Analysis of Toxicity of Recombinant Pneumolysin from Streptococcus pneumoniae.

We studied toxicity of recombinant Streptococcus pneumoniae pneumolysin protein in experiments on mice and its cytopathogenic effect on cultures of Vero green monkey kidney cells and human lung carcinoma A549 cells in vitro. In vivo and in vitro experiments proved the absence of compromised toxicity and direct cytopathogenic action of the recombinant protein.

[Influence of siRNA complexes on the reproduction of influenza A virus (Orthomyxoviridae: Alphainfluenzavirus) in vivo].

INTRODUCTION: Influenza is one of the most pressing global health problems. Despite the wide range of available anti-influenza drugs, the viral drug resistance is an increasing concern and requires the search for new approaches to overcome it. A promising solution is the development of drugs with action that is based on the inhibition of the activity of cellular genes through RNA interference. AIM: Evaluation in vivo of the preventive potential of miRNAs directed to the cellular genes FLT4, Nup98 and Nup205 against influenza infection. MATERIALS AND METHODS: The A/California/7/09 strain of influenza virus (H1N1) and BALB/c mice were used in the study. The administration of siRNA and experimental infection of animals were performed intranasally. The results of the experiment were analyzed using molecular genetic and virological methods. RESULTS: The use of siRNA complexes Nup98.1 and Nup205.1 led to a significant decrease in viral reproduction and concentration of viral RNA on the 3rd day after infection. When two siRNA complexes (Nup98.1 and Nup205.1) were administered simultaneously, a significant decrease in viral titer and concentration of viral RNA was also noted compared with the control groups. CONCLUSIONS: The use of siRNAs in vivo can lead to an antiviral effect when the activity of single or several cellular genes is suppressed. The results indicate that the use of siRNAs targeting the cellular genes whose expression products are involved in viral reproduction is one of the promising methods for the prevention and treatment of not only influenza, but also other respiratory infections.

Preparation a Recombinant Form of Pneumolysin Protein from Streptococcus pneumoniae.

A recombinant form of pneumolysin from Streptococcus pneumoniae was obtained. By using Vector NTI Advance 11.0 bioinformatic analysis software, specific primers were designed in order to amplify the genome fragment of strain No. 3358 S. pneumoniae serotype 19F containing the nucleotide sequence encoding the full-length pneumolysin protein. A PCR product with a molecular weight corresponding to the nucleotide sequence of the S. pneumoniae genome fragment encoding the full-length pneumolysin was obtained. An expression system for recombinant pneumolysin in E. coli was constructed. Sequencing confirmed the identity of the inserted nucleotide sequence encoding the full-length recombinant pneumolysin synthesized in E. coli M15 strain. Purification of the recombinant protein was performed by affinity chromatography using Ni-Sepharose in 8 M urea buffer solution. Confirmation of the recombinant protein was performed by immunoblotting with monoclonal antibodies to pneumolysin.

Effect of colchicine on physiological and biochemical properties of Rhodococcus qingshengii.

The genus Rhodococcus includes polymorphic non-spore-forming gram-positive bacteria belonging to the class Actinobacteria. Together with Mycobacterium and Corynebacterium, Rhodococcus belongs to the Mycolata group. Due to their relatively high growth rate and ability to form biof ilms, Rhodococcus are a convenient model for studying the effect of biologically active compounds on pathogenic Mycolata. Colchicine was previously found to reduce biof ilm formation by P. carotovorum VKM B-1247 and R. qingshengii VKM Ac-2784D. To understand the mechanism of action of this alkaloid on the bacterial cell, we have studied the change in the fatty acid composition and microviscosity of the R. qingshengii VKM Ac-2784D membrane. Nystatin, which is known to reduce membrane microviscosity, is used as a positive control. It has been found that colchicine at concentrations of 0.01 and 0.03 g/l and nystatin (0.03 g/l) have no signif icant effect on the survival of R. qingshengii VKM Ac-2784D cultivated in a buffered saline solution with 0.5 % glucose (GBSS). However, colchicine (0.03 g/l) signif icantly inhibits biof ilm formation. Rhodococcus cells cultivated for 24 hours in GBSS with colchicine acquire a rounded shape. Colchicine at 0.01 g/l concentration increases C16:1(n-7), C17:0, C20:1(n-9) and C21:0 fatty acids. The microviscosity of the membrane of individual cells was distributed from the lowest to the highest values of the generalized laurdan f luorescence polarization index (GP), which indicates a variety of adaptive responses to this alkaloid. At a higher concentration of colchicine (0.03 g/l) in the membranes of R. qingshengii VKM Ac-2784D cells, the content of saturated fatty acids increases and the content of branched fatty acids decreases. This contributes to an increase in membrane microviscosity, which is conf irmed by the data on the GP f luorescence of laurdan. All of the above indicates that colchicine induces a rearrangement of the Rhodococcus cell membrane, probably in the direction of increasing its microviscosity. This may be one of the reasons for the negative effect of colchicine on the formation of R. qingshengii VKM Ac-2784D biof ilms.

Gasdynamic electron cyclotron ion sources: Basic physics, applications, and diagnostic techniques.

The gasdynamic electron cyclotron resonance (ECR) ion source is a type of the device in which the ionization efficiency is achieved primarily due to a high plasma density. Because of a high particle collision rate, the confinement is determined by a gasdynamic plasma outflow from a magnetic trap. Due to high efficiency of resonant heating, electrons gain energy significantly higher than that in inductively or capacitively coupled plasmas. As a consequence of such a parameter combination, the gasdynamic ECR plasma can be a unique source of low to medium charged ions, providing a high current and an ultimate quality of an ion beam. One of the most demanded directions of its application today is a development of high-current proton injectors for modern accelerators and neutron sources of different intensities. Special plasma parameters allow for the use of diagnostic techniques, traditional for multiply charged ECR plasmas as well as for other types of discharges with a high plasma density. Among the additional techniques, one can mention the methods of numerical simulation and reconstruction of the plasma density and temperature from the parameters of the extracted ion beams. Another point is that the high plasma density makes it possible to measure it from the Stark broadening of hydrogen lines by spectroscopy of plasma emission in the visible range, which is a fairly convenient non-invasive diagnostic method. The present paper discusses the main physical aspects of the gasdynamic ECR plasma, suitable diagnostic techniques, and possibilities and future prospects for its various applications.

Immunization with Recombinant Pneumolysin Induces the Production of Antibodies and Protects Mice in a Model of Systemic Infection Caused by Streptococcus pneumoniae.

Immunogenic and protective activity of recombinant pneumolysin was studied in experiments on male BALB/c mice. The mice were immunized intraperitoneally with recombinant pneumolysin sorbed on Al(OH)3 (200 µg per mouse). In 2 weeks after immunization, the isotypes of antibodies to recombinant pneumolysin in the serum of immunized mice were determined by ELISA. The animals were infected with Streptococcus pneumoniae serotype 3. Immunization with recombinant pneumolysin induced the production of anti-pneumolysin antibodies, mainly of IgG1 subisotype. On day 21 after intraperitoneal infection with S. pneumoniae serotype 3 in a dose of 106 microbial cells, the survival rate of animals immunized with recombinant pneumolysin in a dose of 25 µg/mouse was 67% vs. 0% in the control (p<0.001). Recombinant pneumolysin could be considered as a promising protective antigen for inclusion in the serotype-independent vaccine against S. pneumoniae.

A powerful pulsed "point-like" neutron source based on the high-current ECR ion source.

The paper presents recent results of a "pointlike" neutron source development based on a D-D fusion in a D-loaded target caused by its bombardment with a sharply focused deuterium ion beam. These developments are undergoing at the Institute of Applied Physics of Russian Academy of Sciences in order to study a possibility to create an effective and compact device for fast-neutron radiography. The last experiments with a beam produced by a gasdynamic high-current ECR ion source and its focusing with a magnetic lens demonstrated that 60 mA of deuterium ions may be constricted to a transversal size of ∼1 mm at the focal plane. With a purpose to improve this result in terms of the beam current and its size, a combined electrostatic and magnetic focusing system is proposed and analyzed. It is shown that the combined system may enhance the total beam current and reduce its footprint down to 0.13 mm. All numerical analysis was performed using the IBSimu code.

Wide-aperture dense plasma fluxes production based on ECR discharge in a single solenoid magnetic field.

Results of experimental investigation of the ECR discharge in a single coil magnetic field as an alternative to rf and helicon discharges for wide-aperture dense plasma fluxes production are presented. A possibility of obtaining wide-aperture high density hydrogen plasma fluxes with homogeneous transverse distribution was demonstrated in such a system. The prospects of using this system for obtaining high current ion beams are discussed.

[Oncolytic Properties of a Mumps Virus Vaccine Strain in Human Melanoma Cell Lines].

The oncolytic potential of the attenuated mumps virus (MV) vaccine strain Leningrad-3 (L-3) was evaluated in a panel of four human metastatic melanoma cell lines. The lines were shown to be susceptible and permissive to MV infection. Efficient MV replication led to death of melanoma cells, but the effect differed among the cell lines. Possible mechanisms mediating the selectivity of MV L-3 towards the cell lines were explored. Replicative and oncolytic activity of MV was found to depend on the expression pattern of type I interferon genes. None of the melanoma cell lines showed induction of expression of the total spectrum of genes required to inhibit virus replication. Based on the results, MV L-3 was assumed to be a promising oncolytic agent for human melanoma cells.

Study of hydrogen ECR plasma in a simple mirror magnetic trap heated by 75 GHz pulsed gyrotron radiation.

Plasma of electron cyclotron resonance (ECR) discharge sustained by millimeter wave radiation is widely used for production of ion beams of different kind. The main trend in ECR ion sources development nowadays is an increase of frequency and power of microwave heating. The most advanced systems use gyrotrons in 24-60 GHz frequency range. In previous studies at IAP RAS it was demonstrated that ECR source SMIS 37 (Simple Mirror Ion Source) with 37.5 GHz heating operating in quasigasdynamic regime of plasma confinement is able to produce proton and deuteron beams with ion current density about 700 mA/cm2. As the next step of these investigations plasma properties of the discharge sustained by 75 GHz radiation have been studied. Plasma density and electron temperature were determined using spectroscopic and Langmuir probe techniques. It was demonstrated that plasma density could reach values close to 1014 cm-3 and that is of great interest for further development of high current ion sources for various applications.

[The concentration of cerebral natriuretic peptide in tear and blood serum.]

The natriuretic peptides regulate pressure and water metabolism in human organism. The concentration of N-thermal cerebral natriuretic propeptide (NT-proBNP) in tear and blood serum was analyzed. The sampling included 49 patients divided on three groups. The group I (main) included 14 patients with primary open-angle sub-compensated glaucoma degree I-III. The group II (comparative group) included 15 patients with age-related immature cataract. The group III (control) included volunteers without any visual organ diseases. The concentration of NT-proBNP in tear and blood serum was detected by solid-phase enzyme-linked immunosorbent assay technique using test system NT-proBNP - IFA - BEST¼ («Vektor-Best¼, Russia). The median of content of NT-proBNP in tear turned out to be less than in blood serum in all groups (р1 = 0,00763; р2 = 0,00452; р3 = 0,00029) and made up to in patients with glaucoma 41,3 pg/ml, with cataract - 37.5 pg/ml, in healthy people 0 25.0 pg/ml (inter-quartile range made up to 20-60 pg/ml, 20-65 pg/ml, 10-49,5 pg/ml correspondingly). No differences in concentration of NT-proBNP were established in all groups both for blood serum (p = 0,494) and tear (p = 0,388). The concentration of NT-proBNP in tear correlated with its concentration in blood serum (r = 0,694). However, no dependencies were established from value of intra-ocular pressure (r = 0,168). Therefore, the concentration of NTproBNP in tear is not a perspective laboratory arker for diagnostic of increased intra-ocular pressure and stratification of development of glaucoma.

[POSSIBILITY OF USING NESTED POLYMERASE CHAIN REACTION FOR DIAGNOS- TICS OF DISEASES CAUSED BY VARICELLA ZOSTER VIRUS].

AIM: Demonstrate the possibility of using nested PCR method for determination of Varicella Zoster virus (VZV) in clinical samples of peripheral blood of patients. MATERIALS AND METHODS: Material from 35 patients with clinical manifestations of herpes zoster and control group of 20 healthy donors was used in the study. Monocyte fraction of venous blood cells, pretreated with heparin, was isolated by centrifugation in ficoll-verografin density gradient, total DNA was then isolated from cells by phenol-chloroform extraction with subsequent precipitation with alcohol. Polymerase chain reaction was carried out in thermocyclers Tercyc and TProfessional Gradient (Biometra), amplified DNA was analyzed by electrophoresis on 1.6% agarose gel in the presence of ethidium bromide. RESULTS: Data on detection of viral DNA in blood monocytes in 17 (49%) of ill patients, as well as in 1 (out of 20 in control group) practically healthy donor were obtained. A possibility of a subclinical reactivation of the virus is discussed in the latter case. CONCLUSION: A possibility of viral DNA determination in monocytes of patient blood without using expensive equipment is shown, that could find application in clinical practice, especially for diagnostics of patients with non-characteristic clinical manifestations, as well as patients with subclinical forms of the disease.

[Plasma lipid spectrum and survival in rats with heart failure varied by severity under the influence of neurohormonal modulators].

In rats with relatively mild chronic heart failure (CHF) there was an increase in plasma LDL levels. In turn, in severe CHF it was revealed a significant decrease in total cholesterol concentration due to LDL levels decline. ACE inhibitors (captopril, enalapril and lisinopril) and the most selective beta1-adrenoblockers (metoprolol, nebivolol) had not any significant influence on plasma lipids. Less selective and especially nonselective beta-adrenoblockers pindolol and propranolol brought up the atherogenic potential of plasma because of HDL levels reduction in mild CHF whereas in case of severe model--due to the trends toward decrease in HDL concentration and toward increase in LDL levels. Lipophilic ACE-inhibitors and beta-adrenoblockers without intrinsic sympathomimetic activity improved survival in rats with mild CHF to the equal extent. On the contrary in severe CHF beta-adrenoblockers, especially non-selective, had an advantage over ACE inhibitors on the survival effect.

Effect of long-term treatment with ß-adrenoceptor antagonists on blood lipid composition and survival of rats with heart failure of different severity.

Plasma LDL content increased in rats with mild chronic heart failure, but decreased in animals with severe pathology. The lipid profile of ß-adrenoceptor antagonists depended on their selectivity. The most selective antagonists of ß-adrenoceptors had little effect on the lipid composition. Nonselective ß-adrenoceptor antagonists increased the index of plasma atherogenicity, which did not depend on the severity of a pathological process. In mild chronic heart failure, these changes were associated with a decrease in HDL concentration, while in severe chronic heart failure; they were related to a tendency towards a decrease in HDL content and an increase in LDL concentration. The increase in survival rate was associated with true antagonism of the agent to ß-adrenoceptors (the absence of intrinsic sympathomimetic activity) and, to a lesser extent, with nonselective blockade of ß-adrenoceptors.

[Effect of angiotensin-converting enzyme inhibitors, beta-adrenoblockers, and their combinations on survival and plasma catecholamine levels in rats with chronic heart failure with induced exacerbations].

By using model of chronic heart failure (CHF) occurring with induced decompensations, it was shown that inhibitors of angiotensin-converting enzyme (ACE) and beta-adrenoblockers promoted to different degree the correction of hypercatecholaminemia and an increase of survival of rats. The most pronounced effect was characteristic of beta-adrenoblockers atenolol, propranolol, and carvedilol. The lower activity was demonstrated by other adrenoblockers that had no intrinsic sympathomimetic activity, as well as captopril. However, positive effect of these drugs on rat survival occurred regardless of their action on the sympathoadrenal system. The ACE inhibitors, particularly hydrophilic lysinopril, to the lesser degree than beta-adrenoblockers, prevented death of animals at the period of the CHF decompensation. The combined use of ACE inhibitors with beta-adrenoblockers, especially with carvedilol, promoted the maximally expressed decrease of the sympathoadrenal activity and an increase of the rat survival, including that at the CHF decompensation.

[Changes in lipid spectrum in rats with chronic heart failure of different severity on the background of angiotensin-converting enzyme inhibitors and beta-adrenoblockers].

The character of lipid spectrum changes in rats with experimental heart failure of non-ischemic genesis depended on its severity. In mild heart failure there was found an increase in the low density lipoprotein levels, whereas in the severe one--a decrease of total cholesterol levels at the expense of the low density lipoproteins. The ACE inhibitors produced no significant changes in the lipid spectrum. The effect of beta-adrenoblockers was determined by their selectivity for beta1-adrenoreceptors. The most selective drugs were distinguished by the metabolically neutral activity. On the contrary, nonselective beta-adrenoblockers increased the degree of plasma atherogeneity.

[Evolution of intercellular comminication and structural brain organization].

The review considers key problems connected with historical development of the nervous system, including intercellular contacts and brain neurotransmitter systems. A particular attention is paid to structural-functional organization of the central nervous system of the fresh-water pulmonary mollusc Lymnaea stagnalis.

[Temperature dependence of monoamine-induced pulmonary respiration of mollusc Lymnaea stagnalis].

Pulmonary respiration of molluscs (spontaneous and mediated by intracavital injection of monoamines) was studied at different environmental temperatures (5, 15, and 25 degrees C). It was established that monoamines (dopamine, serotonin, adrenalin) did not enlarge the temperature diapason, in which the respiratory behavior was realized. Microelectrode studies of spontaneous electrical activity of neurons from the respiratory network of Lymnaea stagnalis (RPeD1, VD4, cells of the Vi cluster) have shown that the respiratory program, both spontaneous and the monoamine-induced, is terminated in hypothermia. The indicated effects are suggested to be due to temperature dependence of the chemical, predominantly peptidergical, transmission of signal between neurons of the central pattern generator of respiratory pattern in Lymnaea.

Heart rate variability in conscious and anesthetized rats under the action of angiotensin converting enzyme inhibitors.

We studied the effects of three various angiotensin converting enzyme inhibitors (enalapril, lisinopril and quinapril) on heart rhythm variability in anesthetized and immobilized rats. In all cases (except for quinapril in experiments on anesthetized animals), the preparations reduced the total rhythm variability and, according to spectrum analysis, increased activity of the parasympathetic autonomic nervous system to different degrees and decreased sympathetic tone. Quinapril and lisinopril produced the most pronounced influence on heart rhythm in anesthetized rats; enalapril was less potent in this respect. In immobilized animals, quinapril and enalapril showed the greatest activity and lisinopril the lowest. The more pronounced effect of quinapril both under anesthesia and during immobilization appears to be linked to the highest affinity of quinaprilat to circulatory and tissue compartments of the renin-angiotensin-aldosterone system.

Influence of the shear flow on electron cyclotron resonance plasma confinement in an axisymmetric magnetic mirror trap of the electron cyclotron resonance ion source.

Influence of shear flows of the dense plasma created under conditions of the electron cyclotron resonance (ECR) gas breakdown on the plasma confinement in the axisymmetric mirror trap ("vortex" confinement) was studied experimentally and theoretically. A limiter with bias potential was set inside the mirror trap for plasma rotation. The limiter construction and the optimal value of the potential were chosen according to the results of the preliminary theoretical analysis. This method of "vortex" confinement realization in an axisymmetric mirror trap for non-equilibrium heavy-ion plasmas seems to be promising for creation of ECR multicharged ion sources with high magnetic fields, more than 1 T.