[Enzymatic transglycosylation of natural and modified nucleosides by immobilized thermostable nucleoside phosphorylases from Geobacillus stearothermophilus].

Natural and modified purine nucleosides have been synthesized using the recombinant thermostable enzymes purine nucleoside phosphorylase II (EC 2.4.2.1) and pyrimidine nucleoside phosphorylase (EC 2.4.2.2) from Geobacillus stearothermophilus B-2194. The enzymes were produced in recombinant E. coli strains and covalently immobilized on aminopropylsilochrom AP-CPG-170 after heating the cell lysates and the removal of coagulated thermolabile proteins. The resulting preparations of thermostable nucleoside phosphorylases retained a high activity after 20 reuses in nucleoside transglycosylation reactions at 70-75 degrees C with a yield of the target products as high as 96%. Owing to the high catalytic activity, thermal stability, the ease of application, and the possibility of repeated use, the immobilized preparations of thermostable nucleoside phosphorylases are suitable for the production of pharmacologically important natural and modified nucleosides.

[Synthesis of 2-chloro-2'-deoxyadenosine by microbiological transglycosylation using a recombinant Escherichia coli strain].

Cladribine (2-chloro-2'-deoxyadenosine) was synthesized using intact cells of the recombinant Escherichia coli strain producing Geobacillus stearothermophilus B-2194 thermostable purine-nucleoside phosphorylase II (EC 2.4.2.1). Use of the cells containing this thermostable enzyme allowed the process to be conducted at a temperature of 70 degrees C, which provided the maximal concentrations of sparingly soluble substrates. The best results were obtained with 2-chloroadenine as a modified base. The highest yield of the target 2-chloro-2'-deoxyadenosine (up to 95% in the case of deoxyguanosine) was reached when using 2'-deoxypurines as donors of deoxyribose. Use of thymidine for these purposes required its considerable molar excess over 2-chloroadenine (up to 6 : 1), which is connected with a nonoptimal amount of endogenous thymidine phosphorylase, necessary for synthesis of deoxyribose-1-phosphate, in the transglycosylation reaction.

[Biotechnological synthesis of ribavirin. Effect of ribavirin and its various combinations on the reproduction of Vaccinia virus]. / Biotekhnologicheskii sposob polucheniia ribavirina. Deistvie ribavirina i nekotorykh ego kombinatsii na reproduktsiiu virusa ospovaktsiny (Vaccinia virus).

The biotechnological method of synthesis of ribavirin, vidarabin, and 6-azauridine by the use of immobilized recombinant enzymatic preparations of nucleoside phosphorylase was improved. The effect of ribavirin and its combinations with the other synthesized nucleosides on the reproduction of Vaccinia virus was studied using cultures of Vero cells. The combination of ribavirin and vidarabin was shown to provide an antiviral effect at lesser concentrations than when these compounds were taken separately. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 6; see also http://www.maik.ru.

[Design of an expression vector for delivery of in vivo recombinant biologically active proteins. 1. Synthesis of the antigenic determinant of HIV-1 gp120 and gp41 proteins in enterobacteria]. / Konstruirovanie ékspressionnogo plazmidnogo vektora dlia osushchestvleniia dostavki in vivo rekombinantnykh biologicheski aktivnykh belkov. 1. Sintez antigennykh determinant belkov gp120 i gp41 VICh-1 v énterobakteriiakh.

High-level expression vector pAZ was constructed for in vivo delivery of bioactive recombinant proteins, antigenic determinants, among other things. This vector meets the requirements to construction of recombinant bacteria as live oral carriers. It has a strong constitutive promoter, high stability in E.coli and vaccine strain Salmonella cells, and, moreover, encodes in addition for the marker protein (beta-galactosidase) which will later help follow up the fate of bacterial carriers and their interactions in the microorganism. Several recombinant plasmids encoding for beta-galactosidase variants with insertions of short fragments of HIV-1 gp41 and gp120 proteins, which were previously shown to be antigenic determinants, have been constructed on the basis of pAZ. E.coli and vaccine strain Salmonella cells were transformed by recombinant plasmids. To a considerable extent the level of hybrid protein synthesis depends on the structure of the antigenic determinant inserted. The maximal level of synthesis in E.coli is 16%. This hybrid protein could be isolated and purified (up to 90%) with the yield of 4 to 6 mg/g of wet cells. Almost all the hybrid proteins were immunologically reactive, as shown by ELISA with nonfractionated lysates and purified hybrids. In both strains in vitro stability of the vector and recombinant plasmids was at least 90% after 10 passages (about 140 generations) under random conditions. This paper sums up the first stage of construction of recombinant bacteria as live oral carriers.

[Changes in the parameters of nonspecific protection of mice upon inoculation of double-stranded RNA from Saccharomyces cerevisiae yeasts]. / Izmenie pokazatelei nespetsificheskoi zashchity organizma myshei pri vvedenii dvuspiral'noi RNK iz drozhzhei Saccharomyces cerevisiae.

The results of the study of the effect of double-stranded (ds) RNA from killer yeasts Sac. cerevisiae on the parameters of nonspecific protection are presented. Inoculation of dsRNA in a dose of 5 mg/kg intramuscularly was shown to induce interferon production, to increase body temperature, the activity of adrenal cortex, phagocytic activity of macrophages of the peritoneal exudate and blood neutrophils, the level of lysozyme in the blood, and the activity of macrophage acid phosphatase. The increase in the functional activity of the system components was observed at similar intervals, within 3-6 hours after administration of the preparation. The mutual induction of factors of nonspecific protection and their cooperative participation in the formation of antiviral resistance under the effect of dsRNA is discussed.

[High-polymeric xenogenic RNA as an antitumor immunity stimulator]. / Vysokopolimernaia ksenogennaia RNK kak stimuliator protivoopukholevogo immuniteta.

To immunize CC57BR mice a suspension of live cells of Krebs-2 ascites tumour was administered intradermally into the tail partially amputated afterwards. The growth of the tumour transplanted intraperitoneally was inhibited by 23% only after twofold immunization. Single immunization with tumour cell incubated with the cattle liver RNA preparation in conjunction with intraperitoneal administration of RNA following tumour transplantation inhibited its growth by 43--53%, while twofold administration by 84--88%. The high polymeric fraction of the preparation enhanced the immunization effect to the same measures the initial overall preparation. The treatment of the preparation with RNAase and partial depolymerization of RNA in the course of isolation resulted in the activity loss. It is concluded that the capacity of the RNA preparation for stimulating antitumour immunity is due to high polymeric fraction of RNA.

[Effect of exogenous RNA on the activity of poly(A).oligo(dT)-dependent DNA-polymerase in mouse tumors]. / Vliianie ekzogennoi RNK na aktivnost' poli (A). Oligo(dT)-zavisimoi DNK-polimerazy v opukholiakh myshei.

The effects of exogenous RNA on the poly(A).oligo(dT)-dependent DNA-polymerase activity of factor and factor-free carcinomas of the mammary gland of C3H mice and Ehrlich ascite carcinoma were studied. It was shown that after intraparenteral injection of the RNA the enzyme activity is increased in the oncornavirus-containing carcinomas and remains unchanged in the virus-free tumours. The effect of RNA is not due to the glucocorticoid release into the blood and is suppressed by actinomycin D one hour prior to the addition of RNA. The increase of the poly(A).oligo(dT)-dependent DNA-polymerase activity of the cytoplasmic fraction of tumour cells is correlated with the rise of cytoplasmic RNA-polymerase activity and the increased incorporation of the precursors into the cytoplasmic RNA. Thus, in virus-containing tumours exogenous RNA induces the synthesis of cytoplasmic RNAs responsible for the synthesis of RNA-dependent DNA-polymerase.