[Somatic mutations of the P53 gene in stomach cancer].

The paper deals with a study of p53 gene somatic mutations in tumor cell genomes from patients with stomach cancers of different histological patterns. It used sequential and molecular cloning methods. The former involved amplicones characterized by abnormal volatility following SSCP analysis of plasmids from 9 tumors. Replacement nucleotides were identified in 4 tumors (intestinal--2, diffuse--2). Among 8 mutations were 1 single-nucleotide deletion in codon-249 with shifting sensing frame and one targeted mutation. Five of the former were missens-mutations which caused amino acid replacement while the other two silent mutations did not. Exon-assisted analysis of p53 ("wild") gene identified cells with stable structure in each tumor (1 mutation--2; 3 mutations--2 including genuinely-paired mutations in 1 exon). All mutations occurred in structurally and functionally important codons. Our evidence corroborated earlier data of SSCP analysis on tumor cell presence in populations with variable p53 genomes.

[Genetic and clinical and pathological characteristics of breast cancer in premenopausal and postmenopausal women].

This study involved 525 breast cancer (BC) patients of T2-4N0-2M0 stages at the age of 35 years and older. Significant differences in clinical and pathological characteristics between premenopausal and postmenopausal BC patients were found. Mostly marked differences were shown for positive lymph node correlation with distant metastasis, multicentric growth and local recurrence depending on menopause status. The prevalence of various morphological structures in primary tumors was appeared to be associated with different forms of tumor progression in pre- and postmenopausal women. We have studied polymorphisms in 15 genes involved in major cancer related pathways (apoptosis, interleukins, folate metabolism enzymes genes). We found that variant genotypes of MTHFR and DHFR genes were associated with an increased BC risk among premenopausal women while polymorphism in IL-18, p53 genes were associated with BC among postmenopausal women. These results demonstrate novel biological information, which points the different mechanisms contributed to breast cancer progression in premenopausal and postmenopausal women.

[Functional polymorphism of p53 and CCR5 genes in the long-lived of the Siberian region]. / Funktsional'nyi polimorfizm genov p53 i CCR5 u dolgozhitelei Sibirskogo regiona.

The P53 protein is a key regulator of modified-cell apoptosis. The functional oligonucleotide polymorphism of the p53 gene causes the substitution of arginine (Arg) for praline (Pro) in the codon 72. A reduced apoptotic activity of p53 and, as a consequence, development of oncology pathology is associated with the above polymorphism. CCR5 is a compound transmembrane receptor-protein, which apart from chemokines, binds with some molecules and is a coreceptor for HIV-1. 32 bp deletion within the CCR5 encoding region results in the loss of the protein's receptor function. It has been demonstrated that the transmission of the "external" (in respect to cell) stimulus, via the CCR5 system, induces expression of the p53 gene and initiates apoptosis. Allele variants and p53 and CCR5 genotypes (separately and in combinations) were investigated, within the present case study, for 131 long-livers from Novosibirsk and Tyumen Regions. A trend was detected towards accumulation of the p53 Pro alleles in association with the CCR5del32 allele in the study group, which, as the authors believe, can enhance the genome resistance to variable factors that cut the life span.

[Biological effects of interferon, produced by recombinant bacteria of the probiotic preparation subalin]. / Biologicheskie éffekty interferona, produtsiruemogo rekombinantnymi bakteriiami preparata-probiotika subalin.

The present review deals with the analysis of biological and functional activities of recombinant bacteria Bacillus subtilis IF-alpha 2335 are producing a human interferon. The interferon-producing bacteria are constructed on a basis commercial probiotic strain B.subtilis 2335, carrying a recombinant plasmid pMBM 105 with the gene of human alpha-2 interferon. The implementation of the recombinant strain in the preparation probiotic, received a designation "Subalin", necessitates to verify a number of immunologic activities and to perform successive protective effects. Interferon, synthesized by recombinant bacteria shows the activity on macroorganism at oral and rectal application of preparation. Subalin was shown antivirus and antitumor activity and preservation by recombinat bacteria of antagonistic properties. The mechanisms of the positive effect of subalin were considered: this effect was shown to be due to the action of interferon excreted by recombinant bacteria into the mucous of different biotopes of host.

[Use of recombinant Salmonella strains for supplying the macro-organism with biologically active molecules]. / Ispol'zovanie rekombinantnykh shtammov sal'monell dlia dostavki v organizm biologicheski aktivnykh molekul.

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.

[Experimental evaluation of the biological safety of gene-engineered bacteria using a model strain Bacillus subtilis interferon-producing strain]. / Eksperimental'naia otsenka biobezopasnosti genno-inzhenernykh bakterii na modeli shtamma Bacillus subtilis, produtsiruiushchego interferon.

The in vitro and in vivo evaluation of the biological and ecological safety of genetically modified bacteria (GMB) was carried out on B. subtilis recombinant strain 2335/105, capable of producing human interferon alpha-2, used as experimental model. As shown in this investigation made with the use of bacteriological analysis and polymerase chain reaction, the oral administration of GMB to calves, chickens and white mice produced no disturbances in the microbial ecology of the gastrointestinal tract of warm-blooded animals and did not lead to the appearance of spontaneous transformants. The present work is the first experimental evaluation of the biological safety of genetically modified microorganisms, used as the component of Subalin, a probiotic preparation intended for use in veterinary practice.

[Use of phage display for detecting single-nucleotide differences in genes]. / Ispol'zovanie fagovogo displeia dlia obnaruzheniia odnonukleotidnykh razlichii v genakh.

Experimental strategy has been developed for selection of mismatched DNA binding phages from library of E. coli f1 filamentous phages carrying random peptide inserts on the surface of bacteriophage particles. The strategy is based on the use of phage display technique, DNA heteroduplexes (with single nucleotide variations), and paramagnetic beads. DNA heteroduplexes have been obtained from biotin-labeled PCR product. During the first stage the phage particles were incubated with DNA heteroduplexes possessing mismatched nucleotides. The next step after elimination of free phages and separation of bound phages from DNA heteroduplexes was subtraction of phages binding with DNA heteroduplexes (without mismatched nucleotides). Phages selected by this method were capable of discriminating DNA heteroduplexes with single nucleotide variations from DNA homoduplexes. Phages immobilized on solid base retain their activity and specificity, and therefore can be used for developing a new screening automated method for detecting point mutations and gene polymorphism.

[Role of macrophages in the antitumor action of the recombinant probiotic Subalin]. / Rol' makrofagov v realizatsii antiblastomnogo deistviia rekombinantnogo probiotika Subalina.

Course administration of the recombinant probiotic subalin was shown to significantly potentiate the cytotoxic action of macrophages from healthy and tumor-bearing mice against mastocytoma P-815 cells and syngeneic target cells of lung sarcoma of Lewis. Carrageenan, an inhibitor of macrophage activity, significantly cut down the antimetastaic effect of subalin. The crucial role of macrophages in the mechanism of this function of the drug is discussed.

[Stress-inducible bacterial proteins and virulence]. / Stressor-indutsibel'nye bakterial'nye belki i virulentnost'.

Different species of pathogenic bacteria, including Salmonella, Neisseria, Listeria and Francisella have been used to demonstrate relationship between the synthesis of stressor induced proteins by cells and the phenotypic manifestation of their virulence. The impact of such external factors as high temperature, low pH, osmolarity, substrate limitation, the content of active forms of oxygen, etc. is accompanied by the synthesis of different stressor induced proteins playing a complex role. Under unfavorable environmental conditions the synthesis of these proteins ensures the survival of the infective agents. Under conditions of a macroorganism synthesis of some stressor induced proteins promotes the survival of infective agents and their resistance to the action of humoral and cell-mediated protective factors of the host. As is known, the expression of virulence genes is not constitutive. The expression of these genes greatly depends on environmental conditions and its induction is determined by extra- or intracellular location of the infective agent. Several systems of the regulation of bacterial pathogenicity factors have been described that are relatively not numerous, conservative and respond to external signals. The relevance of a number of stressor induced proteins of bacteria to virulence associated factors is discussed.

[Adjuvant properties of subalin, a recombinant interferon-producing probiotic]. / Ad''iuvantnye svoistva rekombinantnogo probiotika Subalina, produtsiruiushchego interferon.

The adjuvant properties of subalin, a recombinant probiotic prepared from live bacteria Bacillus subtilis producing human alpha 2-interferon were studied in the scheme of its use with vaccines against parvovirus enteritis and distemper. Subalin was shown to be capable of preventing immunosuppression caused by the injection of vaccines, accelerating the formation of the antigen-specific clone of memory cells and enhancing antigen-specific immune response. The mechanisms of the adjuvant effect of subalin were considered; this effect was shown to be due to the action of interferon excreted by bacteria of B. subtilis into the lumen of the intestine. The advantages of this method of interferon supply and the prospects of using subalin preparation as adjuvant are discussed.

[Construction of an expression plasmid vector for accomplishing in vivo delivery of recombinant biologically active proteins. 2. Synthesis of HBcAG in a vaccine strain of Salmonella choleraesuis]. / Konstruirovanie ékspressionnogo plazmidnoto vektora dlia osushchestvleniia dostavki in vivo rekombinantnykh biologicheski aktivnykh belkov. 2. Sintez HBcAg v vaktsinnom shtamme Salmonella choleraesuis.

The capacity of a previously described plasmid vector pAZ to deliver bioactive proteins to targets in vivo has been studied. This vector molecule has a strong constitutive promoter, is extremely stable in cells of vaccinal S. choleraesuis strain, and encodes the synthesis of marker protein beta-galactosidase which helps monitor the vector's fate in the host. The gene encoding hepatitis B virus core antigen (HBcAg) has been inserted into pAZ under its constitutive promoter. The resultant recombinant plasmid p19-24 has been used to transform Enterobacteriaceae (E. coli and S. choleraesuis) cells. Transformed cells produce immunologically active HBcAg. p19-24 was stable in S. choleraesuis cells during their culturing and during this strain persistence in mice. Triple oral immunization of rabbits in a dose of 1 x 10(9) S. choleraesuis cells TC177 induced the production of virus-specific antibodies. Successful transformation of cells of another vaccinal strain S. abortus ovis by this plasmid extends the potentialities of the vector. The results demonstrate good prospects of using pAZ vector for the construction of live oral vaccines.

[Cloning and gene expression in lactic acid bacteria]. / Klonirovanie i ékspressia genov v molochnokislykh bakterii.

The possibility of using the genera Lactobacillus and Lactococcus as vector representatives is widely discussed at present. The prospects of the construction of recombinant bacteria are closely connected with the solution of a number of problems: the level of the transcription of cloned genes, the effectiveness of the translation of heterologous mRNA, the stability of protein with respect to bacterial intracellular proteases, the method by protein molecules leave the cell (by secretion or as the result of lysis). To prevent segregation instability, the construction of vector molecules on the basis of stable cryptic plasmids found in wild strains of lactic acid bacteria was proposed. High copying plasmids with low molecular weight were detected in L. plantarum and L. pentosus strains. Several plasmids with molecular weights of 1.7, 1.8 and 2.3 kb were isolated from bacterial cells to be used as the basis for the construction of vector molecules. Genes of chloramphenicol- and erythromycin-resistance from Staphylococcus aureus plasmids were used as marker genes ensuring cell transformation. The vector plasmids thus constructed exhibited high transformation activity in the electroporation of different strains, including L. casei, L. plantarum, L. acidophilus, L. fermentum and L. brevis which could be classified with the replicons of a wide circle of hosts. But the use of these plasmids was limited due to the risk of the uncontrolled dissemination of recombinant plasmids. L. acidophilus were also found to have strictly specific plasmids with good prospects of being used as the basis for the creation of vectors, incapable of dissemination. In addition to the search of strain-specific plasmids, incapable of uncontrolled gene transmission, the use of chromosome-integrated heterologous genes is recommended in cloning to ensure the maximum safety.

[The study of heterogeneity of plasmid-bearing and plasmid-f ree populations of Bacillus subtilis under different environmental conditions]. / Izuchenie geterogennosti v populiatsiiakh plazmidosoderzhashchego i besplazmidnogo shtammov Bacillus subtilis v raznykh usloviiakh su shchestvovaniia.

The population heterogeneity of recombinant and plasmid-free Bacillus subtilis strains introduced into aquatic microcosms was studied. After introduction, the population of the plasmid-free strain B. subtilis 2335 in microcosms has long been represented by both vegetative cells and spores, whereas, already ten days after introduction, the population of the recombinant strain B. subtilis 2335/105 (Km[symbol: see text]nf+) was represented only by spores. The number of plasmid copies in the spore isolates of the recombinant strain was the same as before introduction, but the plasmid abundance in the vegetative isolates of this strain decreased. The isolates of B. subtilis 2335/105 obtained from microcosms and the variants of this strain obtained by ten successive subcultures on M9 and 0.1 x M9 media with and without kanamycin (Km) differed in the number of plasmid copies, Km resistance, and maximum biomass yield during batch cultivation. Irrespective of the presence of Km, more than 50% of the variants subcultured on M9 medium showed reduced plasmid abundance. At the same time, about 70% of the variants subcultured on 0.1 x M9 medium with Km and 90% of the variants subcultured on the same medium without Km retained the initial number of plasmid copies. The variants subcultured on media with Km retained the initial biomass level. In more than 70% of the variants isolated from media without Km, the biomass yield increased.

[Change in regulation of catabolite-dependent lux-operon, cloned in a recombinant plasmid, under the effect of environmental factors]. / Izmenenie reguliatsii katabolitzavisimogo lux-operona, klonirovannogo v rekominantnoi plazmide, pod vozdeistviem faktorov vneshnei sredy.

Expression of the lux-genes cloned on the recombinant plasmid pPHL7 (AprLux+) in Escherichia coli z905 cells was studied in various environments, including model aquatic ecosystems. Expression of the lux-genes strongly depended on the nutritional status of the medium. In particular, the cultivation of cells in nutrient-rich medium favored the maintenance of the initial level of expression of the lux-operon, whereas nutrient limitation induced recombinant cell variants with an impaired control of the catabolite-dependent lux-operon. On the other hand, long-term laboratory cultivation of recombinant strain in nutrient-deficient media or its long-term life in model aquatic ecosystems led to the accumulation of cells with a stringent control on the cloned lux-genes in the bacterial population. The presence of the selective factor (ampicillin) in the medium had no significant effect on the expression of the lux-operon.

[Design of stimulating agents of non-specific resistance system]. / Sozdanie sredstv stimuliatsii sistemy nespetsificheskoi rezistentnosti.

The paper briefly reviews a study on the design of drugs enhancing the body's nonspecific resistance to pathogenic agents. To examine the potential regulatory effects on cytokine function was a main trend. The interferon inductor ridostin, dsRNA of microbiological origin, cytokines, as well as the recombinant probiotic strain yielding interferon alpha-2 synthesis were used as a pharmacological agent. This was shown by using a wide range of experimental models wherein these preparations activated the components of the non-specific resistance system resulting in the host's increased capacity to eliminate invasive agents and transformed cells.

[Modulation of the antitumor effect of cyclophosphamide by the recombinant probiotic Subalin]. / Moduliatsiia protivoopukholevoi aktivnosti tsiklofosfana rekombinantnym probiotikom Subalinom.

A new preparation "subalin" developed on the basis of a recombinant saprophyte strain of Bacillus subtilis 2135/105 producing human interferon alpha-2 has been tested for its ability of increasing the effectiveness of antitumor therapy. The study used Lewis lung carcinoma bearing mice C57B1/6 treated with injections of 60 mg/kg body cyclophosphamide, intraperitoneally, on days 3 and 7 of tumor growth. Subalin was administered per as on days 1-14 after tumor transplantation. Combined treatment with cyclophosphamide and subalin resulted in a significantly higher inhibition of primary tumor growth and metastatic spreading as compared with control receiving cyclophosphamide alone. Also, there were fewer animals with metastasis; the average number of these lesions per animal and the total affected area were less. Subalin proved to exert a moderate antitumor and antimetastatic effect. The cytostatic activity of peritoneal macrophages was higher in mice treated with subalin. It is suggested that the chemo-sensitizing and antitumor effects of subalin are due to its ability to induce endogenous interferon production.

[Recombinant probiotics: problems and prospects of their use for medicine and veterinary practice]. / Recombinantnye probiotiki: problemy i perspektivy ispolzovaniia v meditsine i veterenarii.

Approaches to designing a new probiotic class based on recombinant strains of bacteria that produce the predetermined therapeutic proteins are dealt with. The prospects of the approach are shown via studies of the biological properties of Bacillus subtilis 2335 strain transformed by the plasmid encoding the synthesis of human interferon alpha-2. The recombinant strain was demonstrated to preserve the high antagonistic activity of the parent culture (the bases of the probiotic biosporine) and to acquire marked antiviral properties due to interferon synthesis. The antiviral activity of the designed strain was shown by in vitro and in vivo experiments on experimental viral infections. By using this strain, the authors designed the new probiotic subaline, a promising biological agent for medicine and veterinary practice. Subalin has a number of advantages: it combines antibacterial and antiviral properties, is easy to use and prepare.

[Adaptation of a Bacillus subtilis strain, containing a recombinant plasmid with a human interferon-alpha2 gene, to various environmental conditions]. / Adaptatsiia shtamma Bacillus subtilis, soderzhashchego rekombinantnuiu plazmidu s genom interferona-alpha2 cheloveka, k raznym usloviiam sushchestvovaniia.

Adaptation of the Bacillus subtilis strain 2335/105 (Km Inf+) containing a recombinant plasmid encoding the extracellular human interferon alpha 2 was studied under various conditions. Stability of the plasmid in the population of B. subtilis 2335/105 was estimated under nonselective conditions. The plasmid-free cells and cells with a low number of plasmid copies were found to accumulate progressively, constituting 80% of the population after 10 culture passages, indicating the poor competitiveness of cells carrying a high number of plasmid copies. The behavior of vegetative cells of the recombinant strain introduced into aquatic microcosms differing in trophic chain length was studied. Within the first 10 days, the lysis of vegetative cells of B. subtilis 2335/105 occurred; the number of viable spores was very low but remained constant for half a year.

[Design of an expression vector for delivery of in vivo recombinant biologically active proteins. 1. Synthesis of the antigenic determinant of HIV-1 gp120 and gp41 proteins in enterobacteria]. / Konstruirovanie ékspressionnogo plazmidnogo vektora dlia osushchestvleniia dostavki in vivo rekombinantnykh biologicheski aktivnykh belkov. 1. Sintez antigennykh determinant belkov gp120 i gp41 VICh-1 v énterobakteriiakh.

High-level expression vector pAZ was constructed for in vivo delivery of bioactive recombinant proteins, antigenic determinants, among other things. This vector meets the requirements to construction of recombinant bacteria as live oral carriers. It has a strong constitutive promoter, high stability in E.coli and vaccine strain Salmonella cells, and, moreover, encodes in addition for the marker protein (beta-galactosidase) which will later help follow up the fate of bacterial carriers and their interactions in the microorganism. Several recombinant plasmids encoding for beta-galactosidase variants with insertions of short fragments of HIV-1 gp41 and gp120 proteins, which were previously shown to be antigenic determinants, have been constructed on the basis of pAZ. E.coli and vaccine strain Salmonella cells were transformed by recombinant plasmids. To a considerable extent the level of hybrid protein synthesis depends on the structure of the antigenic determinant inserted. The maximal level of synthesis in E.coli is 16%. This hybrid protein could be isolated and purified (up to 90%) with the yield of 4 to 6 mg/g of wet cells. Almost all the hybrid proteins were immunologically reactive, as shown by ELISA with nonfractionated lysates and purified hybrids. In both strains in vitro stability of the vector and recombinant plasmids was at least 90% after 10 passages (about 140 generations) under random conditions. This paper sums up the first stage of construction of recombinant bacteria as live oral carriers.