[Humoral factors of immunity and the mononuclear cell responses to fetoproteins in patients with pulmonary tuberculosis].

The content of cytokines, the levels of antibodies (Ab) to proinflammatory cytokines and fetoproteins (FP) in serum, as well as the effects of fitohemagglutinin and FP on the level of cell production of cytokines into the conditioned medium were studied in relation to the pattern of a response of mononuclear cells (MNC) to FP. With the positive reaction of MNC to FP, estimated by an increment of CD9* cells and detectable in grades 2-3 dysplasias, the anti-inflammatory effect with lower anti-inflammatory cytokines was shown to be achieved due to elevated Ab levels, which may compensate for the low content of IL-4 and IL-10. With FP, there was an increase in the cell production of IL-10 that is known to stimulate antibody formation in the early phase of tumor growth, as evidenced by the association of the levels of Ab to FP with the grade of dysplasia.

[L-Glutamic acid modulates the cytotoxic effect of tumor necrosis factor in the HL-60 cell line].

L-Glutamic acid was shown to increase the stability of cells of the HL-60 line of human promyelocyte leukemia to the cytotoxic action of tumor necrosis factor alpha (TNF-alpha) due to the inhibition of apoptotic and NF-kappaB-activating cascades induced by this cytokine. At the same time, L-glutamic acid increases the TNF-alpha-mediated differentiating signal and the accompanying enhancement of the phosphatidylinositol-specific phospholipase C activity. Therefore, it is a promising agent for the reduction of total toxicity and inflammatory processes during treatment with TNF-alpha. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 6; see also http://www.maik.ru.

[Reiterative poly(A) synthesis on oligonucleotide templates in an Escherichia coli RNA-polymerase system]. / Rei terativnyi sintez poli(A) na oligonukleotidnykh matritsakh v sisteme RNK-polimerazy Escherichia coli.

The poly(A) synthesis in the E. coli RNA-polymerase system on the oligothymidilates, oligouridilates and oligonucleotides d(5'-OH-CCATCTTTT-3'-OH) and d(5'-HO-TCTTTT-3'-OH) as templates was studied. The reaction was shown to proceed more intensively on the oligothymidilates compared to oligouridilates. The insertion of phosphate residues into 3' end of oligouridilates deteriorates properties of their templates though the length of the synthesized poly(A) is not changed in this case. The synthesis of poly(A) on the nonanucleotide d(CCATCTTTT) proceeds intensively, but there is no AMP incorporation on the hexanucleotide of similar structure. The author's data combined with certain literature results suppose that the template activity of the oligonucleotides containing homopolymer sequences is determined in the RNA-polymerase system in reiterative regime by two factors: by the total amount of oligonucleotide links and by the homopolymer block size. The total amount of links seems to be important for the oligonucleotide enzyme bind stability and must comprise not less than 6 charges of the phosphate groups. The homopolymer block size must be no less than of 4 links.

[Circular transcription map of the plasmid pBR322]. / Kol'tsevaia transkriptsionnaia karta plazmidy pBR322.

The plasmid pBR322 transcription in the isolated E. coli DNA-dependent RNA-polymerase system was studied. Transcription regions as well as transcripts orientation were defined using both the technique of "criss-cross" hybridization and the annealing of RNA-products with L- and H-strands of plasmid. Summing up obtained data together with available data on promoter localization a circular transcription map of plasmid pBR322 was constructed. Effects of heparin, ion strength and E. coli S-30 system on in vitro transcripts were also studied. The 110-long RNA transcript synthesized in Ori region of pBR322 was found to be the most sensitive to all these factors. RNA-transcripts obtained in in vitro system are able to direct protein synthesis in cell-free S-30 system.

[Termination of DNA transcription by a poly(dA).poly(dT) fragment inserted into pBR325 plasmid]. / Terminatsiia transkriptsii DNK fragmentom poli(dA).poli(dT), vstroennym v plazmidu pBR325.

A recombinant DNA was constructed by inserting polynucleotide (dA).(dT) of 80-100 base pairs long into EcoRI site of the pBR325 plasmid DNA. Transcription of this DNA was studied in E. coli RNA polymerase system in vitro. Some transcripts obtained with the recombinant plasmid were shown to have poly(U) clusters at the 3'-ends. Obtained data indicate that poly(dA).poly(dT) sequence acts as a terminator of RNA synthesis. Orientation of this sequence in recombinant DNA was also established.

[Transcription of synthetic oligonucleotides]. / Transkriptsiia sinteticheskikh oligonukleotidov.

The decadeoxynucleotides d(pTTC)3 and d(CCATCTTTT) transcription by E. coli RNA-polymerase was studied. The nucleotide composition of d(pTTC)3 transcript was shown to be consistent with that of the template, but RNA-product was several times longer than the template. With nonanucleotide d(CCATCTTTT) the poly(A) synthesis was observed. This fact may be attributed to the reiteration on the TTTT-cluster. When using the oligonucleotide primers d(pGGA) and r(pAAAA) complementary to the templates d(pTTC)3 and d(CCATCTTTT), accordingly, the nucleosidetriphosphates concentration being reduced and the RNA-polymerase "holo" being replaced by "core", the considerable decrease in the unhomogeneity of the transcript length and in the length itself was found. With d(pTTC)3 as a template the length of RNA-product was found to be of 24-25 nucleotides and with d(CCATCTTTT) it was of 18-19 nucleotides. The sequence of RNA transcribed from both templates in the presence of primers was in accordance with the structure of templates.

[Template properties of the decadeoxynucleotide d(pCCACGAAACC) in the RNA polymerase system of Escherichia coli]. / Issledovanie matrichnykh svoistv dekadezoksinukleotida d(pCCACGAAACC) v sisteme RNK-polimerazy Escherichia coli.

The decadeoxynucleotide d(pCCACGAAACC) transcription by E. coli RNA-polymerase was studied. The transcript was shown to be several times longer than the template. The oligonucleotide GpGpGpGpUp complementary to the "contact" of two neighbouring template molecules was found in the pancreatic RNase digest of the RNA-product. This fact is consistent with our hypothesis reported recently. Pentanucleotide d(pGGTTT) may funtion as a primer in the decadeoxynucleotide transcription being incorporated into RNA.

[Conversion of nucleoside triphosphates in an RNA polymerase system]. / O prevrashcheniiakh nukleozidtrifosfatov v RNK-polimeraznoi sisteme.

The behavior of nucleoside triphosphates (NTP) in the RNA-polymerase system from E. coli was studied. The conversion of NTP to the corresponding nucleoside diphosphate (NDP) was observed. This phenomenon was accounted for a contaminating enzyme activity in the RNA-polymerase preparations. The possibility to remove such a contamination was demonstrated, the best technique being the DNA-cellulose affinity chromatography. The purified enzyme does not catalyze the intermediate formation of NDP's from NTP's during RNA synthesis with poly(U) and poly(dG)-poly(dC) as templates.

[Col E1 DNA transcription in Escherichia coli RNA-polymerase system in vitro]. / Issledovanie transkriptsii DNK plazmidy ColEI v sisteme RNK-polimerazy Escherichia coli.

The ColEI plasmid DNA transcription in E. coli RNA-polymerase system in vitro was studied. Three RNA types: of 110-140 residues long, 1700 residues long and of the length similar to that of DNA-template were synthesized in the reaction, containing 10 mM MgCl2, 50 mM KCl and 15% glycerol. The transcription was shown to be asymmetric, the DNA H-chain being transcribed. The ColEI DNA region transcribed in vitro was located by the blotting techniques. This region comprised most of the ColEI genome except the colicin EI gene. The addition of mitomycin C to the reaction mixture caused only a little stimulation of colicin EI gene transcription. Four fragments of HaeIII digest of ColEI DNA were shown to have affinity to RNA-polymerase in the absence of NTPs and two fragments to have it in the presence of 3 NTPs (in the RNA initiation conditions). These two fragments seem to contain two strong promoters. One of them is in the HaeIIIA fragment, where the colicin EI gene origin is situated, and another one is the HaeIIIB fragment (in the immunity region).

[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.

[Preparation of a mutant human tumor necrosis factor with increased resistance to proteases]. / Poluchenie mutantnogo faktora nekroza opukholei cheloveka s povyshennoi ustoichivost'iu k proteazam.

The potential proteolysis sites of human TNF are considered. By site-directed mutagenesis the Arg-31 residue of mature TNF was substituted by Gln. The analysis of cytotoxicity of initial and mutant (R31Q) proteins on mouse L929 fibroblasts did not reveal any differences in biological activity. For the mutant protein a change in proteolysis dynamics was shown in contrast to the natural variant: mutant TNF displayed increased stability when treated with trypsin.

[Substrate specificity of T4 RNA-ligase. The effect of the nucleotide sequence of the minimum phosphate acceptor on the efficacy of intermolecular ligation]. / Substratnaia spetsifichnost' RNK-ligazy T4. Vliianie nukleotidnoi posledovatel'nosti minimal'nogo aktseptora fosfata na éffektivnost' mezhmolekuliarnogo ligirovaniia.

The 2' (3'),5'-diphosphates of cytidine, uridine, adenosine and the trinucleoside diphosphates, such as NpCpC, NpCpU, NpUpC, NpUpU and ApNpC (where N = U, C, A, G), were used to study the substrate specificity of the T4 RNA ligase. The outcome of ligating various phosphate acceptors to a common phosphate donor was governed not only by nucleotide composition but also the nucleotide sequence of a phosphate acceptor. The effect of the changes in a single position of the phosphate acceptor on the efficiency of the intermolecular ligation cannot be defined independently of the other nucleotide residues. For four examined groups of phosphate acceptors and pCp as a donor, four different ranks were obtained (in order of decreasing reactivity): C greater than A greater than greater than U greater than G (for NpCpC); G greater than U approximately equal to A greater than C (NpCpU); A greater than U greater than C greater than G (NpUpC) and A greater than G greater than C greater than U (NpUpU). The yield of ligation products with various phosphate donors and a common phosphate acceptor significantly depends on the donor structure: pyrimidine phosphate donors are more effective than pAp, while pCp is more effective than pUp.

[Substrate specificity of T4 RNA-ligase. The effect of the nucleotide composition of substrates and the size of phosphate donor on the effectiveness of intermolecular ligation]. / Substratnaia spetsifichnost RNK-ligazy T4. Vliianie nukleotidnogo sostava substratov i razmera donora fosfata na éffektivnost' mezhmolekuliarnogo ligirovaniia.

Nucleoside 2' (3'),5'-diphosphates, dinucleotides pApA, pApC, pApU, pGpC, pCpC, pUpU (phosphate donors), and trinucleoside diphosphates, such as NpCpC, NpCpU, NpUpC, NpUpU and GpApN (N = U, C, A or G; phosphate acceptors) were used to study the substrate specificity of T4 RNA ligase. Relative efficiency of the mono- and dinucleotide donors depends on the 5'-terminal nucleoside moiety of the dinucleotide: upon ligation with the minimal phosphate acceptor GpUpC, dinucleotides pApA, pApC, and pApU are more effective than nucleotide diphosphate pAp; pGpC is more effective than pGp; efficiencies of pCpC and pCp are almost identical, and efficiency of pUpU is slightly lower than that of pUp. In relative efficiency, dinucleotide donors, varying only in 5'-terminal unit, do not correspond to mononucleotides: pApC greater than pCpC greater than pGpC and pCp greater than pUp approximately pAp much greater than pGp. The effects observed for homooligomeric substrates cannot be extra-polated on heterooligomers.

[Synthesis of oligoribonucleotides with the use of RNA polymerases of E. coli and immobilized synthetic DNA-templates]. / Sintez oligoribonukleotidov s ispol'zovaniem RNK-polimerazy E. coli i immobilizovannykh sinteticheskikh DNK-matrits.

A new approach to synthesis of oligoribonucleotides is suggested, based on transcription by E. coli RNA polymerase of synthetic immobilized DNA-templates with AUG as primer. The approach has been experimentally verified by synthesis of two oligonucleotides, viz., a RNA fragment of the fr phage (16 nucleotides long) and a RNA fragment of the tickborne encephalitis virus (18 nucleotides long). Fraction of the synthesized RNA fragments in the whole nucleotide material is about 20%. The templates can be used repeatedly. Sequences of the oligoribonucleotides were confirmed. Advantages of this approach and its usefulness for SP6 DNA-dependent RNA polymerase are discussed.

[Preparation and bacterial expression of a mutant gene for human lymphotoxin]. / Poluchenie i bakteral'naia ékspressiia mutantnogo gena limfotoksina cheloveka.

Using the oligonucleotide directed mutagenesis, a human lymphotoxin (TNF beta) mutant gene lacking 21 N-terminal codons has been obtained. Recombinant plasmid pLT21 for expression of the mutant gene has been constructed. The mutant gene in the plasmid was placed under control of a tandem of constitutive promoters from coliphage T7. A simple procedure for isolation of recombinant protein was developed. The procedure allows to obtain the highly purified biologically active mutant protein with a good yield. During biosynthesis the recombinant protein undergoes a posttranslational processing resulted in the cleavage of N-terminal methionine and leucine residues.

[Biosynthesis of alpha2-interferon and tumor necrosis factor at different stages of culturing]. / Issledovanie biosinteza alpha2-interferona i faktorov nekroza opukholei na stadiiakh kul'tivirovaniia.

Study of the biosynthesis of human alpha 2-interferon and tumor necrosis factor in derivatives of E. coli SG200-50 recipient strain containing pIF16, pTNF311, and pLT21 plasmids demonstrated elimination of the plasmids from the cells without degradation thereof in the course of culturing. Methods increasing the content of the end products in the biomass are proposed.

[Site-specific restriction endonuclease RME211 from Rhizobium meliloti]. / Saitspetsificheskaia éndonukleaza restriktsii RME211 iz Rhizobium meliloti.

A new site-specific restriction endonuclease Rme211 from Rhizobium meliloti has been shown to recognize the following nucleotide sequence 5'-ATCGAT-3' in the double-stranded DNA. Thus, the enzyme is a true isoschizomer for restriction endonucleases Bsu151 and ClaI.

[Design of stimulating agents of non-specific resistance system]. / Sozdanie sredstv stimuliatsii sistemy nespetsificheskoi rezistentnosti.

The paper briefly reviews a study on the design of drugs enhancing the body's nonspecific resistance to pathogenic agents. To examine the potential regulatory effects on cytokine function was a main trend. The interferon inductor ridostin, dsRNA of microbiological origin, cytokines, as well as the recombinant probiotic strain yielding interferon alpha-2 synthesis were used as a pharmacological agent. This was shown by using a wide range of experimental models wherein these preparations activated the components of the non-specific resistance system resulting in the host's increased capacity to eliminate invasive agents and transformed cells.

[Study of antiinfectious potential of recombinant tumor necrosis factor-alpha]. / Izucheniie antiinfekstionnogo potentiala rekombinantnogo faktora nekroza opukholi alpha.

Tumor necrosis factor-alpha (TNF) was shown to have an antiinfectious effect on murine and rat models of viral, bacterial and parasitic infections. The effective antiinfectious dose of TNF was determined to be 10(2)-10(3) U/kg body weight. TNF, interferon, and antibiotics were demonstrated to potentiate the antibacterial effect of each. Clinical trials of TNF in the treatment of slowly progressive, latent, and chronic infections and in the emergency prevention of acute infections are advisable.

[Results of Befnorin clinical trails in healthy volunteers]. / Rezul'taty klinicheskogo ispytaniia preparata benforina na zdorovykh dobrovol'tsakh.

Clinical trails of Befnorin based on the human recombinant TNF-beta elaborated at the Research Design and Technology Institute of Biologically Active Substances, "Vector" State Research Center of Virology and Biotechnology, were carried out on healthy volunteers in compliance with a decision passed by the Committee of Medical and Immunobiological Preparations, Russia's Health Ministry. Single Befnorin doses of 5-10(4) U, 10(5) U, 5-10(5) U, and 10(6) U were administered as intramuscular injections. Clinical, biochemical and immunological parameters were registered for 7 days after a single dose. The drug had an impact on the below immunity indices: Fc-phagocytosis of monocytes, migration index and migration inhibition index. The dose of 10(5) U was proven to be most effective and safe. Supposedly, the drug can be effective in the treatment of herpetic diseases.