[Use of Escherichia coli recF mutant strains for analyzing the genotoxicity of complex platinum compounds]. / Primenenie redF mutantnykh shtammov Escherichia coli dlia analiza genotoksichnosti kompleksnykh soedinenii platiny.

The bioluminescence method of assessing SOS response in Escherichia coli cells was applied to test the genotoxicity of five complex platinum compounds: cis-diamminedichloroplatinum, cycloplatam, trans-diamminedichloroplatinum, and two trans-binuclear complexes. Strains AB1157 (pPLS-1) and JC9239 (pPLS-1), a variant of AB1157 carrying the recF143 mutation, were used in the test procedure. SOS response in JC9239 cells was shown to be induced by significantly lower concentrations of cis-DDP and cycloplatam than in AB1157 cells. A drastic increase in cis-DDP toxicity for the JC9239 strain was shown. However, the presence of the recF mutation in JC9239 cells retarded the SOS response induced by trans-platinum compounds. The results obtained indicated that the E. coli recF system plays an essential role in repair and utilization of cis-DDP-DNA adducts.

[Genetic approaches to Itsenko-Cushing disease]. / O geneticheskikh podkhodakh k bolezni Itsenko--Kushinga.

107 patients with Itsenko-Cushing disease were examined for heredity: family history was analyzed in 75 cases, dermatoglyphics was assessed in 44 cases, I- and II-class HLA antigens were studied in 68 cases. The patients were found to have hereditary loading both by Itsenko-Cushing and other diseases (hypertension, atherosclerosis, autoimmune disorders). Clinico-genealogical evaluation made it possible to identify forms of the disease which are inherited autosome-recessively and autosome-dominantly. However, in the majority of patients the disease onset had multifactorial nature, as there were HLA-antigen associations by DR4, DR5, DR7, DRw53, DQw3. Pilot experience with genetic study of the disease showed its genetic determination in some forms, its association with hypertension and atherosclerosis, approaches to prevention, prognosis, classification. Practical recommendations on detailed family history collection in patients with Itsenko-Cushing disease have been developed.

[Expression of a synthetic gene for human interleukin-4 in E. coli cells. Preparation of a biologically active protein]. / Ekspressiia sinteticheskogo gena interleikina-4 cheloveka v kletkakh E. coli. Poluchenie biologicheski aktivnogo belka.

Expression E. coli plasmid were constructed in which the human interleukin-4 (hIL4) synthetic gene is controlled by tac promoter. The expression level of the gene depends on the distance between RBS and the initial codon ATG, with the maximal production in case of the nine base pair distance. The recombinant protein, accumulated in the inclusion bodies, was solubilized, renaturated, and purified to homogeneous, biologically active preparation, the yield being 2 mg/g wet cells.

[Recombinant interleukin-3 expression system in E. coli]. / Sistemy ékspressii v E. coli rekombinantnogo interleikina-3.

Plasmid pTOTE2IL3 (III) has been constructed, expressing an artificial human interleukin-3 (hIL3) gene under conditions of the induced protein biosynthesis. Levels of the recombinant protein synthesis have been compared in several E. coli strains containing expression plasmids pTE2IL3 (I) (constitutive biosynthesis) and (III) (induced biosynthesis). Optimal combinations of the expression plasmids and the bacterial strains are of importance. A simple and effective method has been elaborated for isolation, purification and renaturation of the recombinant protein accumulated in inclusion bodies.

[Separation and analysis of immunochemical properties of multiple forms of cytochrome P-450 from the rat liver]. / Razdelenie i issledovanie immunokhimicheskikh svoistv mnozhestvennykh form tsitokhroma P-450 pecheni krys.

Four cytochromes P-450 induced by phenobarbital (PB-1--PB-4) and two cytochromes P-450 induced by S-methylcholanthrene (MC-1, MC-2) were purified to electrophoretic homogeneity from rat liver microsomes. The purification procedure involved sequential chromatography on n-aminooctyl-Sepharose 4B, DEAE-Sephacel and hydroxylapatite columns. The spectral and immunochemical properties of the cytochromes P-450 were estimated. All, but MC-1, cytochromes P-450 were found to exist in a low spin state. Using the Ouchterlony double diffusion method, it was shown that all cytochromes P-450 under study can be divided into two groups, i. e., PB-1--PB-2 and PB-3--PB-4, sharing common antigenic determinants inside the groups. High performance liquid chromatography of PB-3 and MC-2 on anion-exchangers yielded two additional peaks from the PB-induced major cytochrome P-450 PB-3 and three peaks from the MC-induced major cytochrome P-450 MC-2. The multiplicity of cytochrome P-450 forms is discussed.

[Identification and characteristics of mRNA for cytochrome P-450 in the rat liver induced by phenobarbital and 3-methylcholanthrene]. / Identifikatsiia i kharakteristika mRNK tsitokhroma P-450 iz pecheni krys, indutsiruemogo fenobarbitalom i 3-metilkholantrenom.

Using two consecutive oligo(dT)-cellulose column chromatography steps, the total poly(A)RNA was isolated from the livers of rats injected with phenobarbital (PB) or 3-methylcholanthrene (MC). During translation of the PB-induced mRNA in the reticulocyte lysate cell-free protein-synthesizing system, a single polypeptide with an apparent molecular weight of 50,000 was synthesized which was specifically immunoprecipitated by antibodies to major PB-inducible cytochrome P-450 PB-3. In contrast, after completion of MC-mRNA translation, the antibodies to major MC-induced cytochrome MC-2 precipitated from the incubation mixture 4-5 polypeptides, of which the largest one with an apparent molecular weight of 58,000 corresponded to cytochrome P-450 MC-2. During sucrose density gradient centrifugation, the PB- and MS-mRNAs with sedimentation coefficients of about 18S and 20S, respectively, were precipitated.

[Bacteriophage P22 H5 transfection and infection of plasmid Salmonella strains]. / Transfektsiia i infektsiia bakteriofagom P22 H5 plazmidnykh shtammov sal'monell.

The results of the Ca2+-dependent transfection of the DNA of bacteriophage P22 H5 to constructed Salmonella typhimurium F'- and R+-strains LT2 WT-R and SA118 demonstrated that in these salmonellae the effectiveness of transfection depended on the specificity of the interrelation of plasmids with host strains. Plasmids RA1, R538-1 and RP1 stimulated the transfection of S. typhimurium strain LT2 WT-R, but suppressed the transfection ability of S. typhimurium strain SA118. At the same time the expression of the function of plasmids R446b and R64-11 did not depend on the host strain, as the former did not affect and the latter suppressed the release of transfectants in both Salmonella strains. The presence of plasmids R124, RA1, R64-11 and R724 in strain SA118, heat-sensitive in respect to the synthesis of cell-wall lipopolysaccharide, not only led to a decrease in the effectiveness of transfection; the effectiveness of the inoculation of bacteriophage P22 H5 was also suppressed 10(4) times in the presence of plasmid R124 and at least 10(10) times in the presence of 3 other plasmids. The development of resistance to S-specific bacteriophage P22 H5 was not linked with disturbances in the adsorption of this bacteriophage. Besides, the addition of CaCl2 into the medium completely removed the limitation of infection with bacteriophage P22 H5, determined by plasmid R124.

[Characteristics of Salmonella mutants competent for isolated nucleic acids]. / Kharakteristika mutantov sal'monell, kompetentnykh dlia izolirovannykh nukleinovykh kislot.

The method for the isolation of transfectable salmonellae by growing them in a liquid medium for a long time and selecting R-clones, subsequently tested in the Ca2+-dependent transfection of the DNA of bacteriophage P22 H5, was developed. The probability of obtaining transfectable clones varied between 18.3% and 48.3% in different Salmonella strains. Testing their sensitivity to specific bacteriophages used for the determination of structural distortions in the lipopolysaccharide layer of the cell wall made it possible to divide transfectable Salmonella clones into 3 phenotypical groups. The effectiveness of the formation of transfectants in the Ca2+-dependent transfection of the DNA of bacteriophage P22 H5 increased to 4.0 X 10(-8) - 8.0 X 10(-8) in the isolated R-clones of S. typhimurium strain SU453. In contrast to the initial strain, these R-clones were characterized not only by more effective transfection, but also by the possibility of plasmid transformation.

[Transfectable strains of Salmonella]. / Transfektabel'nye shtammy Sal'monell.

It the Ca2+-dependent system of an intact bacterial recipient the efficacy of DNA transfection of P22 H5 bacteriophage was determined in 48 strains of bacteria belonging to the Salmonella genus and in 5 Escherichia coli strains with known genetic characteristics and phenotypical properties. The sensitivity of the salmonella strains under study to R- and RS-specific bacteriophages, and also their capacity to ferment galactose were determined. Transfectable mutants of bacteria belongin to the Salmonella genus were referred to the Ra-type. The salmonella P22 H5 bacteriophage of the heterologous E. coli recipient isolated DNA transfection was demonstrated.