[TiO2-DNA nanocomposites capable of penetrating into cells].

Methods of noncovalent immobilization of DNA fragments onto titanium dioxide nanoparticles (TiO2) were developed, which led to TiO2-DNA nanocomposites capable of penetrating through cell membranes. TiO2 nanoparticles of different forms (amorphous, anatase, brookit) with enhanced agglomeration stability were synthesized. The particles were characterized by X-ray diffraction, small angle X-ray scattering, infrared spectroscopy and atomic force microscopy. Three approaches to the preparation of nanocomposites are described: (1) sorption of polylysine-containing oligonucleotides onto TiO2-nanoparticles, (2) the electrostatic binding of oligonucleotides to TiO2 nanoparticles bearing immobilized polylysine, and (3) sorption of oligonucleotides on TiO2 nanoparticles in the presence of cetavlon. All three methods provide an efficient and stable immobilization of DNA fragments onto nanoparticles, which leads to nanocomposites with a density for an oligonucleotide up to 40 nmol/mg. It is shown that DNA fragments in nanocomposites retain their ability to form complementary complexes and can be delivered into cells without transfection agents and other methods of exposure.

[Cloning of Escherichia coli K12 xylose isomerase (glucose isomerase) and studying the enzymatic properties of its expression product].

The coding region of Escherichia coli K12 xylose (glucose) isomerase gene was inserted into the pRAC expression vector and cloned in E. coli BL21 (DE3) cells. After induction of expression of the cloned gene, the proportion of recombinant xylose isomerase accounted for 40% of the total protein content. As a result of one-stage purification by affinity chromatography, a protein preparation of 90% purity was obtained. The recombinant enzyme catalyzed the isomerization of glucose to fructose and exhibited maximum activity (0.8 U/mg) at 45 degrees C and pH 6.8. The enzyme required Mg2+ ions as a cofactor. When Mg2+ and Co2+ ions were simultaneously present in the reaction medium, the enzyme activity increased by 15-20%. Complete replacement of Mg2+ with Co2+ decreased the enzyme activity. In the presence of Ca2+ at concentrations comparable to the concentration of Mg2+, the enzyme was not inhibited, although published data reported inhibition of similar enzymes by Ca2+. The recombinant enzyme exhibited a very low thermostability: it underwent a slow inactivation when incubated at 45 degrees C and was completely inactivated after incubation at 65 degrees C for 1 h.

An examination of the ability of titanium dioxide nanoparticles and its conjugates with oligonucleotides to penetrate into eucariotis cells.

In this study we examine the possibility that TiO2 nanoparticles and their conjugates can penetrate into cultivated cells without any special transfection procedures. Oligonucleotides and their derivates were conjugated with the TiO2 nanoparticles, which were obtained as colloidal solutions at a concentration of TiO2 0.3M by TiCl4 hydrolysis. The electronic microscopy of various cell cultures (KCT, Vero, and MDCK) treated with nanoparticle solutions (20 µg/µl) showed that nanoparticles could enter the cells and accumulate in the vacuoles and phagosomes and form inclusions in cytoplasm. Thus, we demonstrated the penetration of TiO2 nanoparticles and their oligonucleotide conjugates into intracellular space without any auxiliary operations. Most other researches used electroporation techniques for similar purposes [1, 2, 5].

[Consumption of hydrocarbons by psychrotolerant degrader strains].

Oil-oxidizing microorganisms have been sampled in various regions of Siberia and used in strain associations, which degrade n-alkanes of oil from various fields by 64-92% after 6 days of growth in a wide temperature range. These strains are salt-tolerant and psychrotolerant. They are compatible with aboriginal soil microflora. Promising results have been obtained in experiments on growing plants on oil-polluted soil purified with a biodegrader of this series.

[Isolation of total RNA from baker's yeast].

A simple method of production of total RNA from baker's yeast was developed. Total RNA was isolated from yeast (Saccharomyces cerevisiae) biomass using lysis with sodium dodecyl sulfate at 100 degrees C for 40-60 min and subsequent precipitation of the target product with 3 M NaCl. The preparation obtained was characterized in detail: yield of total RNA from 1 kg of pressed yeast, 9.25 g; optical density at 260 nm of 1 mg of RNA dissolved in 1 ml of water, 20.2 U; content of the acid-soluble fraction, 2.02%; and protein content, 1.8%. Total tRNA was isolated from total RNA by fractional precipitation with ethanol followed by gel filtration.

[Influence of the salt concentration in culture on the copy number of plasmid pSH1 in cells of Micrococcus sp. 9].

The study of heterotrophic bacteria isolated from the brackish waters of Lake Shira has shown that some of them contain plasmid pSH1 of approximately 2.7 kb in size. The number of plasmid copies in plasmid-containing strains cultivated at a minimal concentration of sodium chloride is found to be low, whereas the subculturing of these strains at high salt concentrations increases the plasmid number. The role of natural pSH1 plasmid in the osmotolerance of host bacteria is discussed.

[Construction of an expression plasmid vector for accomplishing in vivo delivery of recombinant biologically active proteins. 2. Synthesis of HBcAG in a vaccine strain of Salmonella choleraesuis]. / Konstruirovanie ékspressionnogo plazmidnoto vektora dlia osushchestvleniia dostavki in vivo rekombinantnykh biologicheski aktivnykh belkov. 2. Sintez HBcAg v vaktsinnom shtamme Salmonella choleraesuis.

The capacity of a previously described plasmid vector pAZ to deliver bioactive proteins to targets in vivo has been studied. This vector molecule has a strong constitutive promoter, is extremely stable in cells of vaccinal S. choleraesuis strain, and encodes the synthesis of marker protein beta-galactosidase which helps monitor the vector's fate in the host. The gene encoding hepatitis B virus core antigen (HBcAg) has been inserted into pAZ under its constitutive promoter. The resultant recombinant plasmid p19-24 has been used to transform Enterobacteriaceae (E. coli and S. choleraesuis) cells. Transformed cells produce immunologically active HBcAg. p19-24 was stable in S. choleraesuis cells during their culturing and during this strain persistence in mice. Triple oral immunization of rabbits in a dose of 1 x 10(9) S. choleraesuis cells TC177 induced the production of virus-specific antibodies. Successful transformation of cells of another vaccinal strain S. abortus ovis by this plasmid extends the potentialities of the vector. The results demonstrate good prospects of using pAZ vector for the construction of live oral vaccines.

[Antigenic determinants of proteins. The humoral immune response]. / Antigennye determinanty belkov. Gumoral'nyi immunnyi otvet.

There exist two opposing points of view on the organization of antigenic determinants of proteins: 1) the determinants are stringently predetermined regions of the protein molecule; 2) all the surface of the protein globule is potentially antigenic, and the immune response is generated occasionally in every individual, although some sites of the protein surface could be preferable for eliciting the immune response. In this work, taking into account the results of recent investigations, we propose some reconciliation: a correlation between the rigidity of the protein architecture and the antigenicity of the particular sites of the antigen molecule. Some general principles of the antigenic determinants of proteins are formulated.

[The principles of creating standard serum panels with a low concentration of HIV-1 antibodies for the quality control of commercial immunoenzyme test systems]. / Printsipy sozdaniia standartnykh panelei syvorotok s nizkoi kontsentratsiei antitel k VICh-1 dlia kontrolia kachestva kommercheskikh immunofermentnykh test-sistem.

The main approaches are defined to the production of sera with the required concentration of antibodies to HIV by the dilution method and to formation on their basis of serum panels according to theoretical distribution of optic density (OD) values. It was shown to be principally possible to prepare panels of positive sera with low concentration of HIV-specific antibodies in a dry stable form, and their practical importance in the evaluation of the sensitivity of enzyme immunoassays was demonstrated. The evaluation of the quality of commercial test systems is based on the position and shape of the histogram of OD values distribution for panel sera for the controlled test systems.

[In vitro processing of the artificial analog of beta-lipotropin]. / Issledovanie protsessinga in vitro iskusstvennogo analoga beta-lipotropina.

The processing of the recombinant analogue of beta-lipotropin (beta-LHP) having 11 additional N-terminal amino acid residues and separated from the hormone by the processing signal, was investigated using rat adrenal secretory granule lysate as a test system of processing "in vitro". It was found that incubation of the beta-LPH analogue with secretory granule enzymes leads to its limited specific degradation with a release of native beta-endorphin. It is concluded that the additional N-terminal amino acids induced no qualitative changes in beta-LPH processing.

[Solid-phase immunoenzyme analysis of apoprotein A-1]. / Tverdofaznyi immunofermentnyi analiz apoproteina A-1.

The authors have optimized indirect solid-phase enzyme immunoassay for measuring human blood serum apoA-1. Immunochemical identification of the obtained apoA-1 and specific antibodies has been carried out, working regimens for the components and conjugates employed have been selected. Relationship between the method of preliminary treatment of the serum and the completeness of detected apoA-1 antigenic determinants is demonstrated. Normal blood serum apoA-1 level is 1.06 +/- 0.1 g/l in females and 1.05 +/- 0.1 g/l in male subjects.

[Effect of poly(dA).poly(dT) sequence inserted into EcoR1 site of pBr322 plasmid on the expression of tet and bla genes]. / Vliianie posledovatel'nosti poli(dA).poli(dT), vstroennoi v EcoR1-uchastok plazmidy pBR322, na ékspressiiu genov tet i bla.

The 99 base pair poly(dA).poly(dT) stretch was inserted into EcoRI site of pBR plasmid. The effect of this insertion on tet-gene expression from P2 promoter, and bla-gene expression from P1 and P3 promoters was studied. The insertion results in a slight enhancement of the resistance of the cells to tetracycline and a decreased sensitivity to ampicillin. It is suggested that these findings reflect the effect of insertion on the transcription of the corresponding genes.

[Effectiveness of the promoter Ptac' in vitro and in mini-cells]. / Izuchenie éffektivnosti promotora Ptac' in vitro i v mini-kletkakh.

The efficiency of hybrid promotor Ptac', comprising a synthetic trp-promoter and lacUV5 "-10" sequence, was studied. By means of electrophoresis and hybridization of RNA-products obtained in vitro and in minicells, with promotor-containing plasmids; the hybrid promotor was found to be 6 times and 3-4 times as efficient as trp and lacUV5 promoters, respectively.

[Behavior of the large fragment of DNA polymerase I (the Klenow fragment) during fractionation of a cell-free extract of E. coli MRE-600]. / Povedenie bol'shogo fragmenta DNK-polimerazy I (fragmenta Klenova) pri fraktsionirovanii beskletochnogo ékstrakta E. coli MRE-600.

Distribution of the DNA polymerase I large fragment (Klenow fragment) was studied during fractionation of the E. coli MRE-600 cell-free extract with polyethylenimine. On the basis of the results obtained a simple procedure is proposed that enables the Klenow fragment to be obtained as a coproduct of DNA polymerase I, RNA polymerase, polynucleotide phosphorylase, nucleotide kinases with acetokinase and nucleoside deoxy-ribosyltransferase in the framework of a combined technological scheme.

[Isolation and properties of immobilized alkaline phosphatase from E. coli]. / Poluchenie i izuchenie svoistv immobilizovannoi shchelochnoi fosfatazy E. coli.

Preparations of alkaline phosphatase from E. coli, immobilized on Sepharose, with a specific activity of 40-60 U/g wet weight were obtained. The immobilized enzyme was stable up to 50 degrees C; at higher temperatures it was inactivated. At 70 degrees most of the activity was lost for 1 h. The substrate (AMP) stabilized the enzyme. In the temperature range from 30 to 40 degrees C activation of the enzyme was observed, especially pronounced in the presence of the substrate. The pH optimum of the immobilized enzyme activity (7.8-8.2) is shifted towards the acid region, as compared to the soluble enzyme (8.0-8.6). The kinetic parameters for inhibition by the reaction product were determined using the integral Michaelis-Menten equation. KmAMP was found to be higher in case of the immobilized enzyme as compared to the soluble one (5.02 X 10(-4) M and 1.85 X 10(-5) M, respectively), which seems to be associated with diffusion limitations.

[Distribution of enzymes of nucleic acid biosynthesis and their precursors during fractionation of Escherichia coli MRE-600 extract]. / Raspredelenie fermentov biosinteza nukleinovykh kislot i ikh predshestvennikov pri fraktsionirovanii ékstrakta Escherichia coli MRE-600.

Distribution of the enzymes of template-dependent and template-independent polynucleotide syntheses (DNA-polymerase I, RNA-polymerase, polynucleotide phosphorylase) as well as those of the biosynthesis of nucleic acids precursors (nucleotide kinases, acetokinase and nucleoside deoxyribosyltransferases) during fractionation of Escherichia coli MRE-600 cell extract was studied. On the basis of the results obtained a technological scheme was developed that enabled to combine routine procedures of purification of the above mentioned enzymes.

[Mechanism of reaction catalyzed by RNA-ligase from bacteriophage T4]. / Izuchenie mekhanizma reaktsii, kataliziruemoi RNK-ligazoi bakteriofaga T4.

The dissociation constants of the complexes of RNA-ligase with acceptors, donors and the adenylylated donor A(5')ppAp have been determined on the basis of the inhibition of ATP-pyrophosphate exchange reaction. The dissociation constants of the complexes of the enzyme with "poor" acceptors (oligouridilates) have been shown to be slightly different from those with "good" acceptors (oligoadenylates). The dependence of the reaction velocity of the formation of ligation products on the concentration of acceptors (pA)4, (pU)4 and the adenylylated donor A(5)ppAp has been studied. On the basis of the data obtained the conclusion about the random addition mechanism has been drawn. The reaction takes place in the steady-state conditions in the case of (pA)4 and in the equilibrium conditions--in the case of (pU)4.

[Study of ATP-independent stages of reaction catalyzed by phage T4 RNA-ligase]. / Izuchenie ATP-nezavisimykh stadii reaktsii, kataliziruemoi RNK-ligazoi bakteriofaga T4.

The isotope exchange between [5'-32P]pAP and A(5')ppAp catalyzed by enzyme was shown not to take place in the absence of the acceptor; i. e. the necessity of the acceptor presence during the second step of the process was demonstrated. The isotope exchange reaction between [5'32P]pAp and (pA)5p was studied. It was demonstrated that acceptor (pA)4, slightly whereas the acceptor (pU)4 completely inhibits the isotope reaction. The isotope reaction exchange between [5'-32P]pAp and (pU)4pAp does not take place. The question of existence of adenylated donor elimination mechanism in the presence of "poor" acceptors is considered on the basis of the data obtained.