[The comparative evalution of informativeness of immunologic and molecular genetic methods and means during stages of specific indication of melioidosis agent].

The reference-center of monitoring of agents of glanders and melioidosis carried out testing of reagents kits for diagnostic of agent of melioidosis and other close-related species of Burkholderiae in vitro. At the stage of specific identification of pathogenic Burkholderiae the diagnostic possibilities of commercial and experimental kits of reagents for express- and rapid analysis were evaluated. The criteria of evaluation of diagnostic value of kits of reagents were sensitivity, specificity and time of implementation of studies. The analysis with application of mono- and multi-locus amplification systems, including real-time polymerase chain reaction permitted during 5-6 hours to implement identification and differentiation of Burkholderia pseufomallei, B. thailandensis and B. cepacia.

Mutations in fd phage major coat protein modulate affinity of the displayed peptide.

Multibillion-clone libraries of phages displaying guest peptides fused to the major coat protein pVIII (landscape libraries) are a rich source of probes for proteinaceous and non-proteinaceous targets. As opposed to the pIII-type fusion phages, which display peptides as independent structural domains, the guest peptides in the pVIII-fusion phages can be structurally and functionally influenced by contiguous subunits. To decipher the impact of the locale of a guest peptide on its affinity characteristics, we constructed a library of phages carrying beta-galactosidase-binding peptide ADTFAKSMQ at the N-terminus of the pVIII protein surrounded by random amino acids. It was found that mutagenesis of amino acids 12-19 (domain C) has polar effects on target binding affinity of the displayed peptide. The phages with highest affinity are characterized by: (i) a net electrostatic charge around -1 of domain C of the mutated phages at pH 7.0; (ii) a lower radius of cylinder coaxial to alpha-helix formed by domain C; (iii) a lower higher occupied molecular orbital (HOMO) of domain C leading to a decreased formation of hydrogen bonds and (iv) positively charged surface and torsion energy of domain C, which may require a conformational transition of N-terminal peptide ADTFAKSMQ for its binding with beta-galactosidase. Influence of the guest peptide on the diversity of mutations in the neighboring landscape area was also observed.

[A study of conformational states of substrates of cytochrome P450 3A4].

A multiconformational study of substrates of cytochrome P450 3A4 has been carried out within the BiS/MC algorithm. The method allowed one to create a pseudoatomic model of the cytochrome and to find the substrate conformers responsible for the interaction with the cytochrome. It has been found that, in most cases, the geometry of the acting conformer is much different from the geometry of the global minimum conformer. It has been shown that the mirror conformational antipodes ("enantioconformers") are characterized as a rule by different Michaelis constants. A quantitative relationship between the Michaelis constants and the parameters of interactions in "model 3A4 isoform-substrate" complexes has been determined. The relationship describes the experimental value of Michaelis constant with the squared cross-validation correlation coefficient of 0.88.

[Identification of the agents of coccidioidomycosis using polymerase chain reaction].

Two pairs of primers for diagnosis of coccidioidomycosis using the method of PCR were constructed. One pair was used for identification of the two species of Coccidioides (C. immitis and C. posadasil) on the basis of MBP-1 gene. The other pair was chosen on the basis of SOWgp82 gene, which encodes an immunodominant, spherule outer wall glycoprotein for detecting only C. posadasii. The used primers allowed the agents of coccidioidomycosis to be detected using PCR with high sensitivity and specificity. The effective method of isolation of fungus DNA from soil contaminated with arthroconidia of Coccidioides spp. was developed. It includes guanidinthiocyanate-phenol-chloroform deproteinization followed by DNA purification using nuclear sorption.

[The modeling of interactions of the CYP2E1 isoform of human cytochrome P450 with substrates].

The interactions between the substrates of the 2E1 isoform of the human cytochrome P450 and receptor were simulated. It was found that the CP4 isoform of the cytochrome of the bacterial cell is highly homologous to the 2E1 isoform of the human cytochrome P450. The orientation of the substrates of the 2E1 isoform in the CP4 isoform of the bacterial cell cytochrome was performed. A cavity in the receptor was found that is responsible for the binding of the substrate. Amino acid residues Phe87, Pro89, Val119, Thr185, Leu244, Leu245, Leu246, Val247, Gly248, Gly249, Thr252, Val295, Asp297, Cys357, Ile395, and Val396, the heme, and water molecules are involved in the formation of the cavity. The mode of the interactions of the substrate molecule with cytochrome was analyzed. Active sites of the receptor, and a part of the substrate molecule responsible for the binding to cytochrome were found. Equations for the dependence of the Michaelis constant on the structural parameters of complexes of substrates with cytochrome were derived.