Synthesis of 2-chloro-2′-deoxyadenosine by microbiological transglycosylation using a recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> 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°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.     

An analysis of the neuronal processes in the activating brain systems of the rat during the acquisition of an instrumental defense reflex

In chronic experiment, a defensive conditioned reflex was elaborated in rats with electrodes implanted in the reticular oral pons nucleus, cortico-medial group of amygdalar nuclei, ventromedial hypothalamic nucleus and central grey substance of the midbrain. Synchronization of the activity of neuronal groups of emotiogenic formations in the studied brain structures, became enhanced at formation of the conditioned reflex. A dependence was revealed of the level of correlation of limbic neuronal groups activity on functional state of the reticular formation, as well as neurochemical correlation mechanisms. The increase (decrease) of coefficients of correlation of the activity of neuronal ensembles of limbic structures was accompanied by a change of evoked potentials parameters recorded in them.     

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 (E. C. 2.4.2.1) and pyrimidine nucleoside phosphorylase (E. C. 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°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

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.     

Ribavirin: Biotechnological Synthesis and Effect on the Reproduction of 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 on the culture of Vero cells. The combination of ribavirin and vidarabin was shown to provide the antiviral effect at lesser concentrations than with these compounds taken separately.     

High-polymeric xenogenic RNA as an antitumor immunity stimulator

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.