Possible Function of the ribT Gene of Bacillus subtilis: Theoretical Prediction, Cloning, and Expression.

The complete decipherment of the functions and interactions of the elements of the riboflavin biosynthesis operon (rib operon) of Bacillus subtilis are necessary for the development of superproducers of this important vitamin. The function of its terminal ribT gene has not been established to date. In this work, a search for homologs of the hypothetical amino acid sequence of the gene product through databases, as well as an analysis of the homolgs, was performed; the distribution of secondary structure elements was theoretically predicted; and the tertiary structure of the RibT protein was proposed. The ribT gene nucleotide sequence was amplified and cloned into the standard high-copy expression vector pET15b and then expressed after induction with IPTG in E. coli BL21 (DE3) strain cells containing the inducible phage T7 RNA polymerase gene. The ribT gene expression was confirmed by SDS-PAGE. The protein product of the expression was purified by affinity chromatography. Therefore, the real possibility of RibT protein production in quantities sufficient for further investigation of its structure and functional activity was demonstrated.

[Destabilization and stabilization of poly(A).poly(U) structure by platinum(II) compounds].

On the basis of molecular biophysics, a methodology for the analysis of intramolecular structural order of the polynucleotide duplex poly(A).poly(U) has been developed. It was shown that the combination of circular dichroism spectroscopy with differential scanning calorimetry is an optimal approach, which ensures the screening of a wide set of substances and interaction conditions and the choice of compound(s) that can stabilize the structure and increase the biological activity of this duplex. The study is aimed at obtaining a new and highly active antiviral remedy.

[Broadening of the organ specificity of the action of polynucleotide interferon inducers]. / Rasshirenie organospetsifichnosti deistviia polinukleotidnykh induktorov interferona.

Interferon titers in the blood and brain of mice and their protection from the herpes virus were compared after the animal exposure to poly(G).poly(C) duplex, both native and modified with cis-diammine dichloroplatinum (II). It was shown that the duplex platination especially at the level of the poly(G) strand resulted in sharp rising of the interferon titers in the extracts of the animal brain and rearrangement of the types of interferon induced in the brain to predominance of gamma-interferon. The interferonogenesis indices correlated with the duplex protective activity against the herpes virus. It was concluded that the platinum binding could increase the membrane specificity of the duplex and stimulate its penetration through the hematoencephalic barrier. Possible structural changes in the duplex under the action of platinum (II) resulting in the observed effect are discussed.

[Antiviral activity of poly(A)-poly(U) duplexes modified with cis-diammine-dichloroplatinum (II)]. / Protivovirusnaia aktivnost' dupleksov poli(A)-poli(U), modifitsirovannykh tsis-diammin-dikhloplatinoi(II).

Polyribonucleotide duplex poly(A).poly(U) was modified with cis-diammine dichloroplatinum (II) (cis-DDP). It was shown that the antiinfluenza protective activity of the modified duplex in mice increased with the degree of modification (rb) rising up to 0.2. The effect was different from that for poly(I).poly(C) and poly(G).poly(C). The interferon titers in the murine brain increased in parallel with increasing of the antiviral activity. It was assumed that the structural specificity of the poly(A).poly(U) duplex was responsible for the phenomenon and that cis-DDP interaction with N(7) atoms of the adenine heterocycles blocked the "abnormal" Hoogsteen pairing of adenines with uracils. As a result the antiviral activity increased because of lowering the quantity of the intramolecular defects and increasing the length of the regular double-stranded regions.

Template-dependent biosynthesis of poly(G) x poly (C) and its antiviral activity in vitro and in vivo.

Experimental conditions for poly(G) synthesis from GTP on a poly(C) template with the aid of Escherichia coli DNA-dependent RNA polymerase were investigated. The reaction product was purified without the use of RNase. On the basis of spectral data, gel permeation chromatography, affinity adsorption and electron microscopic visualization, the poly(G) x poly(C) product was assumed to possess a high degree of structural regularity. Its in vitro and in vivo antiviral activities were compared with those of traditional poly(G) x poly(C) and poly(I) x poly(C). Template-dependent poly(G) x poly(C) was similar in its in vitro activity to poly(I) x poly(C) or even surpassed it, whereas the 'traditional' poly(G) x poly(C) was only slightly active in vitro. However, 'traditional' poly(G) x poly(C) and poly(I) x poly(C) had similar activity in vivo, whereas template-dependent poly(G) x poly(C) was much less active in vivo. The role of intramolecular structural regularity in the in vitro and in vivo antiviral activity of polyribonucleotide duplexes is discussed.

[Biological activity of the poly(G).poly(C) complex, modified by divalent platinum compounds]. / Biologicheskaia aktivnost' kompleksa poli(G).poli(C), modifitsirovannogo soedinenimi dvukhvalentnoi platiny.

Modification of poly(G).poly(C) with cys-diaminodichloroplatinum (cys-DDP) at the level of rb = 0.02 increased the in vivo antiviral and interferon-inducing activity of the complex, in contrast to the data reported for complex poly(G).poly(C). Antiinfluenza activity in this case depends on the method of modification and increases more intensively when a ready complex is treated with cys-DDP, as against treatment of poly(G) alone before the formation of a complex with poly(C). If rb is increased, the activity reduces again. Modification with trans-DDP at rb = 0.02 also leads to an increase of antiinfluenza activity of poly(G).poly(C), but mainly after pretreatment of poly(G).

[Analysis of defects in the structure of the complex poly(G).poly(C)]. / Analiz defektov struktury kompleksa poli(G).poli(C).

The concentration of free poly(C) in solution in the course of its interaction with poly(G) as well as in the presence of preformed complex poly(G).poly(C) was measured by differential pulse polarography (DPP) at a mercury dropping electrode. Poly(C) binding with poly(G) was shown to hamper its electrochemical interaction with the mercury electrode and registration by DPP. It was concluded that the extremely low DPP signal from poly(C) in the presence of preformed complex was the result of its interaction with the distortions in the secondary structure of complex molecules containing free guanines. For quantitative testing of these defects, measurement of Tb3+ ion fluorescence was applied. It was shown that preliminary denaturation of the poly(G) secondary structure reduced the amount of structural defects in the complex and restored of complete DPP registration of redundant poly(C) added to this complex. These results show that the combination of DPP and Tb3+ fluorescence measurements permits one to detect at the quantitative level the structural defects in the poly(G).poly(C) complex.

[Interrelationship between the chain length of poly-L-lysine and the degree of protection of polyribonucleotide interferon inducers from human blood nucleases]. / Vzaimosviaz' mezhdu dlinoi tsepi ékraniruiushchego poli-L-lizina i stepen'iu zashchity poliribonukleotidnykh induktorob interferona ot nukleas krovi cheloveka.

The action of the human total blood serum on polynucleotide interferon inducers, larifan and ridostin (natural double-stranded RNAs) and poly(I).poly(C) (a double-stranded complex of synthetic polyribonucleotides) used both in the free state and in the state shielded with poly-L-lysine was studied. The rate of the accumulation of the acid soluble products was compared with the residual interferon-inducing activity in mice. All the unshielded inducers were shown to completely loose their activity after a 4-hour contact with the serum. The protective activity of poly-L-lysine increased in parallel with the increase of its molecular weight and was maximal for the preparation with the molecular weight of 12300 +/- 1000 Da. Differences in the structure of the inducers and the mechanism of their biosynthesis and degradation must be taken into account.

[Resistance of natural and synthetic polyribonucleotide inducers of interferon to human blood ribonucleases]. / Ustoichivost' prirodnykh i sinteticheskikh poliribonukleotidnykh induktorov interferona k ribonukleazam chelovecheskoi krovi.

The resistance of polyribonucleotide inductors of interferon to blood ribonucleases was studied. Blood resistance of larifan and ridostin in the free and shielded state as well as that of the complexes of poly(I)-poly(C) and poly(G)-poly(C) were also investigated. A protective action of polylysine against the inductors was detected which, in case it had no effect on the biological activity of the drugs, could provide its recommendation as a compound for shielding the inductors.

[Evaluation of the size of the continuous poly(G) site necessary for the biological activity of the poly(G).poly(C) complex]. / Otsenka razmera nepreryvnogo uchastka poli(G), neobkhodimogo dlia biologicheskoi aktivnosti kompleksa (G).poli(Ts).

On the basis of synthesis of a series of poly(G, A).poly(C) copolymers with changing G:A ratio from 15:1 to 90:1 and trials of their biological activity in comparison with poly(G).poly(C), the size of poly(G) in it was evaluated within the range of a continuous double-stranded area necessary for the activity. The antiviral activity close to that of poly(G).poly(C) in experimental tick-borne encephalitis of mice and vesicular stomatitis virus infection of chick embryo cells was found only in poly(G,A).poly(C) complexes with a G:A ratio equal to or higher than 90:1. Consequently, the high activity of poly(G).poly(C) is present at an average length of poly(G) equal to 90-100 nucleotides within the limits of the continuous double-stranded area.

[Combined antiviral effect of synthetic polyribonucleotide interferonogens and specific antiviral antibodies in arbovirus infections]. / Kombinirovannyi protivovirusnyi éffekt sinteticheskikh poliribonukleotidnykh interferonogenov i spetsificheskikh protivovirusnykh antitel pri arbovirusnykh infektsiiakh.

A model of tick-borne encephalitis in BALB/c mice was used to investigate the protective anti-viral effect of an interferon inducer, poly(G).poly(C), and specific gamma-globulin administered to the animals together or separately in small doses 24 hours before or after virus inoculation. Administration to the animals of poly(G).poly(C) alone or gamma-globulin alone was shown to produce a poor protective effect. Simultaneous administration of both preparations resulted in a significant decrease of mouse mortality after infection. As a result of the pretreatment of chick embryo cell cultures with poly(G).poly(C) before inoculation and the addition of specific immune serum to the agar overlay after the Sindbis virus inoculation, its multiplication was inhibited much more than after treatment of the cells with interferon inducer alone or antibody alone. Possible mechanisms of the observed additive antiviral effects of the interferon inducer and antibody, including those associated with the influence on the virus-induced interferon production, as well as the possibility of their combined use for the prevention and treatment of viral infections are discussed.

[Thermoactivation of poly(G).poly(C): a description of the effect and its hypothetical mechanism]. / Termoaktivatsiia poli(G).poli(Ts): opisanie éffekta i ego predpolagaemyi mekhanizm.

Heating of poly(G).poly(C) complex solutions at a temperature about 100 degrees C was shown to overcome a decrease in the antiviral and interferon-inducing activity of the preparations which were obtained at relatively high concentrations of polynucleotides from poly(G) stored in solution, or were stored frozen themselves. These unfavourable conditions contributed to stabilization of the poly(g) secondary structure and decrease in the degree of regularity of the complex molecules. The results suggest that thermal activation of such poly (G). poly(C) preparations occurred in 2 stages by melting residual free regions of poly(G) and their subsequent interaction with poly(C) with formation of a more regular complex.

[Modification of the poly G-poly C complex by incorporation of adenosine in the purine chain]. / O modifikatsii kompleksa poli(G)-poli(Ts) vkliucheniem adenozina v purinovuiu nit'.

Antiviral and interferonogenic activity of the complexes of poly(G,A) . poly(C) and poly(G) . poly(C) was studied in mice and cell cultures. Three out of 4 complexes of poly(G,A) . poly(C) had insignificant antiviral and interferonogenic activity in chick embryo cells. One of the complexes induced low levels of interferon production in mice and decreased the rate of their death from experimental forest-spring encephalitis. The activity of poly(G) . poly(C) in the above cell systems was much more pronounced. Unlike this complex, some complexes of poly(G,A) . poly(C) showed a noticeable activity in the cells of Primates. The effect of the noncomplementary base in the purine thread of poly(G) . poly(C) on its biological activity and nucleotide composition is discussed.

[Effect of different polyribonucleotide interferonogens on acute and latent viral infections in mice]. / Vliianie razlichnykh poliribonukleotidnykh interferonogenov na ostruiu i latentnuiu virusnuiu infektsiiu myshei.

Poly(G) . poly(C) and poly(I) . poly(C) complexes administered soon after the viral challenge induced a high survival rate in mice with experimental tick-borne encephalitis. The protective effect was still noted when the treatment was given 24 hours after the infection. If the therapy was conducted at the end of the incubation period, at the peak of the virus reproduction in the mouse brain, poly(I) . poly(C) intensified the infection development and increased the animal death rate, while poly(G) . poly(C) had no such effect. Poly(I) . poly(C) injected 12 hours after the peak of the virus-induced interferonogenesis led to death of 80% animals inoculated with non-pathogenous Newcastle disease virus. The action of various samples of poly(I) . poly(C) was diverse. Poly(G) . poly(C) failed to effect the outcome of latent viral infection. The death of infected mice induced by polyribonucleotide complexes was not connected with their anti-viral interferonogenous activity, but correlated with the level of their toxicity for the intact animals. The results of the study have confirmed the risk of using poly(I) . poly(C) for the therapy of viral infections, especially during their clinical manifestation, and proved the safety of application of poly(G) . poly(C) and of some other polyribonucleotide interferonogens.

[Hydrolysis and the inactivation of double-stranded polyribonucleotides by monkey blood serum]. / Gidroliz i inaktivatsiia dvukhnitevykh poliribonukleotidov syvorotki krovi obez'ian.

The effect of Macaca rhesus monkey blood serum on double-stranded polyribonucleotide complexes poly (I).poly (C), poly (G).poly (C), and poly (G,I).poly (C) was studied. The poly (I).poly (C) complex was found to be the most sensitive to hydrolysis as indicated by a decrease of the molecular weight, accumulation of acid-soluble products and a sharp decline of the antiviral and interferon-inducing activities in tissue culture after incubation of the complex in the presence of the serum at 37 degrees C for 1 hour. The poly (G).poly (C) complex was the most stable, and retained its original activity in tissue culture and a high molecular weight after 3-hour incubation with the serum. The interferon-inducing activity of all the complexes under study assayed by intravenous injection in a dose of 2 mg to M. rhesus monkeys was similarly low irrespective of their sensitivity to the serum. Conjectural species features of the interferon induction system in monkeys are discussed.

[Antiviral activity of the complexes obtained at different ratios of complementary homopolyribonucleotides]. / Protivovirusnaia aktivnost' kompleksov, poluchennykh pri razlichnykh sootnosheniiakh komplmentarnykh gomopoliribonukleotidov.

Antiviral activity of the complexes of synthetic polyribonucleotides, i.e. poly (I).poly (C) and poly (G).poly (C) obtained at non-equimolar ratios of homopolymers was studied. The system of chick embryon fibroblasts and horse Venezuellan eguine encephalitis virus served as the model. It was shown that the active and stable complexes poly (I).poly (C) and poly (G).poly (C) were formed at some excess of poly (C), i.e. at the ratio of poly G) or poly (I) to poly (C) equal to 40/60 to 20/80 molar per cent. The role of the excessive poly (C) in formation of the stable secondary structure of the nucleotide complexes and its significance as one of the means for affecting the fine structure of double-stranded RNA were discussed.