[Inheritable phenotypic normalization of rodent cells transformed by simian adenovirus SA7 E1 oncogenes by singled-stranded oligonucleotides complementary to a long region of integrated oncogenes]. / Nasleduemaia fenotipicheskaia normalizatsiia transformirovannykh E1 onkogenami adenovirusa obez'ian SA7 kletok gryzunov odnotiazhevymi oligonukleotidami, komplementarnymi protiazhennym uchastkam integrirovannykh onkogenov.

G11 mouse cells and SH2 rat cells transformed with simian adenovirus SA7 DNA showed inheritable oncogen-specific phenotypic normalization when treated with sense and antisense oligonucleotides complementary to long RNA sequences, plus or minus strands of the integrated adenovirus oncogenes E1A and E1B. Transitory treatment of the cells with the oligonucleotides in the absence of serum was shown to cause the appearance of normalized cell lines with fibroblastlike morphology, slower cell proliferation, and lack of ability to form colonies in soft agar. Proliferative activity and adhesion of the normalized cells that established cell lines were found to depend on the concentration of growth factors in the cultural medium. In some of the cell lines, an inhibition of transcription of the E1 oncogenes was observed. The normalization also produced cells that divided 2 - 5 times and died and cells that reverted to a transformed phenotype in 2 - 10 days. The latter appeared predominantly upon the action of the antisense oligonucleotides.

[Mapping of cloned fragments of the macrophage-stimulating factor receptor gene (FMS) from a non-amplified library of human leukocyte genes]. / Kartirovanie klonirovannykh fragmentov gena retseptora koloniestimuliruiushchego faktora makrofagov (FMS) iz neamplifitsirovannoi biblioteki genov leikotsitov cheloveka.

For investigation of FMS gene polymorphism and mutations that reveal functionally meaning in leukemia and myelodysplastic disorders the overlapping recombinants lambda-clones inserted by FMS gene fragments have been obtained from human leukocyte genomic library in the EMBL 3A phage by using oligonucleotide prode (27 nucleotides) based on 12 exon of the FMS gene. 15 DNA probes were prepared by subcloning the lambda-clones obtained in the pBSKS+ plasmid. The probes obtained allow to analyse extracellular, transmembrane and tyrosine kinase regions of the FMS gene independently.

[Oncogene-directed mutagenesis in vivo. Polyalkylating derivatives of short single-stranded polynucleotides, complementary E1-adeno-oncogene, in the normalization of adenovirus-transformed rodent cell lines]. / Onkogen-naprovlennyi mutagenez in vivo. Polialkiliruiushchie proizvodnye korotkikh odnotiazhevykh polinukleotidov, komplementarnykh E1- adenoonkogenu, v normalizatsii transformirovannykh adenovirusom kloetochnykh linii gryzunov.

Polyalkylating derivatives of single-stranded polynucleotides (30-200-mers) complementary to the long E1 oncogene sequences of simian adenovirus SA7 cause inherited normalization of SH2 and G11 cells transformed with adenovirus SA7; certain deletions in the integrated proviral E1A oncogene were observed in several cases during this process. The transformed cells are indifferent to reagents noncomplementary to the E1 region. Thus polyalkylating derivatives of single-stranded 30-200-mers act as addressed mutagenes which react in a specific way with the integrated complementary DNA sequences of E1 oncogene in transformed rodent cells and realize oncogene-directed mutagenesis in vivo. During this treatment temporary normalized cells reverting to the initial transformed phenotype are also produced.

[Penetration of oligo/polynucleotides and their polyalkylating derivatives into rat cells transformed by simian adenovirus DNA]. / Proniknovenie oligo/polinukleotidov i ikh poliakriliruiushchikh proizvodnykh v kletki krysy, trnasformirovannye DNK adenovirusa obez'ian.

The transport regularity of the [32P]-oligo/polynucleotides and their polyalkylating derivatives into SH2 rat cells transformed with SA7 adenovirus DNA was investigated. Derivatives penetrate the SH2 cells and their distribution in the subcellular fractions are proportional to the concentration of reagent in the medium. The transport efficiency of the derivatives is inhibited sharply with cell concentration increase and practically does not depend on the action of cell metabolism inhibitors. The data obtained assumes the mechanism of the derivatives transport to be liquid endocytosis. Being distributed in the cell components the polyalkylating derivatives were accumulated by the cell nuclei up to 10(5)-10(7) molecules per nucleus. Transport efficiency is much greater in the anchored cells than in the suspended ones. Though essential dephosphorylation of the utilized substances is observed in the SH2 cells, part of them maintain native chain length and the 5'-phosphate group after 1 h incubation in nucleic acids obtained from the cell nuclei.

Complementary directed modifications of nucleic acids and oncogene-directed mutagenesis in vivo.

High reactivity of the polyalkylating ss oligomers that were sense or antisense 30-200-mers containing sequences complementary to E1 oncogenes of simian adenovirus SA7 and one alkylating residue -CH2CH2N(C2H5OH) (CH2)3N(Ph-p-CH2OH)CH2CH2Cl per each 25 bases of oligomers was demonstrated in vitro by alkylation of ss DNA of recombinant M 13 mp8E1 and mp9E1 phages with inserted E1 sequences of adenovirus oncogene and then by followed complete and selective elimination of E1 sequences from recombinant ss DNA. Treatment of rodent cell cultures transformed by oncogenic SA7 with polyalkylating oligomers which are complementary to the long region of the minus or plus chains of E1 DNA of SA7 revealed a rather high extent of mutant cell clones formation. The cells formed were normalized; they had lost some properties of the transformed cells. Dividing cell clones inherited the new phenotypic properties: morphology, slower and more limited proliferation, and higher dependence on bovine serum growth factors. Some of the mutant cell DNAs demonstrated different mutations in the E1A sequences of the integrated proviral oncogene. There were exchanges G to C (leu to val) in the 525 and C to A (asp to tyr) in the 555 positions of E1A oncogene. Besides a deletions in the 1057-1477 E1A region or/and a mutation in the 1457-1477 of E1A were observed. Thus the inherited cell normalization observed is performed due to oncogene-directed mutagenesis in vivo.

[Preparation and characteristics of a new transformed cell line G11 by transfection of NIH/3T3 mouse fibroblasts with DNA from the SA7 oncogene of simian adenovirus]. / Poluchenie i kharakteristika novoi transformirovannoi kletochnoi linii G11 pri transfektsii fibroblastov myshei NIH/3T3 DNK onkogena adenovirusa obez'ian SA7.

Murine fibroblasts NIH 3T3 were transfected with the plasmid pASP containing simian adenovirus oncogene insertion. Focus forming transformants were cloned with a final dilution technique and a new cell line G11 was created as a result. Transformed status of this cell line is evidenced by changes in morphology, specific cytochemical and adhesion properties, ability to grow in semisolid agar and FCS concentration growth independence. Presence of intact integrated E1a-region of adenovirus SA7 oncogene was shown by blot-hybridization technique. Transformed status of G11 cells can be explained by integration of SA7 oncogene, that is evidenced indirectly by the increased resistance to heat shock.

[Immunomodulating activity of RNA mononucleotides]. / Immunomoduliruiushchaia aktivnost' mononukleotidov RNK.

The immunological action of RNA mononucleotides was studied in animal experiments. The most pronounced activation of macrophagal glycolysis urea cycle, oxidative phosphorylation, lysosomal hydrolases was induced by uridine 5'-monophosphate (5'-UMP) and guanosine 5'-monophosphate (5'-GMP); 5'-GMP also induced the maximum increase of the expression of FC gamma receptors. 5'-UMP ensured cell activation comparable with the total action of all mononucleotides. 5'-UMP and 5'-GMP, used in combination, produced the highest stimulating effect on macrophages, and the addition of low-active adenosine 5'-monophosphate (5'-AMP) to active 5'-UMP did not decrease the stimulating potency of the latter. The stimulating activity of sodium nucleinate exceeded that of all mononucleotides and their combinations. 5'-GMP and 5'-AMP induced the maximum activation of oxygen metabolism, evaluated by chemiluminescence, while 5'-UMP and cytidine 5'-monophosphate (5'CMP) proved to be inactive. The shift of the phosphate group to the third carbon atom or the production of the oligonucleotide 5'-UMP consisting of 5-15 nucleotides resulted in the appearance of the capacity for stimulating oxygen metabolism in macrophages. Their in vitro cultivation with 5'-GMP and 5'-AMP induced the maximum increase of cell spreading in comparison with other mononucleotides, while the maximum increase of phagocytosis was ensured only by 5'-UMP and 5'-GMP.2+ 5'-UMP and 5'-GMP enhanced nonospecific resistance to Salmonella typhi infection, and sodium nucleinate, to Pseudomonas pseudomallei and Pseudomonas mallei infections.

[Similarity of adhesion to phagocytosis: macrophage interaction with different substrates]. / Skhodstvo adgezii s fagotsitozom: vzaimodeistvie makrofagov s razlichnymi substraktami.

Similarity between adhesion and phagocytosis is shown in the case of macrophages. Both adhesion of macrophages to polystyrene and phagocytosis of polystyrene particles (latex) do not depend much on the temperature and are not inhibited with iodoacetate. In contrast, both cell adhesion to adsorbed immunoglobulin G and phagocytosis of erythrocytes opsonized with immunoglobulin G, which are mediated with Fc-receptors, have some characteristic temperature dependence and are inhibited with iodoacetate. It may be concluded that it is the type of substrate, rather than its size, that determines mechanisms of adhesion and phagocytosis.

[Redistribution and activation of peritoneal macrophages with the peroral use of sodium nucleinate]. / Pereraspredelenie i aktivatsiia peritoneal'nykh makrofagov pri peroral'nom primenenii nukleinata natriia.

Three hours after the administration of sodium nucleinate the activation of glycolysis, the hexosomonophosphate shunt, the urea cycle (determined by the analysis of key enzymes), the expression of FC gamma-receptors and the decrease of oxidizing phosphorylation were noted in the peritoneal macrophages of mice. By 36 hours the gradual decrease of these characteristics occurred. After three oral administrations of the preparation immature macrophages with lower metabolic activity and increased oxidizing phosphorylation appeared in the exudate, while resident and activated cells practically disappeared. The immaturity of the cells was confirmed by their incubation in vitro. The changes in the cells, revealed in this investigation, are supposed to occur due to the redistribution of activated and intact macrophage populations in situ.

The principles of enzyme stabilization. VI. Catalysis by water-soluble enzymes entrapped into reversed micelles of surfactants in organic solvents.

1. The possibility of stabilizing water-soluble enzymes against the inactivation action of organic solvents by means of surfactants has been studied. Several enzymes (alpha-chymotrypsin (EC 3.4.21.1), trypsin (EC 3.4.21.4), pyrophosphatase (EC 3.6.1.1), peroxidase (EC 1.11.1.7), lactate dehydrogenase (EC 1.1.1.27) and pyruvate kinase (EC 2.7.1.40)) were used to demonstrate that enzymes can be entrapped into reversed micelles formed by surfactants (Aerosol OT, cetyltrimethylammonium bromide, Brij 56) in an organic solvent (benzene, chloroform, octane, cyclohexane). The enzymes solubilized in this way retain their catalytic activity and substrate specificity. 2. A kinetic theory has been put forward that describes enzymatic reactions occurring in a micelle-solvent pseudobiphasic system. In terms of this theory, an explanation is given for the experimental dependence of the Michaelis-Menten equation parameters on the concentrations of the components of a medium (water, organic solvent, surfactant) and also on the combination of the signs of charges in the substrate molecule and on interphase (++, +-, --). 3. The results obtained by us may prove important for applications of enzymes in organic synthesis and for studying the state and role of water in the structure of biomembranes and active centres of enzymes.