Pathological Changes in the Gastrocnemius Muscle throughout Hind Limb Ischemia Modeling in Rats.

We present a two-stage model for the study of chronic hind limb ischemia in rats. In the area of ischemia, sclerotic changes with atrophic rhabdomyocytes and reduced vascularization were revealed. CD31 expression in the endothelium increased proportionally to the number of vessels in the ischemic zone, and at the same time, focal expression of ßIII-tubulin was detected in the newly formed nerve fibers. These histological features are equivalent to the development of peripheral arterial disease in humans, which allows using our model in the search for new therapeutic strategies.

The Study of TRPV1 Channels of the Central Nervous System and Their Effect on Anxiety in ICR Mice.

The TRPV1 channel is actively involved in various neuronal processes and is found in various structures of the nervous system, including peripheral and central neurons, sensory ganglia, spinal cord, and various parts of the brain. Due to its ability to respond to various stimuli, TRPV1 can have a significant impact on the body's responses to stress. Studies indicate the involvement of TRPV1 in the regulation of anxiety behavior. Suppression of TRPV1 activity leads to a decrease in the level of anxiety in animals, which indicates the importance of this channel in psychoemotional regulation. A promising compound for inhibiting this channel is the APHC3 peptide, which is a selective receptor antagonist. The results obtained this study show that this peptide has a pronounced anxiolytic effect, reducing the level of anxiety in the studied animals.

Characterization of Risk Factors for Modeling of Type 2 Diabetes Mellitus Induced by a High-Fat Diet in C57BL/6 Mice.

Type 2 diabetes mellitus develops due to a combination of genetic and environmental factors. C57BL/6 mice prone to obesity and leptin resistance were kept on a high-fat diet for 21 weeks. The animals showed a significant increase in fasting and postprandial glucose levels and body weight, the development of insulin resistance, and by week 18, an increase in the serum TNFα level. Metformin therapy at a dose of 250 mg/kg was effective against the background of disturbances in carbohydrate metabolism: animals showed a significant decrease in insulin resistance and TNFα level.

[CONSTRUCTION AND PROPERTIES OF THE FRANCISELLA TULARENSIS VACCINE STRAIN WITHOUT ONE COPY OF THE IGLC GENE AND WITHOUT RECA GENE].

The live vaccine based on the Francisella tularensis subsp. holarctica vaccine strain 15 NIIEG line is used in Russia against tularemia. This vaccine is highly effective, but fairly unstable. Therefore, development of stable live tularemia vaccine with minimal side effect is rather urgent. The method of allel removal in the F. tularensis vaccine strain was used to remove one copy of the iglC gene, which is required to provide intracellular production of the vaccine strain, as well as removal of the recA gene. The latter is crucial for homological recombination. pGM5 suicide vector based on pHV33 bireplicon plasmid was constructed to provide replacement of intact F. tularensis chromosome segments by modified segments. Modified chromosome segments contain F. Tularensis DNA fragment without iglC structural gene segment 545 p. b. (in pGMΔiglC plasmid), as well as DNA fragment containing no recA structural gene segment 1060 p.b. (pGMΔrecA plasmid). The constructed 15/23-1ΔrecA mutant, in contrast to the vaccine strain 15, was capable of reproducing in the macrophage-like cells J774A.1 line, whereas the efficiency of the reproduction was 8-10 times less. BALB/c mouse responded to immunization by the 15/23-1ΔrecA strain by smaller weight decrease (-2%) as compared to the strain 15 (-14%). Bacteria of the 15/23-1ΔrecA strain were virtually incapable of germinating from the BALB/c murine spleen 14 days after invasion, whereas bacteria of the strain 15 were found in the murine organs even after 21 days. The F. tularensis 15/23-1ΔrecA strain having smaller reaction ability can be used as a basis for construction of stable live safe tularemia vaccine.

[CITRULLINUREIDASE GENE DIVERSITY IN THE GENUS FRANCISELLA].

This work describes the results, of the in silico analysis of the genetic diversity of the citrullinureidase gene (ctu) in two species of bacteria of the genus Francisella: tularensis (ssp. tularensis, holarctica, mediasiatica, novicida) and philomiragia. The strains of the Central Asiatic subspecies possessing the citrullinureidase activity differ in the gene ctu from the ssp tularensis Schu by three nucleotide substitutions leading to two insignificant amino acid substitutions in the encoded polypeptide. In the strain F. tularensis of the ssp. holarctica the gene ctu encodes inactive enzyme, which is probably due to amino acid substitutions: 151 Gly --> Asp, 183 Pro --> Leu, 222 Asp --> Asn. Except for the Japan biovar bacteria, in all strains of the Holarctic subspecies there are two stop codons in the gene ctu. The bacteria of the subspecies novicida contain the ctu gene only in the strain 3523, whereas the other strains contain the gene FTN_0827 encoding the C-N hydrolase, which probably provides the citrullinureidase activity.

[Francisella tularensis strains molecular typing using the multiple-locus variable-number tandem repeat analysis].

Variable-number tandem repeat analysis (VNTR) of 25 loci was used for molecular typing of the Francisella tularensis strains isolated from different regions of Russia and the former Soviet Union. This approach allowed us to subdivide F. tularensis subspecies and determine genotype diversity with regard to the geographical prevalence. All 25 loci were examined for their ability to discriminate subspecies and local geographical group. 42 genotypes among the 58 investigated F. tularensis subsp. holarctica isolates were found using cluster VNTR analysis.

Biological properties and structure of the lipopolysaccharide of a vaccine strain of Francisella tularensis generated by inactivation of a quorum sensing system gene qseC.

A knockout mutant with a deletion in a quorum sensing system gene qseC was generated from the vaccine strain Francisella tularensis 15 by site-directed mutagenesis. The variant with the inactivated gene qseC differed from the parental strain in growth rate on solid nutrient medium but had the same growth dynamics in liquid nutrient medium. The mutation abolished almost completely the resistance of the vaccine strain to normal rabbit serum and its ability to survive in macrophages; in addition, the strain lost the residual virulence. A significant phenotypic alteration was observed in the lipopolysaccharide of F. tularensis. Particularly, the mutant strain synthesized no noticeable amount of the lipopolysaccharide with the high-molecular-mass O-polysaccharide, presumably as a result of impairing biosynthesis of the repeating unit, namely, a loss of the ability to incorporate a formyl group, an N-acyl substituent of 4-amino-4,6-dideoxy-D-glucose.

Cloning and expression of protective antigens of Mycobacterium tuberculosis Ag85B and ESAT-6 in Francisella tularensis 15/10.

The possibility of expression of genes encoding mycobacterial antigens in Francisella tularensis 15/10 vaccine strain cells has been shown for the first time. To obtain stable and effective expression of mycobacterial antigens in the F. tularensis cells, the plasmid vector pPMC1 and hybrid genes consisting of the leader part FL of the F. tularensis membrane protein FopA and structural moieties of the mature protein Ag85B or the fused protein Ag85B-ESAT-6 were constructed. Recombinant strains F. tularensis RVp17 and RVp18 expressing protective mycobacterial antigens in the fused proteins FL-Ag85B and FL-Ag85B-ESAT-6, respectively, were obtained. Expression of the protective mycobacterial antigens in F. tularensis was analyzed using specific antisera to the recombinant proteins Ag85-(His)6 and ESAT-6-(His)6 isolated from Escherichia coli producer strains created on the basis of the pET23b(+) and pET24b(+) vectors. The expression of heterologous protective antigens in F. tularensis 15/10 is promising for creation of live recombinant anti-tuberculosis vaccines on the basis of the tularemia vaccine strain.

Genetic organization of the Francisella plasmid pFNL10.

We report here the molecular characterization of pFNL10, a 3990-bp cryptic plasmid of Francisella novicida-like F6168. The plasmid was maintained in F. novicida Utah 112 and F. tularensis LVS strains. We sequenced the entire plasmid and found six open reading frames (ORFs)-ORF1, ORF2, ORF3, ORF4, ORF5, and ORFm. ORF3, ORF4, ORF5, and ORFm are located on the same strand, and we designated it the plus strand. ORF1 and ORF2 are on the complementary strand. The ORFs appear to be arranged in two operons, one comprising ORF5 and ORF4 and the other ORF1 and ORF2. There exist two distinct promoters similar to the Escherichia coli sigma(70) promoter, one 5' to ORF1-ORF2 operon and the other 5' to ORF5-ORF4 operon. We found that in both promoters the transcriptional start is an adenosine. ORF3 is positioned in tandem with ORF5-ORF4, but has its own transcriptional start, a thymidine. However, sequence analysis revealed no recognizable promoter in physical proximity to ORF3. Sequence analysis revealed transcriptional terminators immediately downstream of the two operons. Experimental results showed that the ORF1-ORF2 terminator is authentic. But we could not definitively confirm the ORF5-ORF4 terminator. Two sets of direct repeats, one 31 and the other 13 bp, characteristic of ori are positioned between the two promoters. ORF1 encodes a protein that bears homology to the replication initiation protein RepA of various bacteria, and disruption of this ORF indeed blocked pFNL10 replication. In contrast, ORF2 disruption caused formation of plasmid multimers, suggesting aberrant replication. Our analysis also suggests that pFNL10 replicates by the theta mode. The ORF5-ORF4 operon resembles the phd-doc operon of Escherichia coli bacteriophage P1, but the significance of this similarity is unclear.

[pCSE4 plasmid for cloning of promoter-containing DNA fragments in francisella tularensis]. / Plazmida pCSE4 dlia klonirovaniia promotorsoderzhashchikh fragmentov DNK v francisella tularensis.

Recombinant pCSE4 plasmid has been constructed, which contains the cat gene of the Tn9 without its own promoter and with the restriction BamH1 site in front of the Shine-Delgarno region of this gene. pCSE4 is consistently inherited in the cells of F. tularensis and E. coli and makes the cells of both microorganisms to be resistant to tetracycline. By cloning the Sau3A fragments of F. tularensis chromosomal DNA at the BamH1 site of pCSE4. Promoter-containing DNA fragments were selected. The plasmids with such chromosomal DNA fragments retained their structural integrity and functional activity in F. tularensis.

[Tularemia vaccine strain is a potential vector]. / Tuliaremiínyí vaktsinnyí shtamm-potentsial'nyí bakterial'nyí vektor.

The immunological and genetic properties of Francisella tularensis vaccine strain are discussion with regard to its use to produce recombinant vaccines. This bacterium-based vector is supposed to be an excellent object for investigating the role of protective antigens in the development of immunity against intracellular bacteria.

Cryptic plasmid pFNL10 from Francisella novicida-like F6168: the base of plasmid vectors for Francisella tularensis.

The plasmid pFNL1OO was created by ligation of Escherichia coli plasmid pBR328 and plasmid pFNL1O from Francisella novicida-like strain F6168. This plasmid was able to replicate and to express the genes for chloramphenicol and tetracycline resistance in both E. coli and F. tularensis. The origin of replication of pFNL1O, needed for the replication of pFNL1OO in F. tularensis, was mapped. A Sau3A-deletion derivative of pFNL1OO, designated pFNL2OO, was constructed. This plasmid could replicate only in F. tularensis and was found to be stably inherited during cultivation both on solid medium and in liquid cultures.

[Isolation and molecular-genetic characteristic of a cryptic plasmid from the Francisella novicida like F6168 strain]. / Vydelenie i molekuliarno-geneticheskaia kharakteristika kripticheskoi plazmidy iz shtamma Francisella novicida like F6168.

A 4 Kb plasmid DNA has been isolated from Francisella novicida like strain F6168. Restriction map of the plasmid was constructed for restriction endonucleases HindIII, XbaI, EcoRV, BgIII. The plasmid pFN10 has been shown to be stably inherited by F. tularensis. The use of pFN10 for the construction of plasmid vectors for microorganisms of the genus Francisella is discussed.

Specific fragmentation of T7 phage DNA at low-melting sites.

A method has been developed for selective fragmentation of T7 DNA at AT-rich regions. The molecules have been subjected to complete digestion with single-strand-specific SI endonuclease after fixation of DNA AT-rich regions in the denatured state by glyoxal. The treatment resulted in three fragments having molecular weights of 13.6 +/- 0.4, 8.2 +/- 0.4 and 3.5 +/- 0.16 megadaltons as determined by electron microscopy. The position of these fragments along the T7 DNA molecule has been determined by means of analysis of the intermediates during SI-cleavage.

[Changes in hemodynamics in patients with chronic alcholism]. / Izmeneniia gemodinamiki u bol'nykh khronicheskim alkogolizmom

The indices of total hamodynamics were studied in 52 patients with chronic alcoholism and without any clear signs of cardiac pathology and with normal arterial pressure. When examined at rest in a supine position they exhibited normal values of the cardiac and stroke index, peripheral vascular resistance, circulating blood volume and hematocrit. Along with the increasing period of alcohol abuse the cardiac index gets somewhat decreased, indicating a decreasing contractile capacity of the myocardium. This is further supported by the changing structure of the phases of the systole: prolongation of the phase of isometric contraction, of the tension phase, the tension index, reduction of the intrasystolic index and of the speed of intraventricular pressure increase. The orthostatic test revealed certain disorders in the regulation of the cardiovascular system in an upright position which manifested themselves in a more distinct, than in normal individuals, reduction of the cardiac and stroke indices, and in a more significant growth of cardiac contractions rate and peripheral vascular resistance. The latter indicates an increased mobilization of the sympathoadrenal system in an orthostatic position.

[Disorders of cardiac rhythm in chronic alcoholics]. / Narusheniia serdechnogo ritma u bol'nykh khronicheskim alkogolizmom

The friquency and type of cardiac rhythm and conduction disturbances were studied in 200 patients with chronic alcoholism. Attention was payed to the character and duration of dipsomania. Patients with a long abuse period were shown to have auricular fibrillation in 10 per cent of the cases. Patients with chronic alcoholism and particularly with rhythm disturbances demonstrated the decrease of potassium concentration in blood serum.