Bacteriophage MS2 As a Tool for Targeted Delivery in Solid Tumor Chemotherapy.

Bacteriophage MS2 was employed for targeted delivery of an apoptosis-inducing agent, Tl+, into a tumor tissue. The targeted delivery was ensured by iRGD peptide, a ligand of integrins presumably located on the surface of endotheliocytes of the tumor tissue neovasculature and certain tumor cells. The synthesized peptide was conjugated to MS2 capsid proteins. Tl+ ions from TlNO3 penetrated the phage particles and tightly bound to phage RNA. Peptide-modified MS2 preparations filled with Tl+ caused cell death in two types of cultivated human breast cancer cells and effected necrosis of these tumor xenografts in mice. Neither peptide-conjugated bacteriophage MS2 without Tl+ nor the phage filled with Tl+ but without the peptide or the same phage with the non-conjugated peptide in solution produced such effects. The preparation exhibited no acute toxicity at a therapeutic dose.

[Functional activity of transposase of Bordetella pertussis IS element in Escherichia coli cells].

An Escherichia coli strain producing transposase of a repeated sequence of Bordetella pertussis chromosome (RSBP) was constructed. A defective MGE-helper plasmid method, which allowed the determination of transposase functional activity was developed. It was shown that transposase synthesized in E. coli cells ensures transposition of "defective" RSBP into the host chromosome. Overexpression of transposase was shown to markedly decrease the vital activity of E. coli cells under selective cultivation conditions. Reasons for a decrease in viability transposase-producing cells are discussed. Results showing the impact of transposase on replication of recombinant plasmids and E. coli cell division were obtained.

[Identification of the open reading frame coding transposase of Bordetella pertussis RS-element]. / Identifikatsiia otkrytoi ramki schityvaniia, kodiruiushchei transpozazu RS-élementa Bordetella pertussis.

A computer-aided analysis of the repeating sequence of Bordetella pertussis chromosome (RSBP3) revealed 3 open reading frames, one of whose (ORF1) can code a protein whose structure and properties are similar to those of transposasas, i.e. enzymes in charges for the traveling of migrating genetic elements of pro- and eukaryote. Mutants of the RSBP3 insertion sequence with the affected and unaffected ORF1 sequence were constructed in order to substantiate the above assumption. Two independent experimental models (formation of inter-plasmid co-integrates and of co-integrates between plasmid and E. coli chromosome) were used to show that the RSBP3-stimulated formation of co-integrates is only true for plasmids containing RSBP3 with the unaffected ORF1 sequence. An activity of the Hpr protein (a component of the phosphoenolpyruvate-dependent phosphotransferase) was proven to influence the formation process of inter-plasmid co-integrates.

Selection of E. coli strains for stable transformation with recombinant plasmids containing full-length genome of clinical HIV-1 isolates.

Strain chi6007 obtained from the parent E. coli strain chi5097 is a result of ptsH5 mutation, which allowed cells to grow without common components of the phosphoenolpyruvate-dependent phosphotransferase system. Segregants of strain chi6007 retaining the Pol+ gene responsible for inability to grow at 37 degrees C, but gaining rifampicin resistance (RifR) were used for cloning of cointegrate plasmids. Pre-integration complexes of HIV-1 were co-integrated with the pBR-322 plasmid and transformed strain chi6018. Sequencing showed that the pPIC91 hybrid plasmid contains full-length genome of HIV-1 with shortened 5-terminal LTR and full-length copy of pBR322. Elimination of the pPIC91 plasmid from chi6018 cells was followed by the appearance of auxotrophic insertion mutants. Sequencing of the insert region showed that chromosome DNA of the host cell includes integrated genomes of pBR-322 and HIV-1.

[RNA transduction by phage MS2 noninfectious virions]. / RNK-transduktsiia neinfektsionnymi virionami faga MS2.

Two fragments (1.9 and 1.8 kb) including, respectively, "structural" genes (A, B, and lysis polypeptide) and "replicase" gene, were obtained by reverse transcription PCR on the template of bacteriophage MS2 genome RNA. The fragments contained a common sequence which allowed the assembly of the entire functional genome sDNA. 1.8 kb fragment was subjected to two independent modifications: 1) so that the replicase gene could be replaced by other sequences no more than 1.5 kb in size and 2) it was inserted (on an artifician Tn-like structure) into E. coli chromosome. A system for replication of MS2-like corpuscles was thus created, containing a "shortened" genome and having extra genes. For extra genes, aminoglycoside-3'-phosphotransferase (kanamycin resistance) or a sequence of antiviral genetic program directed against hepatitis C virus were used.

[Integration and intramolecular transposition of the TnBP3 Bordetella pertussis transposon in the Escherichia coli K-12 cells -- mutant for the phosphoenolpyruvate-dependent phosphotransferase system Hpr protein ]. / Integratsiia i vnutrimolekuliarnoe peremeshchenie transpozona TnBP3 Bordetella pertussis v kletkakh Escherichia coli K-12, mutantnykh po belku Hpr fosfoenolpiruvat-zavisimoi fosfotransferaznoi sistemy.

Integration of a plasmid carrying the TnBP3 transposon of Bordetella pertussis into the chromosome of Escherichia coli and transpositions of the integrated structure within a chromosome in the wild-type and mutant cells ptsH devoid of the major Hpr protein of the phosphoenolpyruvate-dependent phosphotransferase system were studied. When transposed to a new chromosome site, the integrated structure was precisely (or almost precisely) excised from the metY gene sequence, which resulted in restoration of the Met+ phenotype. The integration and transposition events were only observed in the E. coli cells carrying the ptsH+ allele. The ptsH mutations inhibited integration and intramolecular transposition, which were restored after phenotypic or genetic suppression of the ptsH mutation. The intensity of the processes studied were suggested to depend on the integrity of a chain that ensures transferring of the phosphoryl residue by proteins of the phosphotransferase system in E. coli K12. The results obtained indicate that the ptsH mutants of E. coli can serve as the optimal host for cloning of fragments carrying repeated sequences of B. pertussis, which may apply to the repeated sequences of other microorganisms.

[Integration of plasmids into E. coli K12 chromosomes, caused by a Bordetella transposon]. / Integratsiia plazmidy v khroosomu E. coli K12, obuslovlennaia transpozonom Bordetella.

Transposition of Bordetella pertussis transposon in E. coli chromosome has been studied on a model of exclusion of donor multicopy pKK3 plasmid with coumermicin. TnBP3 induced the formation of co-integrates between the plasmid and chromosome. The structure of co-integrate was determined. Facts of exclusion of integrated structure and transposon transposition within integrated plasmid into new sites on a recipient chromosome were detected. Relationship between these processes and activity of bacterial cell recombination system has been determined.

[Transposition and inheritance of Bordetella Tn-element in Escherichia coli K12]. / Transpozitsiia i nasledovanie Tn-élementa Bordetell v kletkakh Escherichia coli K12.

B. pertussis genetically mobile element TnBp3 integrates the plasmid in E. coli chromosome. During culturing under nonselective conditions the majority of cells of some E. coli strains lose the kanamycin resistance marker, which indicates the instability of TnBp3 inheriting. The stability of inheriting the integrated structure is higher in E. coli cells with recB-21 recC-12 sbcB-2 mutations. The role of RecBC recombination system in extrusion of TnBp3 is discussed.

[The antidiphtheria activity of the pre-EGF fragment]. / Antidifteriinaia aktivnost' fragmenta pre-EGF.

The DNA fragment, coding a part of the protein molecule--the precursor of the epidermal growth factor (pre-EGF76-208)--and containing the sequence of 133 N-end amino acid residues, was obtained with the use of gene engineering and molecular biological techniques. For this purpose a fraction of poly(A+) = RNA was isolated from the kidney of a newborn infant; on this fraction the "library" of cDNA fragments whose coding capacity corresponded to the required sequence of mRNA in the pre-EGF gene was obtained in the reaction of reverse transcription, conjugated with the polymerase chain reaction (PCR). After the determination of the nucleotide sequences in 11 fragments only 1 fragment which contained practically no mutations appearing in PCR as the result of amplifications was chosen. This fragment was used for the construction of a hybrid plasmid controlling the synthesis of hybrid fusion protein with the sequence of pre-EGF76-208. Fusion protein was synthesized with very low effectiveness, but the use of metallo-affinity chromatography permitted to isolate and purify it, its purity reaching 98%. Then its antitoxic activity was determined in the skin test on guinea pigs and found to be not less then 10(6) I.U./mg of protein.

[Effect of cyclic 3,5-adenosine monophosphate on the virulence of Salmonella typhimurium]. / Vliianie tsiklicheskogo 3,5-adenozin-monofosfata na virulentnost' Salmonella typhimurium.

To study the role of cAMP in the virulence of S. typhimurium, cAMP-producing plasmid pTG 4 was transferred to cAMP-deficient S. typhimurium mutant. The transfer of the plasmid enhanced the virulence of the microorganisms due to the increased destruction of macrophages and the intensified multiplication of salmonellae in the spleen of mice.

[Bacteriostatic action of penicillin on Escherichia coli K12]. / Bakteriostaticheskoe deistvie penitsillina na Escherichia coli K12.

Penicillin has been found to have a bacteriostatic effect on logarithmically growing E. coli K-12 cells. The capacity for mitosis is restored due to the aggregation of cells. The bacteriostatic effect of this antibiotic is not linked with morphological changes in the cells.