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Applied Biochemistry and Microbiology (v.48, #9)

Impact of metagenomics on biotechnological development by S. V. Shestakov (pp. 705-715).

This review analyzes the methodology available for the creation and analysis of metagenomic libraries, targeted search for genes encoding certain types of enzymes, and also phenotypic selection of clones containing functioning genes with consequent identification by sequencing, cloning, and heterological expression in surrogate hosts in order to study gene products. Special attention was paid to the description of new enzyme families (lipases, esterases, oxidases, cellulases, proteases, and others) that are characterized by valuable biotechnological properties, such as thermostability, resistance to cold, alkaline pH, organic solvents, salinity, xenobiotics and multifunctionality, substrate specificity, and stereoselectivity.

Keywords: antibiotics; biotechnology; genomic libraries; microorganisms; metagenomics; enzymes; expression of heterologous gene

Uridine phosphorylase from Shewanella oneidensis MR-1: Heterological expression, regulation, transcription, and properties by N. N. Mordkovich; V. A. Manuvera; V. P. Veiko; V. G. Debabov (pp. 716-722).

A DNA fragment containing a promoter-operator and structural parts of the uridine phosphorylase gene from Shewanella oneidensis MR-1 was cloned. Cross-heterological expression of the udp genes from Sh. oneidensis MR-1 and Escherichia coli under the control of authentic regulatory regions is shown. The UDP protein accumulates in an active form in the cytoplasmic fraction of cells. The recombinant UDP protein from Sh. oneidensis MR-1 obtained by heterological expression was isolated and characterized. E. coli udp gene promoter activity was observed during heterological expression in Sh. oneidensis MR-1 cells under both aerobic and anaerobic conditions.

Keywords: heterological expression; uridine phosphorylase (UDP); Escherichia coli ; Shewanella oneidensis

In vitro and in silico investigation of interferonogenic analogues of nucleic acids, artificially programmed to block the initial stages of HIV infection of cells by A. V. Serbin; A. V. Veselovskii; V. B. Tsvetkov (pp. 723-739).

Approaches to the molecular design of antiviral agents based on the principals of adequacy and mimicry to biopolymers have been discussed. The approaches permit for the reproduction of pharmaceutically valuable properties of the polymeric basis (core) and to create novel nongenetic “programs” of bioactivity of macromolecules by means of grafting specific side group combinations to this core. In vivo experiments have shown that the immunostimulating (in particular, interferon-inducing) antiviral potency of nucleic acids (NAs) is a characteristic of carboxylic acid analogues with an NA-type order of furan and anionic component alteration in the polymer chain. Controlled grafting of virus-targeted side groups of the polymeric basis leads to the direct blockage of viral infection in vitro as an additional antiviral effect. In this work, the inhibition of the early penetration stages of human immunodeficiency virus type 1 (HIV-1) in cells has been studied. Modelling of the macromolecule interaction with the most probable target (the virus-cell fusion mediator (an α-helical complex of the N- heptad repeat regions of three HIV-1 envelope glycoprotein gp41 transmembrane molecules) was performed. Computational docking of the experiment in silico resulted in the clarification of the putative mechanisms and parameters for the programming of site-tropic, axial, and belting blockages of the target via the application of an alicyclic-type side group. The discovered opportunities of multipoint axial cobelting blockage of a target opens up new prospects for the prevention/therapy of AIDS, as well as for other viral infections that use similar systems of envelope fusion peptides (of the first and third types) for penetration into cells.

Keywords: alternating maleic acid copolymers; cage alicycles; docking; gp41; glycoprotein of the HIV envelope; modelling; virus-cell fusion inhibitors

Polystyrene microspheres as bioligand carriers in the determination of Δ-9-tetrahydrocannabinol in human urine by N. M. Solodukhina; L. A. Zlydneva; E. N. Levshenko; M. A. Myagkova; I. A. Gritskova (pp. 740-745).

For the first time, a diagnostic test system for the determination of drugs, in particular, cannabinoids, in human urine was constructed on the basis of the latex agglutination reaction. For this purpose, polystyrene microspheres with a diameter of 0.6 μm and a narrow particle size distribution and carboxylic groups of stabilizer molecules on their surfaces were used. The studies revealed the optimal characteristics of particle suspension, which provide for a high sensitivity and specificity level of the test, in particular, the quantitative range of the antibody content for covalent binding with carboxylic groups on the surface of polystyrene microspheres. The operating titer of Δ-9-tetrahydrocannabinol (Δ-9-THC)-specific antibodies was 1/16-1/512 for a conjugated antigen taken at a concentration of 0.1 mg/mL. The sensitivity of the latex agglutination reaction when determining Δ-9-THC was 150 ng/mL.

Keywords: heterophase polymerization; organosilicon surfactant; latex agglutination; methods for the detection of cannabinoids in human urine; polysterene microspheres; tetrahydrocannabinol

Transgenic tomato plants as supersweet protein thaumatin II producers by A. P. Firsov; A. S. Pushin; I. V. Korneeva; S. V. Dolgov (pp. 746-751).

Yalf tomato plants have been transformed with a gene for thaumatin II from Thaumatococcus daniellii Benth. The nucleotide sequence for thaumatin II cDNA was cloned in the pBI121 vector under the control of the CaMV 35S promoter of cauliflower mosaic virus. Expression of the thaumatin II gene was detected in all of the studied transgenic lines. A quantitative estimation of the thaumatin II accumulation in fruits was performed by ELISA. The highest content of thaumatin in transgenic tomato fruits (line 91) was 46.4 ± 10.5 μg/mg of total soluble protein (4.6%). In the other studied lines, the thaumatin content ranged from 17.6 ± 6.1 to 41.3 ± 12.3 μg/mg of total soluble protein (1.8–4.1%). The fruits of transgenic plants had a well-defined sweet taste with a long aftertaste typical of thaumatin II. Transgenic tomato lines with high expression levels can be potentially used as producers of thaumatin for the food and pharmaceutical industries.

Keywords: sugar substitutes; thaumatin; tomato; transgenic plants

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Applied Biochemistry and Microbiology (v.48, #9)

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Applied Biochemistry and Microbiology (v.48, #9)