground squirrel heart are associated with changes in the ratio of activities of the calcium-transporting system in the hibernation period.

[Quantitative analysis of variability of electrocardiograms typical for polymorphic arrhythmias]. / Kolichestvennyi analiz variabel'nosti élektrokardiogramm, tipichnykh dlia polimorfnykh aritmii.

A new approach to the analysis of variability of electrocardiograms (ECGs) typical of polymorphic arrhythmias is developed. In these ECGs, separate QRS complexes can be often hardly identified. As a result, the mathematical methods that have been elaborated hitherto are not suitable for such arrhythmias. The approach presented here is based on the quantitative estimation of the variability of neighboring parts of the ECG. In this case, the necessity of the identification of separate QRS complexes ceases to be significant. Based on this approach, the analysis of normalized ECG variability is developed in the framework of which two indices that characterize the oscillation variability and its changes in time are related to a part of the ECG and/or the ECG as a whole. Variations of these indices allow both the polymorphism of a separate ECG to be estimated and different ECGs to be compared with each other. The method presented may be useful in studies of the mechanisms and in the diagnosis of polymorphic arrhythmias.

[Effect of insulin on the myocardium of the active, hibernating and wakening ground squirrel Citellus undulatus]. / Vliianie insulina na miokard aktivnogo, giberniruiushchego i probyzhdaemogo suslika Citellus undulatus.

The effect of insulin (0.1-100 nM) on isometric force of contraction in isolated ground squirrel papillary muscle was investigated. In summer, autumn and winter active animals, insulin had a negative inotropic effect on papillary muscles, decreasing the amplitude of contraction by about 30% of the control value. In some cases, predominantly in the summer group of animals, insulin produced different effects on contractility: low doses (0.1-0.5 nM) caused a transient activation of isometric contraction by about 10-15% of control, whereas high doses produced a negative inotropic effect by about 30% of the control level. During deep hibernation (at 5-6 degrees C of heart temperature) and during arousal from hibernation (from 3 to 20 degrees C), insulin had no significant effect on contractility. Opposite inotropic effects of insulin at concentrations of 0.1-50 nM were found during arousal: from 26 to 31 degrees C of heart temperature--a positive inotropic effect by about 20-25% of control, and from 32 to 36 degrees C--a negative one by about 30-40% of the control value.

Weak combined magnetic field affects basic and morphine-induced rat's EEG.

The present study was undertaken to find out, whether weak combined magnetic field (CMF) with intensity comparable to that of the Earth's static magnetic field can influence the EEG activity of the rat's brain at normal (non-treated animals) conditions and after intraperitoneal (i.p.) and intracerebroventricular (i.c.v.) administration of morphine in experimental animals bearing chronically implanted electrodes and cannules. Most of the experiments were performed using CMF containing co-linear static (20.9 microT) and alternating sinusoidal (20.9 microT, 48 Hz) components, i.e., tuned for Ca2+-resonance. The effects of the field were estimated by comparison of the averaged EEG frequency spectra in the range of frequencies between 0.8-23 Hz in experimental and control animals. Statistically significant effects of CMF were observed both in non-treated and morphine-treated rats. However, the most profound effect-the drastic power reduction at most EEG frequencies-appeared in the animals subjected to the i.p.-injection of morphine. These results show that weak CMF can influence the spontaneous electrical brain activity. The data obtained are consistent with the findings of other groups demonstrating that weak magnetic fields may drastically modify the effects of both exogenous and endogenous opioids on different basic functions in vertebrates and invertebrates. Possible mechanisms for the observed effects are discussed.