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Applied Biochemistry and Microbiology (v.47, #8)

Rational design of nano- and micro-size medicinal forms of biologically active substances by A. P. Kaplun; D. A. Bezrukov; V. I. Shvets (pp. 711-717).

In the review, the properties of nano- and microparticles as tools for delivery of medicinal substances (namely, size, shape, and superfacial characteristics) are discussed. The main fields of application of these particles, such as therapy of malignant tumors and intracellular infections, increase in vaccine adjuvanticity, solubilization of poorly soluble compounds, and delivery to the brain, are discussed. Effects provided by substance loading into nano- and microparticles (reduced toxicity, protection from rapid degradation, and deposition) are described. The most progress is expected in active targeting, intracellular targeting, and controllable release.

Keywords: active targeting; adjuvant; controllable release; liposomes; microparticles; nanoparticles; passive targeting; solubilization

Effect of modified DNA and RNA oligonucleotides on telomerase activity and tumor cell survival in vitro by L. V. Svinareva; A. I. Glukhov; E. Yu. Moskaleva; V. I. Shvets (pp. 718-722).

Comparative analysis of the inhibitory effect of modified DNA and RNA oligonucleotides on telomerase activity and tumor cell growth in vitro has been carried out. The study was performed with MCF-7, HeLa, and Mel-10 human cell line extracts. It was shown that PS-TelP5 and PS-TM024 phosphorothioate DNA oligonucleotides inhibited telomerase at nanomolar concentrations and suppressed the growth of tumor cells in vitro.

Keywords: immortality; 2′-O-Me-RNA-oligonucleotides; phosphorothioate DNA oligonucleotides; telomerase; telomerase activity; telomerase inhibitors; telomeres

Immunogenicity of synthetic fragments corresponding to variable and conservative sites of H3N2 influenza virus hemagglutinin heavy chain by E. I. Isaeva; N. A. Mazurkova; R. Ya. Podchernyayeva (pp. 723-729).

Immunogenic properties of synthetic peptides corresponding to regions 122–133, 136–147, 154–164, and 314–328 of the heavy chain (HA1) of A/Aichi/2/68 virus hemagglutinin were studied. Peptides 122–133 and 136–147 together form a nearly complete antigenic determinant A, peptide 154–164 is a part of determinant B, and peptide 314–328 corresponds to the C-terminal HA1 fragment. In a model influenza A/Aichi/2/68 infection in CBA mice, a protective effect of conjugates of BSA with peptides 136–147 and 314–328 was shown. Immunization of animals with conjugates BSA-(136–147) and BSA-(314–328) in combination with interferon inducers (larifan and ridostin) and a plant immunomodulator (immunomax) intensified the protection of mice against the influenza infection.

Keywords: immunogenicity; immunomodulator; influenza virus; interferon inducers; synthetic peptides

Obtaining of the p17 recombinant protein of human immunodeficiency subtype A virus by R. I. Al-Shekhadat; I. V. Dukhovlinov; A. I. Kobatov; N. A. Klimov; A. P. Kozlov (pp. 730-736).

A simple and efficient method for expression in Escherichia coli cells and purification of a recombinant matrix protein, p17, of human immunodeficiency type I virus has been described. HIV-1 subtype A DNA sequence encoding p17 was obtained by amplification of the viral gag gene segment and cloned into an expression vector under the control of T7Lac promoter. The conditions for cell growth and induction of p17 synthesis by lactose and its further purification by metal chelate chromatography were optimized. p17 preparations with 97% purity were obtained; the yield of the protein of 28 mg per 1l of culture was achieved. The obtained protein was capable of binding antibodies from blood serum of a HIV-infected patient during immunoblotting.

Keywords: p17 protein; human immunodeficiency virus; HIV-1; subtype A; Gag

Optimization of roller culturing of influenza virus vaccine strains in MDCK and Vero cell cultures using plant origin components by E. I. Isaeva; N. A. Mazurkova; M. O. Skarnovich; G. P. Troshkova; L. N. Shishkina; R. Ya. Podchernyayeva (pp. 737-743).

Roller culturing of MDCK and Vero cells in an experimental nutrient medium based on soy flour hydrolysate, plant material obtained using the bromelain plant enzyme, was studied. The medium supplemented with 2 or 3% fetal calf serum (FCS) had a strong growth-stimulating effect on Vero and MDCK and cells, respectively, and did not alter the cell morphology. A/Solomon Islands/03/06 (H1N1) and B/Malaysia/2506/04 influenza vaccine viruses were grown on MDCK and Vero cell cultures obtained as a result of culturing in rollers on media containing soy flour hydrolysate and FCS (2 or 3%, respectively). The titer of the viruses was high in the presence of either trypsin (2 μg/ml) or bromelain (20 μg/ml).

Keywords: bromelain; influenza virus; soy flour hydrolysate; cultural vaccine; MDCK and Vero cell cultures; roller

Capacity of N- and C-domains of Saccharomyces cerevisiae flo1p cell wall protein homologue to expose lip2 lipase on cell surface of Yarrowia lipolytica yeast by E. Yu. Yuzbasheva; T. V. Yuzbashev; T. K. Konstantinova; I. A. Laptev; N. I. Perkovskaya; S. P. Sineokii (pp. 744-753).

The closest homologue of the Saccharomyces cerevisiae Flo1p cell wall protein was detected in Yarrowia lipolytica yeast (YALI0C09031p) by the method of genomic analysis, and capacity of its N- and C-domains to expose the Lip2 lipase on the cell surface was studied. The efficient fixation of the enzyme on the Y. lipolytica cell wall surface was demonstrated. The activity of the cell-bound lipase was 9170 and 3200 units per 1 g of dry solid matter when using N- and C-domains of the cell wall protein, respectively. At the same time, in the case of immobilization using the N-domain, approximately 30% of the total lipase activity was detected in the culture medium, whereas when using C-domain of the cell wall protein YALI0C09031p, practically all lipase was in the immobilized state. Obtained values of the level of the cell-bound lipase activity considerably exceed previously published data opening a prospect for new technological solutions which meet industrial needs.

Keywords: Flo1p homologue; N- and C-domains; Lip2 lipase; exposure on cell wall surface; Yarrowia lipolytica

Study of Yersinia pseudotuberculosis antigenic structure to obtain a sensitin for design of species-specific diagnostic kits by G. L. Karbyshev; D. I. Simakova; L. V. Larionova; A. N. Terent’ev; L. K. Lysova; E. M. Sanamyants (pp. 754-761).

A method for isolation of outer membrane proteins from Yersinia pseudotuberculosis, which are perspective for further application as sensitin for design of species-specific pseudotuberculosis antigenic diagnostic kits, has been modified. Common species-specific antigens of nine Y. pseudotuberculosis serovars (with molecular weight from 80.62 to 12.2 kDa) were detected by SDS-PAAGE electrophoresis and immunoblotting of the outer-membrane protein preparations. These antigens react with neither the rabbit experimental antiplague antiserum nor antiserum to 39 Y. enterocolitica serovars and normal rabbit serum.

Keywords: electrophoresis; immunoblotting; outer-membrane proteins; Yersinia pseudotuberculosis

Enhancement of larvicidal activity of Brevibacillus laterosporus by bioincapsulation in Protozoa Tetrahymena pyriformis and Entamoeba moshkovskii by M. V. Zubasheva; L. A. Ganushkina; T. A. Smirnova; R. R. Azizbekyan (pp. 762-766).

Conditions for bioincapsulation of crystal-forming strain Brevibacillus laterosporus LAT 006 spores and crystals by using Tetrahymena pyriformis and Entamoeba moshkovskii Protozoa have been developed. Increase in the larvicidal activity of the incapsulated bacteria was demonstrated. Fractions of pure spores and crystals and intact spore-crystal preparation of LAT 006 were shown not to have toxic effect on the protozoa cells.

Keywords: bioincapsulation; Brevibacillus laterosporus ; mosquitoes; Protozoa

Genetic transformation of wheat using mature seed tissues by D. N. Miroshnichenko; G. N. Poroshin; S. V. Dolgov (pp. 767-775).

A protocol for biolistic transformation of bread wheat based on using mature seed tissues as explants has been developed. Embryogenic callus obtained from mature seed tissues was transformed with a psGFP-BAR plasmid containing gfp reporter gene and bar selectable marker gene. The influence of hormone composition of the medium on the efficiency of transformation of mature wheat seed tissues has been demonstrated. The use of auxin 2,4-D resulted in the formation of transgenic plants with a frequency of 0.75%, while the use of Dicamba auxin for the regeneration of plants did not result in transformant development. The transgenic status of the plants obtained in the experiments has been confirmed by PCR and RT-PCR. Stable inheritance of transgenic features in the following generations of wheat (T₁, T₂) has been demonstrated and transgenic plants exhibiting high resistance to herbicides have been obtained. The protocol developed allows for a simplified transformation of wheat in order to obtain transgenic plants with novel features.

Keywords: biolistic transformation; transgene inheritance; herbicide resistance; gfp fluorescence; Triticum aestivum L.

Estimation of heavy metal resistance in the second generation of creeping bentgrass (Agrostis solonifera) obtained by cell selection for resistance to these contaminants and the ability of this plant to accumulate heavy metals by E. A. Gladkov; O. N. Gladkova; L. S. Glushetskaya (pp. 776-779).

Creeping bentgrass plants have been grown from seeds obtained from plants subjected to cell selection for resistance to heavy metals and NaCl, and the sensitivity of the plants and seeds to cadmium, copper, zinc and lead in the soil has been assessed. The data obtained demonstrate the conservation of tolerance to the ecological factors studied in the seeds of second-generation plants. Cross-resistance to pollutants was observed in some cases. The plants obtained can be recommended for the remediation of heavy-metal contaminated soils in urban ecosystems.

Keywords: heavy metals; resistance; bioremediation

Obtaining recombinant forms of the outer membrane protein F of Pseudomonas aeruginosa and assessment of their immunogenic properties by A. A. Kaloshin; E. V. Gatypova; N. A. Mikhailova (pp. 780-788).

Two recombinant forms of the outer membrane protein F (OprF) of Pseudomonas aeruginosa were obtained, the full-length protein OprF and the C-terminal part of the OprF protein (aa 192–342). As a result of double immunizations, these recombinant proteins provided mice with resistance to experimental intraperitoneal challenge with P. aeruginosa. The best protective effects were observed at a dose of 25 μg for OprF and 50 μg for the truncated OprF variant (indices of efficiency were 3.3 and 2.8, respectively). Rabbit antisera immune to the recombinant proteins were also able to protect mice from the experimental infection with P. aeruginosa. Indices of efficiency were 6.4 for OprF and 6.0 for the OprF C-terminal part; these values were approximately two times as high as the effect of sera from intact test animals (3.2).

Keywords: immunization; outer membrane protein F (OprF); Pseudomonas aeruginosa ; recombinant protein

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Applied Biochemistry and Microbiology (v.47, #8)

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Applied Biochemistry and Microbiology (v.47, #8)