[Actual problems of biosafety].

Actual problems of biosafety are presented. Examination of biosafety as a subsystem of life safety of humans allows to attract methodical apparatus developed by taking into account general terms of safety theory applied to problems of ensuring biosafety. As an actual goal implementation of technologies of risk analysis in evaluation of potentially dangerous biological objects and territories of Russian Federation is determined. Analysis of legislation and normative-methodical documentation in the field of ensuring biological safety during work with pathogenic biological agents revealed a number of problems of technical, organizational and scientific nature. Proposals for their solution are given.

[Conceptual bases of biological safety. Part 1].

Up to date there is a narrow and broad interpretation of the term biological safety (BS) the world over. In the narrow sense it is defined as availability of international regulations applied to diagnostic, manufacturing, or experimental works with pathogenic biological agents (PBA) in accordance with specified levels of biological hazard and BS. In a broader context it has no national, conceptual, terminological or defying basis. Therewith, establishment of this framework has become the core issue of the study. Investigations have revealed that BS should conceptually cover the whole sphere of sanitary-and-epidemiological welfare as well as related fields such as veterinary-sanitary, phytosanitary provision, ecological safety, environmental conditions (occupational, socio-economic and geopolitical infrastructures, ecological system), and be exercised to prevent and control emergency situations (ES) of biological character. It is demonstrated that this type of ES differs from ES in the sphere of public health care of international concern which is formalized in IHR (2005), in the way that it is characterized by high socio-economic and geopolitical significance of the negative influence on human vital activities, comparable with national and international security hazard. Elaborated is the conceptual, terminological and defying toolkit of the BS broad interpretation (27 terms).

[Topical issues of biological safety under current conditions. Part 2. Conceptual, terminological, and definitive framework of biological safety].

In accordance with the established conceptual base for the up-to-date broad interpretation of biological safety, and IHR (2005), developed is the notional, terminological, and definitive framework, comprising 33 elements. Key item of the nomenclature is the biological safety that is identified as population safety (individual, social, national) from direct and (or) human environment mediated (occupational, socio-economic, geopolitical infrastructures, ecological system) exposures to hazardous biological factors. Ultimate objective of the biological safety provision is to prevent and liquidate aftermaths of emergency situations of biological character either of natural or human origin (anthropogenic) arising from direct and indirect impact of the biological threats to the public health compatible with national and international security hazard. Elaborated terminological framework allows for the construction of self-sufficient semantic content for biological safety provision, subject to formalization in legislative, normative and methodological respects and indicative of improvement as regards organizational and structural-functional groundwork of the Russian Federation National chemical and biological safety system, which is to become topical issue of Part 3.

[Determination of DNA content in individual Vibrjo cholerae cells by using flow cytofluorimetry method: comparative analysis of inhomogeneity in cells of strains with various biological properties].

AIM: Comparative analysis of DNA content in individual cells of Vibrio cholerae strains with various biological properties. MATERIALS AND METHODS: 24-hour agar cultures of 2 avirulent (lacking cholera toxin gene) and 2 virulent strains and their subcultures obtained by cultivation in 1% peptone water for 1, 3 and 5 hours were studied. DNA of the killed bacteria was dyed by a mixture of ethidium bromide and mitramycin. Ratio of cells with low, intermediate and high relative DNA content in conditional units of specific DNA fluorescence intensity was determined by flow cytofluorimetry method. The degree of inhomogeneity of the studied microbial population cells was evaluated by DNA histogram variation coefficient value. RESULTS: At the level of major statistical samples of individual V. cholerae cells a principally different reaction pattern of the studied toxigenic and non-toxigenic strains on changes of cultivation conditions was registered. CONCLUSION: Populations of cells of toxigenic V. cholerae strains in contrast to non-toxigenic probably shift to polyploid state during starvation. This phenomenon may turn out to be a differential feature in determination of the risk group (hazard) of a strain.

[Determination of DNA content in individual Yersinia pestis cells by using flow cytofluorimetry method: comparative analysis of inhomogeneity in cultures of strains with various biological properties].

AIM: Comparative analysis of Yersinia pestis strains with various biological properties by DNA content in individual cells. MATERIALS AND METHODS: Virulent strain 231, avirulent strain KM 260 (12) [231], that is its isogenic (no-plasmid) derivative, and vaccine strain EV NIIEG were used. 48-hour agar cultures of the studied strains reproduced at 28 degrees C and their subcultures obtained by cultivation of the initial cultures by aeration on liquid nutrient medium from 37 degrees C were prepared. DNA of the fixed bacteria was dyed by a mixture of ethidium bromide and mitramycin, and then the bacteria were studied by using flow cytofluorimeter for the determination of rates of cells with relatively low or high DNA content in the studied bacterial populations. The degree of inhomogeneity of a bacterial population was evaluated by DNA histogram variation coefficient value. RESULTS: In 6 hours of growth at 37 degrees C in optically non-dense bacterial cultures a high degree of DNA content per cell inhomogeneity was established that is related to the activation of DNA replication process in bacteria. In 48 hours of growth this inhomogeneity completely disappeared in the virulent strain cultures and remained in the avirulent strain cultures of the plague pathogen. Based on the studied parameters the vaccine strain held an intermediate position. CONCLUSION: Further studies of the plague culture DNA content per cell inhomogeneity may become a base for the operative strain differentiation based on pathogenicity level (hazard) for humans, and therefore the requirements for the management of safe working conditions with this microorganism.

[Development of a competitive immunoassay based on monoclonal antibodies for the detection of specific antibodies to pseudotuberculosis pathogen]. / Razrabotka konkurentnogo immunofermentnogo analiza na osnove monoklonal'nykh antitel dlia vyiavleniia spetsificheskikh antitel k vozbuditeliu psevdotuberkuleza.

A highly sensitive, effective and rapid method--a competitive alternative to ELISA--was developed for the detection of specific antibodies to Y. pseudotuberculosis. It is based on monoclonal antibodies (MAbs) recognizing the species- or serogroup specific epitopes of Y. pseudotuberculosis. The competitive ELISA was used in testing the murine hyperimmune sera and human antisera sampled from patients with different infectious intestinal diseases including several cases of pseudotuberculosis. The use of the MAbs-based ELISA in the laboratory diagnostics of pseudotuberculosis is under discussion.

[A circulation study of California serogroup and Batai viruses (fam. Bunyaviridae, genus Bunyavirus) on the territory of Saratov Province]. / Izuchenie tsirkuliatsii virusov kaliforniiskoi serogruppy i Batai (sem. Bunyaviridae, rod Bunyavirus) na territorii Saratovskoi oblasti.

Examining 337 sera from Saratov healthy residents in the neutralization test with Tyaginya and Inco viruses has revealed 56 positive results (16.6%), of which 19 (5.6%) reacted only with Tyaginya virus, 13 (3.9%) did only with Inco virus, and 24 (7.1%) simultaneously with these two viruses. Batai virus antibodies were not detected in the population. Among 80 bovine serum samples collected in the Saratov district of the region, type-specific antibodies to Tyaginya virus were found in 10 (12.5%) and a serum (1.2%) reacted with Tyaginya and Inco viruses; 49 sera (61.2%) contained Batai virus antibodies.

[Protein expression of Yersinia pestis during changes due to long-term cultivation in vivo]. / Izuchenie belkovoi ékspressii Yersinia pestis v dinamike dlitel'nogo kul'tivirovaniia in vivo.

The electrophoretic study of Yersinia pestis proteins made possible to find the significant modification of Yersinia pestis polypeptide specters when the bacteria were cultivated in semi-penetrable cells implanted into the guinea pigs peritoneum. The proteinogramms of the isolates from the implanted cells lacked the stained bands characteristic of Yersinia pestis cells grown in vitro and contained the new polypeptides absent from the bacteria grown on the Hottinger agar plates. The difference was found at the late stage of bacteria incubation in implanted cells and had the predominantly reversible characteristics. The protein of Yersinia pestis being changed in vivo is proposed to be the species specific fraction I.

[Use of deletion mutants of plasmid RP4::D3112 for the genetic analysis of Pseudomonas aeruginosa bacteriophage D3112]. / Ispol'zovanie deletsionnykh mutantov plazmidy RP::4D3112 dlia geneticheskogo analiza bakteriofaga D3112 Pseudomonas aeruginosa.

The hybrid plasmid RP4::D3112 becomes unstable in Escherichia coli K-12 cells under certain growth conditions. The deletion mutants of this plasmid are formed at a high frequency. All the deletions selected have a specific feature: they start in the left end, at the point of joining of plasmid and phage DNA, and remove different portions of the phage genome. The deletion mutants have been used for genetic mapping of D3112. We have localized the repressor gene cI (0-1.3 kb), 3 early genes (1.3-14.2 kb) and two groups of late genes (14.2-29.9 and 29.9-38 kb). Electron microscope studies of RP4::D3112 DNA and its deletion derivatives have shown that integration of D3112 genome in RP4 occurs through the ends of the genome, without permutations. It appears that bacterial nucleotide sequences joined to DNA from mature D3112 particles, to the right end of D3112 genome, are lost. Thus, transposable phages D3112 of Pseudomonas aeruginosa and E. coli Mu phage have some similarities in the genome organization and in the way of their integration into the host DNA.

[Submicroscopic features of guanylate cyclase detection in mammalian epithelial and myocardial cells]. / Submikroskopicheskie osobennosti vyiavleniia guanilattsiklazy v épitelial'nykh i miokardial'nykh kletkakh u mlekopitaiushchikh.

The electron cytochemical method of guanylate cyclase demonstrated a number of advantages making possible differentiation between the plasmalemmal, cytoplasmic, nucleoplasmic, and nucleolar localization of the enzyme. This method permitted to reveal that the enzyme possessed organ specificity of distribution in the cell and could be activated by a specific activator--sodium azide. The distribution of guanylate cyclase strictly correlated with the functional state of the cell.

[Electron microscopic demonstration of adenylate cyclase in rabbit small intestine enterocytes doubly stimulated by cholera toxin and sodium fluoride]. / Elektronno-mikroskopicheskoe vyiavlenie adenilattsiklazy énterotsitov tonkogo kishechnika krolikov pri dvoinom stimulirovanii kholernym toksinom i ftoristym natriem.

Cytochemical investigations showed adenylate cyclase in the rabbit small intestine enterocytes to be activated both with cholera toxin and sodium fluoride. Following double stimulation of adenylate cyclase in the intestinal enterocytes by the mentioned two substances maximal critical levels of cAMP were attained resulting in self-inhibition of adenylate cyclase; in this case only a low adenylate cyclase activity, if any, could be demonstrated by electron microscopy.

[Electron microscopic study of the localization of adenylate cyclase in the small intestinal epithelial cells of rabbits with NAG-infections]. / Elektronno-mikroskopicheskoe izuchenie lokalizatsii adenilattsiklazy vépitelial'nykh kletkakh tonkoi kishki krolikov pri NAG-infektsii.

As found, in NAG-infection primary intensification of adenylate cyclase activity occurred at the apical plasmalemma of the villar cells; then the process spread to the lateral and further to the basal plasmalemma of the enterocytes. At more advanced stages of NAG-infection an increased adenylate cyclase activity was observed in the crypt cells. Thus, with increased duration of the toxin action there was a gradual rise of the enzyme activity, and the epithelial cells of the small intestine in the area of local affection become involved in the pathological process.