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The tick-borne encephalitis virus (TBEV) strain C11-13 (GenBank acc. no. OQ565596) of the Siberian genotype was previously isolated from the brain of a deceased person. TBEV C11-13 variants obtained at passages 3 and 8 in SPEV cells were inoculated into the brains of white mice for subsequent passages. Full genome sequences of all virus variants were analyzed by high-throughput sequencing. A total of 41 single nucleotide substitutions were found to occur mainly in the genes for the nonstructural proteins NS3 and NS5 (GenBank MF043953, OP902894, and OP902895), and 12 amino acid substitutions were identified in the deduced protein sequences. Reverse nucleotide and amino acid substitutions were detected after three passages through mouse brains. The substitutions restored the primary structures that were characteristic of the isolate C11-13 from a human patient and changed during the eight subsequent passages in SPEV cells. In addition, the 3'-untranslated region (3'-UTR) of the viral genome increased by 306 nt. The Y3 and Y2 3'-UTR elements were found to contain imperfect L and R repeats, which were probably associated with inhibition of cellular XRN1 RNase and thus involved in the formation of subgenomic flaviviral RNAs (sfRNAs). All TBEV variants showed high-level reproduction in both cell cultures and mouse brains. The genomic changes that occurred during successive passages of TBEV are most likely due to its significant genetic variability, which ensures its efficient reproduction in various hosts and its broad distribution in various climatic zones.

The WHO has declared a SARS-CoV-2 pandemic. During a pandemic, the researches aimed at finding the new treatments for SARS-CoV-2 become relevant. The review focuses on studies of androgens and antiandrogens in this disease. Since the beginning of the COVID-19 epidemic, it has been noted that men have more severe forms of infection and higher mortality. The main cause of both the severity of the disease and the high mortality of men from COVID-19 are associated with androgens. It was found that patients receiving androgen deprivation are less likely to become infected and easily tolerate COVID-19. The researchers explain the effect of the therapy by the effect on the TMPRSS2 protein. It was found that both TMPRSS2 expression and a more severe course of coronavirus infection are observed in men with hyperandrogenism - androgenic alopecia, acne, excessive facial hair growth and increased skin oiliness. In this regard, some researchers suggest to use androgen deprivation for men at high risk of developing COVID-19. Steroid and non-steroidal antiandrogens are used for androgen deprivation. At the same time, obtaned scientific data on the relationship of severe forms and mortality of COVID-19 with low testosterone levels leads to a hypothesis about the possibility of a positive effect not of androgen devrivation therapy but of androgen replacement therapy in case of hypogonadism have diagnosed. These studies have not been completed recently, and data on the effectiveness and safety of antiandrogens and androgens in the treatment of a new coronavirus infection require clarification.

There is a decrease in the expression of the reelin gene (RELN) in the brain of schizophrenia patients, which can underlie observed cognitive abnormalities. It is suggested that this decrease is caused by the hypermethylation of the RELN promoter. The aim of the study was to investigate methylation of the RELN promoter in the peripheral blood of schizophrenia patients and its association with their cognitive deficits. A modified SMRT-BS (single-molecule real-time bisulfite sequencing) was used. We determined the methylation rate of 170 CpG sites within a 1465 bp DNA region containing the entire CpG island in the RELN promoter in 51 schizophrenia patients and 52 healthy controls. All subjects completed a battery of neuropsychological tests. There were no DNA methylation changes associated with schizophrenia. Most CpGs sites were unmethylated in both groups. At the same time, there was a variability in the methylation level of different regions within the promoter. The methylation level in the area from -258 to -151 bp relative to RELN transcription start site was a significant predictor of the index of patients' cognitive functioning if sex, age, smoking, education, and polymorphism rsl858815 had been considered. The positive correlation between the methylation rate in this region and cognitive index suggests that the hypomethylation of the RELN promoter could contribute to the development of cognitive deficits in schizophrenia.

Aim: To study the role of polymorphism rs7341475 and methylation of the reelin gene in symptoms of schizophrenia and semantic verbal fluency., Material and Methods: Genotypes at the locus rs7341475 were identified in 556 patients with schizophrenic disorders. PANSS scores were obtained for 549 patients and 221 patients performed a test for semantic verbal fluency. The association of the reelin promoter methylation with the PANSS and verbal fluency measures was evaluated in 35 patients. A five-factor model of the PANSS was used., Results: The interaction effect of sex with genotype on the PANSS scores was found (F=2.70, p=0.020). Schizophrenic men homozygous for a common allele G had the lowest scores of the positive syndrome. Verbal fluency was related to the reelin promoter methylation., Conclusion: The results suggest that polymorphism rs7341475 may be associated with the variability of positive symptomatology in schizophrenic men. At the same time, the reelin gene methylation pattern, which consists of a higher methylation level in the region of the transcription start site and a lower one in the distal region of the promoter, may be beneficial for verbal fluency.

The expression of cancer-testis (CT) genes varies with tumor type. There are tumors with high, low, and intermediate gene expressions. Tumor cells of different origin are characterized by ST gene co-expression. The expression of ST genes increases in later stages of tumor development in the presence of metastases. In colon cancer, the tumor samples showed most frequently MAGE-A and SSX mRNA. The peripheral blood samples displayed most commonly XAGE, MAGE-C, and SSX mRNA. In patients with colon cancer, the expression of TSP50, MAGE-A(1-6), and SSX1,2,4 genes was associated with a poor prognosis, that of MAGE-C1 and XAGE1 was related to a favorable prognosis.

Aim: to study reelin expression in the neocortex of fetuses and newborns with internal hydrocephalus (HC) and to reveal its features in relation to the etiology of HC., Material and Methods: The brains of fetuses and newborn at 22-40 weeks' gestation with internal HC associated with Sylvian aqueduct malformation (n=9), post-inflammatory (n=4) and post-hemorrhagic (n=5) HC were examined. In a comparison group, the fragments of brain tissue with a slit-like lumen of the lateral ventricles were no more than 0.5 cm (n=20). A standard immunohistochemical method was used to reveal reelin expression in the neocortex of the anterior third of the precentral gyrus., Results: HC associated with Sylvian aqueduct malformation is characterized by a negative reelin immunoexpression. In postinflammatory HC, the expression of reelin decreases significantly (p=0.025) with respect to the conditional norm and, in posthemorrhagic HC it is comparable with that in the comparison group., Conclusion: The features of reelin expression in the brain of fetuses and newborns at 22-40 weeks' gestation with internal HC should be considered as morphological differential and diagnostic criteria for the disease in relation to its etiology.

This review was devoted to the use of the versatile component oftumoral stroma (fibroblast activation protein, FAP) as a target of the versatile tumor therapy. The tumor is a coevolution system, which includes the microenvironment or reactive stroma differing from the normal tissue by the phenotypic and genotypic features. Important elements of the tumor microenvironment are cancer-associated fibroblasts (CAFs), which contain typical marker FAP (serine proteinase with the enzymatic activity of dipeptidyl peptidase and endopeptidase). According to the literature, more than 90% of tumors contain FAP-positive activated fibroblasts. FAP is virtually absent in normal tissues, but it is present in the embryonic and tumor tissues, which makes it a selective and versatile model. In this work, basic approaches to affecting the CAF using FAP as a target were discussed. The use of FAP as a target provides an important advantage: its proteolytic activity can be used along with the protein-targeted agents. The main directions in the therapeutic use of FAP were discussed in this work.

Objective: to estimate the diagnostic and prognostic value of analyzing the abnormal overexpression of the chimeric protein ERG, encoded by the chimeric gene TMPRSS2/ERG, in prostatic neoplasias., Material and Methods: A total of 100 prostate adenocarcinoma samples were examined. The presence of tumor and high-grade prostatic intraepithelial neoplasia (hPIN) was verified by immunohistochemical tests using anti-P504S and anti-34βE12 antibodies in serial sections; RT-PCR was employed to analyze the chimeric transcript TMPRSS2/ERG in 30 prostate adenocarcinoma samples., Results: ERG expression was noted in 46% of the adenocarcinomas and in 21% of hPIN. Eight (8%) patients were observed to have heterogeneous ERG expression: the marked reaction in some tumor portions was concurrent with its complete absence in others. Furthermore, there was ERG expression in all cases of intraductal (noninvasive) carcinoma (the foci of intraductal carcinoma were assessed as atypical cribriform lesions by light microscopy). The prognostic value of ERG expression could not be determined at the current stage of the investigation., Conclusion: The relatively low rate of ERG-positive hPIN counts in favor of the limited role of this marker in the differential diagnosis of hPIN. ERG in combination with P504S and 34βE12 is an informative marker for the differential diagnosis of hPIN with intraductal carcinoma.

The review provides an update on the pathogenesis and new treatment modalities for neovascular age-related macular degeneration (AMD). The impact of polymorphism in particular genes, including complement factor H (CFH), age-related maculopathy susceptibility 2 (ARMS2/LOC387715), and serine peptidase (HTRA1), on AMD development is discussed. Clinical presentations of different forms of exudative AMD, that is classic, occult, or more often mixed choroidal neovascularization, retinal angiomatous proliferation, and choroidal polypoidal vasculopathy, are described. Particular attention is paid to the results of recent clinical trials and safety issues around the therapy.

We have developed a biochip for the analysis of polymorphisms in candidate genes for schizophrenia: DISC1, RELN, ZNF804A, PLXNA2, COMT, SLC18A41, CACNA1C, ANK3, TPH1, PLAA and SNAP-25. Using biochip the allele and genotype frequencies in 198 patients with schizophrenia and 192 healthy individuals have been obtained. For SLC18A1 polymorphism rs2270641 A>C, the frequencies of A allele (p = 0.007) and AA genotype (p = 0.002) were lower in patients compared with healthy individuals. A significant association was found between AA genotype (p = 0.036) of the TPH1 polymorphism rs1800532 C>A and schizophrenia. The C allele (p = 0.039) of the RELNpolymorphism rs7341475 C>T were lower in patients with schizophrenia compared with healthy individuals in a tatar population. Genotype AA of the TPH1 polymorphism rs1800532 C>A were more frequent in patients with schizophrenia compared with healthy individuals. Ithas been shown that the C allele (p = 0.0001) and GC (p = = 0.0001) genotype of the PLXNA2 polymorphism rs1327175 G>C are associated with the family history in patients with paranoid schizophrenia. The obtained data suggest that SLC18A1, TPH1 and RELN gene polymorphisms are associated with the risk of paranoid schizophrenia.

The study of enzymatic and protective properties of recombinant IgA1 protease in active and mutant form showed that active form of IgA1 protease exhibited species - and type-specificity for mouse and human immunoglobulins. Mutant form, which did not exhibit enzymatic activity, had protective properties against meningococcal infection, induced by meningococcus serogroup A, B and C protecting the mice from lethal infection by living virulent culture of heterologous serogroups of meningococcus. Obtained results make it possible to consider IgA1 protease as a perspective preparation at the stages of development of polyvalent vaccine for protection the people from meningococcal infection of various etiology.

Unlabelled: Familial hypercholesterolemia (FHC) is a genetic disorder manifest as a rise in serum cholesterol level responsible for the development ofcardiovascular diseases., Aim: To study genetic peculiarities of FHC in Kareliya., Materials and Methods: 109 patients of the 196 ones with FHC (124 families) were subjected to genetic examination. Other parameters studied included the lipid spectrum, blood glucose level, ECG, 24 hr ECG monitoring, echocardiography, triplex scanning of brachiocephalic arteries and lower limb vessels, functional tests. Simon Broom criteria were used to diagnose FHC., Results: "Definitive" FHC was diagnosed in 136 (69.4%) patients, (probable) FHC in 30.6%. The total encoding region of the low density lipoprotein receptor gene was sequenced in 109 (55.6%) patients in parallel with the search for major mutations in the APOB and PCSK9 genes. A total of 13 mutations (p.G20R, c. 192del110/ins8, c.195-196insT, p.S206R, c925- 931del17, p.S447C, p.13981, p.L426P, L511S, c.1686del18/insT, p.L646I, p.N640N, c.2191delG) were identified in low density lipoprotein receptor gene; seven of them are reported for the first time in the world. No major mutations in the APOB and PCSK9 genes were found. The new c.2191delG (p.(Val73 1Serfs*6)) mutation is characterized and its segregation with familial dyslipidemia is shown. The present case is characterized by the absence of clinical picture of coronary heart disease and the family history complicated by cerebral basin lesion. Phenotypic manifestations of atherosclerosis in FHC with gene mutations need further studies.

A study on the role of CFH, HTRA and IL-8 gene polymorphism in age-related macular degeneration (AMD) development has been conducted. At the first stage of the study genetic testing was done in 69 patients with exudative AMD and 370 random Moscow citizens without the disease. The goal of the second stage was to determine the influence of gene polymorphism on patient's response to endovitreal ranibizumab treatment. For that, visual acuity and foveal thickness were assessed before and after ranibizumab injections in 120 patients with wet AMD. All patients were genotyped for the genes of interest. The results showed that the presence of homozygous 402H polymorphism in CFH gene, as well as homozygous (-625)A mutation in HTRA1 gene, determines certain clinical presentations. Moreover, visual acuity below 0.1 and presence of 402H, (-625)A and (-251)A alleles in both copies of all three genes (CFH, HTRA and IL-8) are negative predictors of disease severity and antiangiogenic treatment response.

57 Y pestis bv. caucasica strains were assayed using molecular typing. The results of these assays indicated the presence within this biovar of the three separate clonal clusters and necessity of detachment of the Leninakan mountain mesofocus (subfocus) from the structure of Transcaucasian-highland focus into self-supporting one, as well as inclusion of a part of the Pre-Araks low-mountain natural plague focus in the capacity of the subfocus along with Pre-Sevan mountain and Zanzegur-Karabakh mountain subfoci into the structure of Transcaucasian-highland focus. It was shown that the strains circulating in the East-Caucasian highland plague focus were the most ancient branch of bv. caucasica or even of the entire Y pestis phylogenetic tree.

A detailed analysis of influence of HTRA (serine peptidase) and VEGF (vascular endothelial growth factor) genes mutations is presented. The presence of one gene copy with allele of A- polymorphism rs1120638 of HTRA1 gen, T- polymorphism rs10490924 and de11443in54 of ARMS2 gene increases the risk of CNV in patients with AMD. The feature of clinical presentation in patients with CNV associated with (-625) A mutation of promoter region of HTRA1 gene in two chromosomes was fulminant course of the disease from exudative to scarring processes with fibrous tissue formation not just with sub-, but also intra- and preretinal localization. Genetic screening showed that combination of studied mutations (402H, (-625) A and (-251) A in both gene copies of CFH, HTRA and IL-8) results in the most severe and rapidly progressing form of the disease. Two new mutations were revealed in promoter region of VEGF gene: G > A replacement in position of (-72) nucleotide from transcription start and G > A replacement in 5'-nontranslated region of the 1st gene exon in position of (+31) nucleotide from transcription start.

Plasmid pUC19-recAoc carrying a mutant allele of the recA gene, which plays the key role in the control of the SOS repair system and homologous recombinational repair, causes a 1.5-fold increase in radiation resistance of Escherichia coli DeltarecA cells, as compared to the wild-type recA+ cells. The protective effect of this plasmid is drastically reduced in mutant lexA3 recADelta21 deficient in the LexA protein and in induction of the SOS regulon. Plasmid pUC19-recAoc effectively suppresses UV sensitivity of the DeltarecA mutant. Mutation recAo20 allows constitutive high-level synthesis of the RecA protein. This mutation impairs the SOS box in the operator site of the recA gene and enhances heterology of the dimer LexA binding site. These data confirm that high level of the RecA protein synthesis per se is not sufficient for the expression of gamma-inducible functions and that the derepression of lexA-dependent genes, other than recA gene, is necessary for the complete induction of the SOS repair system.

A site-directed modification in position P1 of the processing site of Bacillus intermedius glutamyl endopeptidase was carried out. Variants of the protease gene were obtained that correspond to the protein with an Ala, Asn, Ser, or Glu residue in this position substituted for the Lys residue. The residue in the P1 position of the processing site was shown to affect substantially the production of the active enzyme; however, none of the mutations leads to the complete termination of active protein production by cells.

This review is focused on recent data on structure and functions of PCSK9 proprotein convertase, a newly identified participant in cholesterol metabolism in mammalian organisms, including humans. Proprotein convertase acts as a molecular chaperone for the low density lipoprotein (LDL) receptor, targeting it to the lysosomal degradation pathway. Various mutations increasing the PCSK9 affinity toward the LDL receptor cause autosomal dominant hypercholesterolemia. In contrast, loss-of-function mutations in PCSK9 gene decrease the blood plasma cholesterol level, thus acting as a protection factor against atherosclerosis and coronary heart disease. It is supposed that pharmacological agents inhibiting the interaction between PCSK9 and LDL receptor may substantially amplify the benefits of drugs--statins and cholesterol absorption blockers--in the treatment of all types of hypercholesterolemia, including its widespread multigenic and multifactorial forms.

The recombinant strain of Bacillus subtilis bearing B. intermedius glutamyl endopeptidase gene in multicopy plasmid delta58.21 secretes the enzyme to the medium at the phase of slowing of growth and the stationary growth phase with accumulation maxima at 24 and 48 h. Enzyme samples were isolated from the culture liquid after 24 and 48 h of culturing of and were purified up to homogeneity by ion exchange chromatography on carboxymethyl cellulose and HPLC on a MonoS column. The molecular weight of the corresponding proteins was 29 kDa. Both preparations had identical structure, but differed in affinity to the specific substrate Z-Glu-pNA. The effects of Ca+ ions and specific low-molecular and protein inhibitors on the activity of the enzyme corresponding to various growth phases has been studied.

The translation initiation site in the extracellular serine subtilisin-like proteinase gene from Bacillus intermedius (aprBi) (AN AY754946) secreting at the stationary growth phase was established. The analysis of aprBi open reading frame revealed three putative translation start sites (TTG, GTG, ATG). Using SignalP online freeware program we have determined the functional activity probability of each of them. To identify the translation start point the modified subtilisin-like protease genes carrying nucleotide replacements in supposed start codons were developed using oligonucleotide-directed mutagenesis. We have investigated the expression of these genetic constructions in protease-deficient strain B. subtilis AJ73. According our results it was concluded that the translation in aprBi gene starts from GTG kodon.

SOS-response system is a cascade of reactions induced by DNA damage in a cell. Genes participate in these reactions are regulated by the LexA protein binding to specific sequence in their upstream regions. The criterion for selection of genes putatively responsible for the SOS-response is the presence of such sequence. Genes with taxon-specific regulation in Enterobacteriales, Pasteurellales, Vibrionales, Pseudomonadales and Alteromonadales were analyzed using comparative genomic approaches. Some genes have conserved sites in regulatory region and suitable function, although their function in SOS-response has not been studied in experiment. The list of such genes includes mfd, which encodes a product repairing the mother chain in case of DNA damage-caused transcription stop; VC0082, which encodes a recombinase, and VP2449, responsible for xenobiotics resistance. Overall, this study characterized the content and evolution of the LexA regulon in gamma-proteobacteria are described here.

Expression of the gene of glutamyl endopeptidase from Bacillus intermedius (gseBi) cloned on the plasmid pV has been studied in Bacillus subtilis recombinant strains with mutations of the regulatory proteins involved in sporogenesis and spore germination. It has been established that inactivation of the regulatory protein Spo0A involved in sporulation initiation resulted in a decrease in the expression of the gseBi gene by 65% on average. A mutation in the gene of the sensor histidine kinase kinA had no effect on the biosynthesis of the enzyme. Inactivation of Ger proteins regulating bacterial spore germination resulted in a 1.5-5-fold decrease in glutamyl endopeptidase activity. It has been concluded that expression of the B. intermedius glutamyl endopeptidase gene from plasmid pV in recombinant cells of B. subtilis is under impaired control by the regulatory system of Spo0F/Spo0A phosphorelay, which participates in sporulation initiation. The regulatory Ger proteins responsible for spore germination also affect expression of the gene of this enzyme.

IgA1-specific proteinases (Igase) are acknowledged as a pivotal pathogenicity factor in meningococcus (Neisseria meningitidis) and in some related bacteria. These enzymes belong to trypsin-like clan of serine proteases. They exhibit high substrate selectivity being able to discriminate between IgA1 and IgA2. On the other hand, these enzymes are able to distinguish the human IgA1 from IgA1 of non-primate species of mammals. In addition to conventional IgA1-processing enzymes, alternative enzymes were recently reported to occur in meningococci. However, the substrate specificity of the conventional Igase, its role in pathogenesis, and ability to complement functionality remains obscure. Within the framework of the present project we studied the structure of the Igase genes and their products in two highly virulent N. meningitidis serogroup A strains M9 and A208. In particular, we succeeded to find both conventional and alternative Igase genes in each genome: nucleotide sequences of these genes were deposited in the NCBI Gene Bank under the access number AY770504, AY558158, AY558159. The DNA sequence of the conventional Igase was almost entirely conserved in the two strains, whereas the recently discovered alternative Igase (formerly known as meningococcal adhesine, type 1) exhibited occurrence of a variable region spanning about 900 bp in the 5'-terminal part of the gene. Conventional genes from both strains were expressed in E. coli rendering inclusion bodies. The recombinant products were used for immunization of rabbits and exhibited reaction with both recombinant and native antigen from the N. meningitidis cultural medium.

The Bacillus intermedius glutamyl endopeptidase is a secretory serine proteinase from the subfamily of chymotrypsin. Its gene was previously cloned and sequenced. The enzyme was thoroughly characterized including 3D structure determination. The present work demonstrates that removal of 3'-noncoding region of the enzyme gene resulted in a decrease of the active glutamyl endopeptidase production level in culture of B. subtilis cells. In this 3'-noncoding region, the sequence with all typical features of transcription terminators of the Firmicutes type was found.

The biosynthesis of the subtilisin-like serine proteinase of Bacillus intermedius 3-19 by the recombinant strain Bacillus subtilis AJ73(pCS9) was found to be enhanced under salt stress conditions (growth in a medium containing 1 M NaCl and 0.25 M sodium citrate). In a recombinant strain of B. subtilis deficient in the regulatory proteins DegS and DegU, which control the synthesis of degradative enzymes, the expression of the proteinase gene was inhibited. In contrast, in the strain B. subtilis degU32 (Hy), which provides for the over-synthesis of proteins positively regulated by the DegS-DegU system, the biosynthesis of the subtilisin-like proteinase of B. intermedius 3-19 increased by 6-10 times. These data suggest that the DegS-DegU system is involved in the positive regulation of the expression of the subtilisin-like B. intermedius proteinase gene in recombinant B. subtilis strains.

Study of individual hepatitis C (HCV) proteins could help to find a molecular structure and conformation, localization of antigenic and immunogenic determinants, to reveal of protective epitopes. It is necessary for practical medicine - development of diagnostic test-systems, vaccines and therapeutics. Linear and conformation dependent epitopes of HCV proteins was localized in this work and immunogenic properties of phage displayed peptides screened on monoclonal antibodies to HCV proteins have been investigated. Eleven epitopes of four HCV proteins have been studied. Three epitopes was found as linear, two epitopes were dependent on secondary structure of proteins and one epitope was dependent on tertiary structure of NS3 protein. Aminoacid sequences of other determinants have been determined and the distinct localization of these determinants will be continued after discovering of tertiary structure of HCV proteins. It was shown, that phage mimotope 3f4 is immunogenic and could induce specific hu- moral immune response to NS5A HCV protein. The data obtained could be useful for improving of HCV diagnostic test-systems, studying of amino acid substitutions and its influence on antigenic properties of the HCV proteins. The results could help to study an immune response in patients infected with different genotypes of HCV. Phage displayed peptides mimicking the antigenic epitopes of HCV proteins could be applied to development of HCV vaccine.

The review gives recent data on the structure of hepatitis C virus genome. Linear RNA comprises one translated and two nontranslated regions: 5'UTR and 3'UTR. Translation of viral RNA gives rise to a single polyprotein precursor that contains structural and nonstructural regions. The structural region comprises one nucleocapsid core-protein and two envelope proteins known as E1 and E2. E2 protein includes two hypervariable regions: HVR1 and HVR2. The nonstructural region consists of 7 proteins: p7 (NS1), NS (metalloproteinase), NS (serine protease/ helicase), NS4A (co-factor protease, co-factor RNA-dependent RNA polymerase), and NS4B, NS5A, NS5B (RNA-dependent RNA polymerase). NS5A includes ISDR (interferon-sensitivity-determining region).

Induction of transcription by the plasmid pKM101 (mutability mediating derivate of the plasmid R46) of the sfiA gene controlling cell division and of the fruA gene encoding the fructose specific enzyme II of the phosphoenolpyruvate-phosphotransferase system in intact cultures of Escherichia coli was studied. The genes under study were fused to the bacteriophage Mu dl (Ap lac). Activation of the sfiA gene, a typical member of the SOS-regulon, was demonstrated to depend on the key genes of the SOS-system-recA and lexA. In contrast, the fruA gene that is non-inducible by the UV-light, a classical SOS-inducing agent, is not activated by the presence of the plasmid pKMIO1 in the bacterial cells. The data obtained suggest that the presence of pKMIO1 plasmid in the Escherichia coli cells induces a SOS-signal as a consequence of the plasmid DNA replication or its conjugative transfer.

We studied the biosynthesis of Bacillus intermedius glutamyl endopeptidase in the recombinant Bacillus subtilis strain AJ73 delta58.21 during the stationary growth phase. We optimized the composition of the culture medium to favor effective enzyme production during the stationary growth phase, and found that the nutritional requirements for glutamyl endopeptidase synthesis were different in the stationary phase and growth retardation phase. Proteinase accumulation was activated by complex organic substrates (casein and gelatin). During final stages of the culture growth, the enzyme production was stimulated by Ca2+, Mn2+, and Co2+ and inhibited by Zn2+, Fe2+, and Cu2+. The synthesis of glutamyl endopeptidase in the late stationary phase was not inhibited by glucose, unlike that in the trophophase during proliferation. We conclude that the regulatory mechanisms of proteinase synthesis during vegetative growth and sporulation are different.

The growth of the recombinant Bacillus subtilis strain AJ73 carrying the Bacillus intermedius 3-19 glutamyl endopeptidase gene on a multicopy plasmid and the effect of some nutrients on the efficiency of extracellular glutamyl endopeptidase production in the stationary growth phase were studied. In this phase, the concentration of glutamyl endopeptidase in the culture liquid peaked at the 48th and 78th h of cultivation and depended on the composition of the cultivation medium. Unlike the synthesis of glutamyl endopeptidase in the trophophase (i.e., during vegetative growth), which was suppressed by glucose, the synthesis of this enzyme during sporulation was resistant to glucose present in the cultivation medium. A multifactorial experimental design allowed optimal proportions between the concentrations of major nutrients (peptone and inorganic phosphate) to be determined. Inorganic phosphate and ammonium ions augmented the production of glutamyl endopeptidase by 30-150%, and complex organic substrates, such as casein and gelatin, enhanced the production of glutamyl endopeptidase by 50-100%. During sporulation, the production of glutamyl endopeptidase was stimulated by some bivalent cations (Ca2+, Mg2+, and Co2+) and inhibited by others (Zn2+, Fe2+, and Cu2+). The inference is drawn that the regulatory mechanisms of glutamyl endopeptidase synthesis during vegetative growth and sporulation are different.

The genetic polymorphism of metabolizers of tobacco smoke carcinogens can influence individual susceptibility to lung cancer. The study was concerned with the Mspl-polymorphism of the CYP1A1 gene responsible for encoding aryl hydrocarbon hydroxylase. It also plays a role in the activation of polycyclic aromatic hydrocarbons (PAH). The CYP1A1 alleles and genotype distribution in 146 lung cancer patients was compared with that in 230 healthy donors. Another control group consisted of 259 "cancer-resistant" subjects, i.e. tumor-free smokers and non-smokers aged 75 and more. The CYP1A1 allele incidence (19%) in patients with squamous lung cancer was significantly higher than in the control cohorts (11%) which is consistent with the leading role of PAH in the etiology of this pathology.

A comparative analysis of the genome of V. cholerae O139 strains isolated in Russia's territory from patients with cholera and from the environment showed essential differences in their structures. The genome of clinical strains possessed all tested genes associated with virulence (ctxAB, zot, ace, rstC, rtxA, hap, toxR and toxT) and the at-tRS site for the CTXp phage DNA integration. As for the O139 V. cholerae chromosome strains isolated from water, 70% of the studied genes (ctxAB, zot, ace, rstC, tcpA, and toxT) and the attRS sequence were not detected in them. A lack of the key virulence genes in O139-serogroup "water" vibrios, including genes of toxin-coregulated adhesion pili. (that are receptors for the CTXp phage), and of the attachment site of the above phage are indicative of that the O139 V. cholerae strains isolated from open water sources located in different Russia's regions are epidemically negligible.

Regulation of activity of the proteolytic sites of Lon protease was studied. It was found that ATP-Mg has the properties of a noncompetitive activator of peptidase sites. The processive mechanism of the hydrolysis of protein substrates by Lon protease was experimentally confirmed under the conditions of ATP hydrolysis. It was shown that the oligomeric state of the enzyme is the necessary prerequisite for the processive proteolysis by the native Lon protease. The study of the properties of the mixed mutant Lon-K362Q/S679A confirmed the existence of the intra- and intersubunit pathways of signal transduction from the ATPase to proteolytic sites. The mutual influence of substrates of Lon protease was studied, and the existence of cooperative interactions between the peptidase sites in the oligomeric enzyme was suggested.

A study was made of the effect of RcsA and RcsB on the Vibrio fischeri lux expression in Escherichia coli. RcsA suppressed the LuxR activity and thereby inhibited expression of the lux genes coding for luciferase and reductase. In osmotic shock, RcsA-RcsB activated lux expression and, consequently, bioluminescence of E. coli cells in the early log phase.

It is established that ATP-dependent protease Lon family belongs to the serine-lysine peptide hydrolases clan. Significant similarity of amino acid sequences of proteases Lon and repressors LexA in the regions including the catalytic serine and lysine residues is revealed by comparing primary structures of different families of the enzymes with Ser-Lys catalytic dyad. The both Lon and LexA families are shown to be divided into two subfamilies in accordance with the nature of amino acids in the catalytically active serine environment. Putative DNA binding sites are revealed in proteolytic domains of Lon A subfamily. Similarities and distinctions of the all families peptide hydrolases of the clan in the regions of their active centers are discussed.

The characterization of known Salmonella vaccine strains and different attenuated mutants used for developing new vaccines is presented. The use of attenuated Salmonella strains as vaccine vector for the supply of heterologous antigens opens prospects for the creation of effective and commonly available vaccines which approximate the "ideal" vaccine in their qualitative characteristics. The possibility of the genetic modification of attenuated strains permits their targeted reconstruction, considering the specific features of the formation of immune response to the definite heterologous antigen supplied to the body by the bacterial vector.

A homozygous insertion mutant with the inactivated clpP2 gene, which encodes the proteolytic subunit of ATP-dependent peptidase, was obtained in the unicellular cyanobacterium Synechocystis sp. PCC 6803. The mutant cannot grow under photoautotrophic conditions, but cells grown under heterotrophic conditions in a glucose-containing medium have active photosystems I and II (PS I and PS II). The loss of capacity for photoautotrophic growth is determined by a high sensitivity of mutant cells to the inactivating effect of light. Their incubation under light with an intensity above 10 microE m-2 s-1 inhibits cell growth in culture and causes degradation of photosynthetic pigments. It is proposed that the ClpP2 peptidase is involved in the protection of Synechocystis 6803 cells from photoinhibition.

The absence of direct correlation between the efficiency of functioning of ATPase and peptidehydrolase sites of Lon protease was revealed. It was shown that Lon protease is an allosteric enzyme, in which the catalytic activity of peptidehydrolase sites is determined by the binding of nucleotides, their magnesium complexes, and free magnesium ions in the enzyme's ATPase sites. It was revealed that complex ADP-Mg, an inhibitor of the native enzyme, is an activator of the Lon-K362Q form of the Lon protease mutant in the ATPase site. Considered are variants of intersite functional contacts realizing in the enzyme. The existence of two ways of signal transduction was established from the ATPase sites to peptidehydrolase ones in the Lon protease oligomer--intra- and intersubunit ways. Location of the enzyme ATPase sites is suggested in the areas of the complementary surfaces of subunits. It is hypothesized that ATP hydrolysis upon degradation of protein substrates by the E. coli Lon protease in vivo acts as a factor of restriction of the enzyme's degrading activity.

It was found that, depending on their frequency, mechanical vibrations (MVs) can either stimulate (4 Hz) or inhibit (50 Hz) the growth and the division of the lon mutant of Escherichia coli K-12. Similar effects were observed when the MV-treated nutrient medium was inoculated with untreated mutant cells. MVs enhanced the motility of mutant cells and the fragmentation of filament cells always present in the populations of lon mutants.

Functional destination of propeptides and precursors in bacillar secretory proteases remains uncertain. Formerly deletion assay demonstrated folding and secretion of subtilisin E, chymotrypsin-like protease SGPB from S. griseus and B. cereus metalloprotease to depend on full-length propeptide in the precursors. Actually an artificial B. amyloliquefaciens metalloprotease gene with deletion of 51 amino acid residues from N-terminus was constructed with regard to carry out functional mapping of secretory metalloprotease propeptides. B. subtilis wprA gene 5'-terminal region spanning promoter and secretory leader was coupled to provide transcription to the truncated gene and secretion to its product. B. subtilis clones bearing a plasmid with the modified gene synthesised an active mature metalloprotease.

A secretory system based on L-form cells of Proteus mirabilis was developed for production of native Bacillus licheniformis glutamylendopeptidase precursor never formerly available. The produced precursor was stable per se under physiological conditions and in presence of trypsin and glutamylendopeptidase from B. intermedius. Complete conversion of the precursor to the mature glutamylendopeptidase was performed by bacillar metalloproteases and subtilisin. The artificially processed glutamylendopeptidase was purified by affinity chromatography on bacitracin-sepharose. Native tertiary structure in the purified glutamylendopeptidase was confirmed by demonstrating its activity towards a specific chromogenous peptide substrate.

Some pathogenic bacteria have been demonstrated to secrete specific IgA1-proteases, the enzymes cleaving the molecules of the first subclass of human immunoglobulin (IgA1) in the single point from the hinge with the formation of Fab- and Fc-fragments. Cleavage generally deprives immunoglobulins having defense properties and the enzymes are considered as pathogenic factors. How to determine the activity, purification, and the promises of use of IgA-proteases is described. Whether inactivated meningococcal IgA1-protease as a vaccine against any of the five (A, B, C, Y, W135) serotypes of pathogenic meningococci is discussed.

A nutrient medium was elaborated for the efficient production of glutamyl endopeptidase by the recombinant Bacillus subtilis strain AJ73 bearing the Bacillus intermedius 3-19 glutamyl endopeptidase gene within a multicopy plasmid. Optimal concentrations of the main nutrients, peptone and inorganic phosphate, were found using a multifactor approach. To provide for active growth and efficient glutamyl endopeptidase production, the cultivation medium of the recombinant strain should be enriched in phosphorus, organic and inorganic nitrogen sources, and yeast extract. Complex protein substrates, such as casein and gelatin, enhanced the biosynthesis of glutamyl endopeptidase. At the same time, easily metabolizable carbon sources suppressed it. The production of glutamyl endopeptidase was stimulated by the bivalent cations Ca2+, Mg2+, and Co2+.

To study the effect of RNA interference (RNAi) on the activity of gene lon in Escherichia coli, genetic constructs were used that could express RNA molecules complementary to the 5' region of lon mRNA in the same direction. These RNAs were termed parallel RNAs (pRNAs). Two approaches were used to control expression. In one approach, lon gene activity was estimated genetically, based on the effect of the Lon protease on bioluminescence determined by the Vibrio fischeri lux regulon. The other approach was direct testing of ATP-dependent proteolysis in vitro. It was found that pRNA considerably suppressed lon expression. The antiparallel RNA (apRNA) was a less effective suppressor of this gene. The specific RNAi was found to decay gradually by the 40th generation. The data obtained indicate that Eubacterium cells have mechanisms for specific regulation of gene activity that are sensitive to the formation of both parallel and antiparallel RNA duplexes involving mRNA of the given gene.

Enzymic and structural peculiarities of the ATP-dependent Lon protease from Escherichia coli and its mutant and modified forms were studied. Amino acid residues important for the function of proteolytic and ATPase sites and for the transmission of the interdomain signals of the activity coupling were found. It was shown that the protein substrates are hydrolyzed only by the full-size enzyme, whereas the isolated proteolytic domain displays a peptide-hydrolyzing activity.

The possibility of application of the bioluminescence method (Lux-test) for studying in vivo functional activity of Escherichia coli protease Lon and its mutants was demonstrated. This assay is based on the capacity of protease Lon and its mutant forms for specific degradation of the LuxR protein, a positive transcriptional activator of the right operon luxICDABE from the marine bacterium Vibrio fischeri, and thus to affect the level of AB luciferase in the cells. A correlation between in vitro activity of the protease Lon mutants and the intensity of bioluminescence measured by the Lux-test was revealed.

A gene coding for thermostable serine protease from Thermoactinomyces sp. K50 is cloned and expressed in Bacillus subtilis cells. Restriction map of cloned DNA fragment is determined. Thermostability and temperature optimum of proteolytic activity of the cloned gene product are lower than those of the natural proteinase of Thermoactinomyces sp. K50. Serine protease, a product of cloned gene, is highly sensitive to proteolysis and its degradation can be prevented by Ca2+ ions.

The functional domain boundaries of the ATP-dependent Lon proteases were identified by comparative analysis of the amino acid sequences of the enzymes from evolutionarily distant organisms. Modified forms of the Escherichia coli Lon protease with the elongated or substituted N-terminal domain and a truncated enzyme lacking the N-terminal domain were obtained through genetic engineering methods. Analysis of the enzymatic properties of the resulting modified forms of Lon protease revealed the importance of the N-terminal domain in its function.