[Incidence of tuberculosis in officials from penitentiaries of the Republic of Udmurtia].

The staff of penitentiaries is at high risk for tuberculosis due to its professional occupation. Tuberculosis morbidity was studied in the officials of the reformatory system in the Republic of Udmurtia over the period of 1997 to 2006. Throughout the follow-up, there were high tuberculosis morbidity rates in the officials of reformatories, in 2000-2002 in particular. In the structure of patients with new-onset tuberculosis, there was a preponderance of young males (75.6%), mainly qualified staff (89.3%). Pulmonary tuberculosis was prevalent (92.9%), extrapulmonary tuberculosis accounted for 7.1%. Infiltrative pulmonary tuberculosis was the major clinical type (76%) in the patients with new-onset tuberculosis. The study indicated the low proportion of patients showing bacteria discharge and lung tissue destruction, suggesting the timeliness of tuberculosis detection in the officials of penitentiaries. The results of treatment for tuberculosis in the officials of the penitentiary system were high, which was associated with their discipline and responsibility for their health.

Investigation of the molecular assembly of beta-cell K(ATP) channels.

We have investigated the protein interactions involved in the assembly of pancreatic beta-cell ATP-sensitive potassium channels. The channels are a heterooligomeric complex of pore-forming Kir6.2 subunits and sulfonylurea receptor (SUR1) subunits. SUR1 belongs to the ATP binding cassette (ABC) family of proteins and has two nucleotide binding domains (NBD1 and NBD2) and 17 putative transmembrane (TM) sequences. Previously we showed that co-expression in a baculovirus expression system of two parts of SUR1 divided at Pro1042 between TM12 and 13 leads to restoration of glibenclamide binding activity, whereas expression of either individual N- or C-terminal domain alone gave no glibenclamide binding activity [M.V. Mikhailov and S.J.H. Ashcroft (2000) J. Biol. Chem. 275, 3360-3364]. Here we show that the two half-molecules formed by division of SUR1 between NBD1 and TM12 or between TM13 and 14 also self-assemble to give glibenclamide binding activity. However, deletion of NBD1 from the N-part of SUR1 abolished SUR1 assembly, indicating a critical role for NBD1 in SUR1 assembly. We found that differences in glibenclamide binding activity obtained after co-expression of different half-molecules are attributable to different amounts of binding sites, but the binding affinities remained nearly the same. Simultaneous expression of Kir6.2 resulted in enhanced glibenclamide binding activity only when the N-half of SUR1 included TM12. We conclude that TM12 and 13 are not essential for SUR1 assembly whereas TM12 takes part in SUR1 Kir6.2 interaction. This interaction is specific for Kir 6.2 because no enhancement of glibenclamide binding was observed when half-molecules were expressed together with Kir4.1. We propose a model of K(ATP) channel organisation based on these data.

Molecular structure of the glibenclamide binding site of the beta-cell K(ATP) channel.

We have investigated the structure of the glibenclamide binding site of pancreatic beta-cell ATP-sensitive potassium (K(ATP)) channels. K(ATP) channels are a complex of four pore-forming Kir6.2 subunits and four sulfonylurea receptor (SUR1) subunits. SUR1 (ABCC8) belongs to the ATP binding cassette family of proteins and has two nucleotide binding domains (NBD1 and NBD2) and 17 putative transmembrane (TM) sequences. Co-expression in a baculovirus expression system of two parts of SUR1 between NBD1 and TM12 leads to restoration of glibenclamide binding activity, whereas expression of either individual N- or C-terminal part alone gave no glibenclamide binding activity, confirming a bivalent structure of the glibenclamide binding site. By using N-terminally truncated recombinant proteins we have shown that CL3 - the cytosolic loop between TM5 and TM6 - plays a key role in formation of the N-terminal component of the glibenclamide binding site. Analysis of deletion variants of the C-terminal part of SUR1 showed that CL8 - the cytosolic loop between TM15 and TM16 - is the only determinant for the C-terminal component of the glibenclamide binding site. We suggest that in SUR1 in the native K(ATP) channel close proximity of CL3 and CL8 leads to formation of the glibenclamide binding site.

Expression of functionally active ATP-sensitive K-channels in insect cells using baculovirus.

We have expressed active ATP-sensitive K-channels (K(ATP) channels) in Spodoptera frugiperda (Sf9) cells using a baculovirus vector. A high yield of active channels was obtained on co-infection with SUR1 and Kir6.2 engineered to contain N- and/or C-terminal tags to permit detection by Western blotting. Channel activity was sensitive to ATP, glibenclamide and diazoxide. Channel activity was also obtained on expression of a C-terminally truncated Kir6.2 (Kir6.2 deltaC26): these channels were blocked by ATP but were insensitive to sulphonylureas. In contrast to Xenopus oocytes and mammalian cells the full length Kir6.2 also gave rise to active channels in Sf9 cells when expressed alone. The highest yield of active K(ATP) channels was obtained on infection with a fusion protein containing SUR1 linked to Kir6.2 deltaC26 via a 6-amino acid linker.

Antigenic determinants synthesized in a library of randomly cloned fragments of the HIV-1 genome.

A DNA expression library of randomly selected fragments of the HIV-1 genome was constructed and used to search for antigenic determinants. A large segment of the HIV-1 provirus was sonicated, and 150-250 bp DNA fragments were cloned in a system of expression vectors developed to obtain high yields of recombinant proteins in Escherichia coli. The expressed library was immunoscreened with sera of AIDS patients. Eleven identified immunoreactive clones were found to correspond to already known and new antigenic regions of HIV-1 proteins gp41, p24, and reverse transcriptase.

Complete sequence of two tick-borne flaviviruses isolated from Siberia and the UK: analysis and significance of the 5' and 3'-UTRs.

The complete nucleotide sequence of two tick-transmitted flaviviruses, Vasilchenko (Vs) from Siberia and louping ill (LI) from the UK, have been determined. The genomes were respectively, 10928 and 10871 nucleotides (nt) in length. The coding strategy and functional protein sequence motifs of tick-borne flaviviruses are presented in both Vs and LI viruses. The phylogenies based on maximum likelihood, maximum parsimony and distance analysis of the polyproteins, identified Vs virus as a member of the tick-borne encephalitis virus subgroup within the tick-borne serocomplex, genus Flavivirus, family Flaviviridae. Comparative alignment of the 3'-untranslated regions revealed deletions of different lengths essentially at the same position downstream of the stop codon for all tick-borne viruses. Two direct 27 nucleotide repeats at the 3'-end were found only for Vs and LI virus. Immediately following the deletions a region of 332-334 nt with relatively conserved primary structure (67-94% identity) was observed at the 3'-non-coding end of the virus genome. Pairwise comparisons of the nucleotide sequence data revealed similar levels of variation between the coding region, and the 5' and 3'-termini of the genome, implying an equivalent strong selective control for translated and untranslated regions. Indeed the predicted folding of the 5' and 3'-untranslated regions revealed patterns of stem and loop structures conserved for all tick-borne flaviviruses suggesting a purifying selection for preservation of essential RNA secondary structures which could be involved in translational control and replication. The possible implications of these findings are discussed.

[Mechanism of AT base pairs recognition by molecules of dye "Hoechst 33258"]. / Mekhanizm "uznavaniia" AT-par v DNK molekulami krasitelia "Hoechst 33258".

Experimental studies are reported on the binding of the dye "Hoechst 33 258" to DNA and synthetic polynucleotides. A stereochemical model of binding of the dye to DNA is proposed. According to the model, the molecules of the dye are located in the minor groove of DNA, each covering 4 base pairs. The benzimidazole backbone of the dye tends to form a helix isogeometric to that of DNA in the B form. The specifisity of "Hoechst 33 258" binding to At pairs involves hydrogen bonding of two NH-groups of benzimidazole rings and CH3N+H group of the piperazine ring to O2 oxygenes of thymines and N3 nitrogens of adenines facing the minor groove of the DNA double helix.

[Binding of actinomycin D analogues containing some substituents at position 7 of the antibiotic chromophore to DNA]. / Sviazyvanie analogov aktinomitsina D, soderzhashchikh zamestiteli v polozhenii 7 khromofora antibiotika, s DKN.

Spectrophotometric methods are used to study the binding to DNA of Actinomycin D (AMD) and its analogues: 7-nitro-AMD; 7-amino-AMD; 7-(Z-Val-Glo-NH)-AMD; 7-(AcO- . +H2-Val-Glo-NH)-AMD; 7-(AcO- . +H2-Val-Glo-Val-Glo-NH)-AMD. The binding constants are calculated from the binding isotherm of AMD and those of the AMD analogues to calf thymus DNA obtained by spectrophotometric titration. Introduction of smaller substituents such as the nitro or amino groups into position 7 of chromophore influences insignificantly the antibiotic binding to DNA, whereas bulky substituents cause a decrease in the affinity of the AMD analogues for DNA, although the spectral characteristics are not affected.

[Determination of the number of GC-pairs "recognized" by actinomycin D during binding with DNA]. / Opredelenie chisla GC-par "uznavaemykh" aktinomitsinom D pri sviazyvanii s DNK.

Experimental studies are reported on the binding of actinomycin D to various synthetic and naturally occurring DNAs. The experimental data obtained agree with a model in which the antibiotic molecule carries only one GC-specific reaction center and covers 5 base pairs upon binding to DNA. Positive cooperative effects are observed for the binding of actinomycin to poly(dG-dC)-poly (dG-dC) duplex.

[Binding of actinomycin D analogs to DNA]. / Svazyvanie s DNK analogov aktinomitsina D.

Equilibrium and kinetic studies are reported on the binding to DNA of actinomycin D analogues containing various substituents at position 7 of the antibiotic chromophore. The binding constants are calculated from the experimental binding isotherms of actinomycin D and its analogues to calf thymus DNA. The rate constants of the association and dissociation processes are measured by stop -- flow method. From these experiments a conclusion is drawn that small substituents (such as nitro or aminogroups) at position 7 of the actinomycin chromophore exhibit a little (if any) influence both on the affinity of antibiotic for DNA and kinetics of association and dissociation processes, while bulky substituents decrease the antibiotic binding affinity and make kinetics behavior slower.

Interactions of the sulfonylurea receptor 1 subunit in the molecular assembly of beta-cell K(ATP) channels.

We have investigated protein interactions involved in pancreatic beta-cell ATP-sensitive potassium channel assembly. These channels, which are of key importance for control of insulin release, are a hetero-oligomeric complex of pore-forming Kir6.2 subunits and sulfonylurea receptor (SUR1) subunits with two nucleotide-binding domains (NBD1 and NBD2). We divided SUR1 into two halves at Pro-1042. Expression of either the individual N- or C-terminal domain in a baculovirus expression system did not lead to glibenclamide binding activity, although studies with green fluorescent protein fusion proteins showed that both half-molecules were inserted into the plasma membrane. However, significant glibenclamide binding activity was observed when the half-molecules were co-expressed (even when NBD2 was deleted from the C-terminal half-molecule). Simultaneous expression of Kir6.2 resulted in enhanced glibenclamide binding activity. We conclude that the glibenclamide-binding site includes amino acid residues from both halves of the molecule, that there is strong interaction between different regions of SUR1, that NBD2 is not essential for glibenclamide binding, and that interactions between Kir6.2 and SUR1 participate in ATP-sensitive potassium channel assembly. Investigation of NBD1-green fluorescent protein fusion protein distribution inside insect cells expressing C-terminal halves of SUR1 demonstrated strong interaction between NBD1 and NBD2. We also expressed and purified NBD1 from Escherichia coli. Purified NBD1 was found to exist as a tetramer indicating strong homomeric attractions and a possible role for NBD1 in SUR1 assembly.

Structure-function relationships in the beta-cell K(ATP) channel.

The ATP-sensitive potassium (K(ATP)) channel plays a key role in controlling beta-cell membrane potential and insulin secretion. The channels are composed of two subunits, Kir6.2, which forms the channel pore, and SUR1, which contains binding sites for nucleotides and sulphonylureas and acts as a channel regulator. Our current studies are aimed at delineating the molecular interactions involved in assembly and ligand binding by K(ATP) channel proteins. We have employed a complementation approach in which SUR1 half-molecules are co-expressed in insect cells using a baculovirus system. Together with data from truncated SUR1 molecules and a fusion protein in which SUR1 is linked to Kir6.2, we have interpreted our findings in terms of a model for the structure of the K(ATP) channel. The main features of the model are: (i) the C-terminal end of SUR1 is close to the N-terminus of Kir6.2; (ii) the two nucleotide binding domains (NBDs) of SUR1--NBD1 and NBD2--are in proximity; (iii) transmembrane helix 12 of SUR1 is orientated in such a way that it can make contact with Kir6.2; (iv) formation of the glibenclamide binding site requires that the two cytosolic loops (CLs) CL3 and CL8 are located close to each other; (v) there are homomeric interactions between the NBD1 domains of neighbouring subunits. We suggest that binding of glibenclamide leads to conformational changes in CL3 and CL8 leading to rearrangement of transmembrane helices. These effects are transmitted to Kir6.2 to result in channel closure.

A new, rapid and simple procedure for direct cloning of PCR products into baculoviruses.

We propose a novel method for direct cloning of foreign genes into baculoviruses which avoids the use of bacterial transfer vectors. The foreign gene to be inserted is derived by PCR using appropriate primers each of which contains an additional 50 nt of baculovirus sequence for homologous recombination between the PCR-derived DNA and the baculovirus DNA, thus accomplishing insertion of the foreign gene into the baculovirus. The direct cloning of green fluorescent protein and beta-glucuronidase in different baculovirus loci is described. The method is simple and avoids the use of cumbersome techniques associated with enzymatic treatment and DNA purification.

Bovine leukemia virus Gag particle assembly in insect cells: formation of chimeric particles by domain-switched leukemia/lentivirus Gag polyprotein.

A key stage in the life cycle of C-type retroviruses is the assembly of Gag precursor protein at the plasma membrane of infected cells. Here we report the assembly of bovine leukemia virus (BLV) gag gene product into virus-like particles (VLPs) using the baculovirus expression system. Expression of BLV Pr44(Gag) resulted in the assembly and release of VLPs, thereby confirming the ability of retroviral Gag polyprotein to assemble and bud from insect cells. Efficient particle formation required a myristoylation signal at the N-terminus of BLV Pr44(Gag). Recombinant baculoviruses expressing matrix (MA) or capsid-nucleocapsid (CA-NC) proteins of BLV were generated but neither of these domains was capable of assembling into particulate structures. To assess the compatibility of Gag domains between leukemia and lentivirus groups three different recombinant chimeras each expressing MA of one virus (e.g., simian immunodeficiency or BLV) and CA-NC of another (e.g., BLV or human T-cell leukemia virus type-I) were constructed. Each of the chimeric proteins assembled efficiently and budded as VLPs, suggesting that the MA and CA domains of these two evolutionary divergent retrovirus groups can be functionally exchanged without perturbation of Gag VLP formation. The lenti-leukemia chimeric Gag approach has potential for studying protein-protein interactions in other retroviruses.