[Quasi-adaptive response to alkylating agents in Escherichia coli and Ada-protein functions].

In 2005 we have described in exponentially growing E. coli cells a new fundamental genetic phenomenon,--quasi-adaptive response to alkylating compounds (quasi-Ada). Phenotypic expression of quasi-Ada is similar to the true Ada response. However, in contrast to the letter, it develops in the course of pretreatment of the cells by a sublethal dose of nonalkylating agent, an NO-containing dinitrosyl iron complex with glutathione (DNICglu). To reveal the mechanisms of quasi-adaptation and its association with the function of the Ada regulatory protein, here we used a unique property of dual gene expression regulation of aidB1 gene, a part of the Ada-regulon, namely its relative independence from Ada protein in anaerobic conditions. Based on the results of aidB1 gene expression analysis an EPR spectra of E. coli MV2176 cells (aidB1::lacZ) in aerobic and anaerobic conditions after the corresponding treatments, we conclude that the function and the spatial structure of meAda and [(Cys-)2Fe+(NO+)2]Ada are identical and thus the nitrosylated protein represents a regulator of the Ada regulon gene expression during quasi-adaptation development.

[The genetic activity of NO-containing agents is regulated by complex formation with intracellular iron].

This work is a part of a directional search for new crystal donors of nitric oxide (NO), which are promising for complex chemotherapy. The relationships between the physico-chemical properties of NO donors, their genotoxic and mutagenic activities, and the dependence on intracellular iron were studied. New crystal NO donors (di- and trinitrosyl iron complexes with synthetic ligands) were examined for the first time and compared with known NO donors containing natural ligands. All but one compound induced expression of the Escherichia coli sfiA gene belonging to the SOS regulon and exerted a mutagenic effect on Salmonella typhimurium TA1535. These effects were fully or significantly inhibited by the iron(II)-chelating agent o-phenanthrolin, depending on the mono- or binuclear structure of the ligands. The rate of donating free NO in solution did not positively correlate with the genotoxic activity of the crystal NO donors. The genetic activity of all NO donors proved to depend on intracellular iron.

[O6-benzylguanine stimulates regulatory functions of the Ada protein in Escherichia coli].

In vitro experiments showed that O6-benzylguanine (O6-benzG, 0.2 microM) fully inhibited the repair activity of human O6-alkylguanine-DNA alkyltransferase (MGMT) due to the formation of S-benzylcytosine in the protein acceptor site. O6-benzG at concentrations increased many times (up to 800 microM) failed to inhibit the repair activity of the Escherichia coli Ada protein, the structural and functional analog of MGMT. It has been shown for the first time that O6-benzG stimulates the regulatory activity of the Ada protein. In experiments with N-nitroso-N-methylurea (NMU), the pretreatment of Escherichia coli cells with O6-benzG at a sublethal concentration of 10 microM led to a twofold enhancement of transcription at the Ada-dependent promoter of the alkA gene in control cells and ensured transcription enhanced 1.6-1.7 times at alkA and alkB promoters in cells with the induced "classical" Ada response. Apparently, an increase in the regulatory activity of the Ada protein was associated with the formation of the stable protein molecule having the strong affinity for alkA and alkB promoters after transfer of the benzyl group from O6-benzG to the acceptor site Cys-69 in the N-terminal domain of Ada protein. O6-benzG did not affect the regulative activity of Ada in alternative quasi-adaptive responses to NMU.

[Quasi-adaptive response to alkylating agents in Escherichia coli: a new phenomenon].

An original hypothesis of a quasi-adaptive response to nitrosomethylurea (NMU) in Escherichia coli cells was verified experimentally. In contrast to the true Ada response, which is induced in cells pretreated with a sublethal dose of NMU, a quasi-adaptive response was induced using NO-containing dinitrosyl iron complex with glutathione (DNICglu). Quasi-adaptation increased expression of the Ada regulon and cell resistance to the cytotoxic and mutagenic effects of NMU. The levels of alkA, alkB, and aidB gene expression in quasi-adaptation were higher than in the true Ada response. Thus, experimental evidence was obtained for the alternative mechanism regulating the function of the Ada sensory protein in controlling expression of the Ada regulon during the adaptive response. The free iron--chelating agent o-phenanthroline (OP) facilitated degradation of DNICglu (by electron paramagnetic resonance (EPR) spectra) and considerably or completely inhibited gene expression in the quasi-adaptive response. The new phenomenon extends the functional range of NO compounds to include a role in genetic signal transduction within the Ada response system in addition to similar roles in the SoxRS, SOS, and OxyR systems in E. coli.

[Connectivity between soxS gene expression and adaptive resistance to the SoxRS-regulon inducers in E. coli cells]. / Vzaimosviaz' ékspressii gana soxS s razvitiem adaptivnoi rezistentnosti k induktoram SoxRS-regulona v kletkakh E. coli.

Development of the adaptive response (AR) to the SoxRS-inducers-menadione (O2(-.)-donor), dinitrosyl-iron complex (NO donor) and their simultaneous action was studied in E. coli. Two AR parameters were used: an increasing in viability and decreasing in the soxS gene (SoxRS-regulon) expression in adapted cells. It was shown that namely peroxynitrite (ONOO-), being formed inside the cells from O2-. and NO, was the most cytotoxic agent among the drugs tested. On the one side, an increase in resistance to menadione treatment was selectively demonstrated in adapted E. coli delta oxyR mutant cells, defective in OxyR-regulon activity. On the other side, a decrease in soxS gene expression was marked in the experiments with menadione, as well So, an AR to O2-. superoxide anion was selectively regulated by the SoxRS DNA-repair pathway. OxyR-regulon that is selectively activated by the most redox-cycling agents and controls AR to these agents doesn't provide development of the AR to O2-..

[The new antitumor drug cycloplatam: cytotoxicity and DNA interstrand cross-linking]. / Novyi protivoopukholevyi preparat tsikoplatam: tsitotoksichnost' i mezhnitevye sshivki DNK.

Cytotoxicity genetic mechanisms such as induction of SOS-repair, excision repair and interstrand coupling induced by cycloplatam or ammine (cyclopentyl amine)-S(-) malatoplatinum (II), a new antitumor drug, were for the first time studied in comparison to those of the known drug cis-diammine dichloroplatinum (II) (DDP) in a model system of Escherichia coli. In the cells of E. coli the cycloplatam cytotoxicity was much lower than that of DDP. Both the drugs induced SOS-repair in E. coli PQ37. In a concentration of 25 microM DDP was 20 times as active as cycloplatam. In concentrations of 40 to 100 microM the difference leveled. Both the drugs induced interstrand coupling in specimens of pure DNA from calf thymus and E. coli. When the cells of the wild type E. coli AB1157 were incubated in the presence of the drugs only DDP induced the DNA interstrand coupling. No correlation between the DNA interstrand coupling induced by cycloplatam or DDP and cytotoxicity of the drugs was observed.

[Expression and functions of adaptive response genes in Escherichia coli treated with mono- and bifunctional alkylating agents. Interference with SOS response]. / Ekspressiia i funktsii genov adaptivnogo otveta v Escherichia coli pri deistvii mono- i bifunktsional'nykh akliliruiushchikh agentov. Interferentsiia s SOS-otvetom.

The expression of genes belonging to the Ada regulon of Escherichia coli under the action of mono- and bifunctional alkylating agents--high-efficiency antitumor HMM, ACNU, and BCNU preparations--was studied. The functional specificity of the alkA, alkB, and aidB1 genes concerning both the structure and volume of DNA alkylation and the specificity of cell preadaptation was revealed. Additional experimental evidence for the role of the aidB1 gene as a unique "hazard gene", a component of the E. coli ada operon, was obtained. A phenomenon of positive interference between alternative SOS and Ada responses was observed for the first time upon gene expression.

[Restoration by T-activin and its subfractions of splenocyte membrane structures in thymectomized animals]. / Vosstanovlenie taktivinom i ego subfraktsiiami struktury membran splenotsitov timéktomirovannykh zhivotnykh.

It was shown that thymectomy induces the injury of plasma membranes of mouse splenocytes. This membrane disorders completely inhibit T-cell response on Concanavalin. A. The incubation of splenocytes with Tactivin or injection of Tactivin or its subfractions to the animals restore the membrane structure and T-cell response.