[Evaluation of biochips for the rifampin resistance detection of M. tuberculosis in strains isolated at the Novosibirsk and Tomsk regions].

Recently in Russia biochips for rifampin resistance detection of M. tuberculosis were developed. To investigate the conformity between rifampin resistance results determined both by the routinely used absolute concentration method and USING the biochips, 272 DNA samples of M. tuberculosis isolated from TB patients at Novosibirsk and Tomsk regions in 2000-2005 were analyzed. The biochip can detect 30 mutations in rpoB gene. The mutations were also tested using the single stranded conformational polymorphism method (SSCP). In addition, 60 DNAs were randomly sampled and sequenced. The results of rifampin resistance detection using biochip and absolute concentration methods were congruent in 86% cases, and were different when analyzed samples consisted of the susceptible and resistant strains of M. tuberculosis mixture. The most frequent mutations in the rpoB gene were S531 (76.2%), H526 (7%), D516 (5.6%), and L511 (5.6%). In 94% of rifampin resistant strains, there was also resistance to isoniazid. Therefore, in Siberia the rifampin resistance is the reliable marker for MDR strains of M. tuberculosis, and biochips can be used also for their detection. To hybridize with biochip the fluorescent-labeled single-stranded DNAs were routinely synthesized by two PCR, and intermediary product after the first PCR should be transferred into another tube. The last stage included high risk of cross-contamination. To exclude the risk, primer concentrations and temperature-time profile of PCR reactions were improved, and both PCR were combined in one tube. The two methods were congruent in 100%. The one tube method would be especially attractive for the routine PCR laboratory.

[The mutagenic activity of N4-(4-aminobutoxy)-2'-deoxycytidine-5'-triphosphate]. / O metagennoi aktivnosti N4-(4-aminobutoksi)-2'-dezoksitsitidin-5'- trifosfata.

We have shown that deoxycytidine-5'-triphosphate modified by O-(4-aminobutyl)hydroxylamine in the pyrimidine ring, is effectively incorporated into DNA synthesizing in vitro, replacing deoxythymidine-5'-triphosphate or deoxycytidine-5'-triphosphate and inducing A-->G and G-->A transitions, respectively. UV spectroscopy and NMR spectroscopy have shown that the modified cytidine-5'-triphosphate is identical to N4-(4-aminobutoxy)-2'-deoxycytidine-5'-triphosphate. When the modified deoxycytidine-5'-triphosphate was inserted into DNA in vitro by DNA polymerase I of E. coli Klenow fragment, retardation sites correlating with poly-A sites (when the modified triphosphate replaced deoxythymidine-5'-triphosphate) or with poly-G sites (when it replaced deoxycytidine-5'-triphosphate) were revealed. Our data show high mutagenic effect of the modified deoxycytidine-5'-triphosphate inserted into DNA, allowing us to recommend this compound for localized static mutagenesis.

[Introduction of statistical mutations into strictly defined areas of the genome using a modified oligonucleotide]. / Vvedenie statisticheskikh mutatsii v strogo opredelennyi raion genoma s pomoshch'iu modifitsirovannogo oligonukleotida.

A method for inducing mutations in the short region of a gene is suggested. The method involves oligonucleotide modification by hydroxylamine derivative, in vitro enzymatic synthesis of double-stranded DNA using modified oligonucleotide as a primer and selection of mutant colonies using the starting unmodified oligonucleotide as a probe.

[Expression of the gene for hybrid beta-lactamase pBR322 with C-terminal fragment containing tuftsin (Thr-Lys-Pro-Arg)]. / Izuchenie ékspressii gena gibridnoi beta-laktamazy pBR322 s C-kontsevym fragmentom, soderzhashchim taftsin (Thr-Lys-Pro-Arg).

A hybrid beta-lactamase gene with a synthetic tuftsin-coding DNA fragment inserted at the Pst I-site of pBR322 plasmid has been obtained and its expression has been studied. Radioactive amino acids have been used to show that in E. coli chi 925 minicells up to 30% of newly synthesized chimeric protein is secreted into periplasm providing the tuftsin transport. After hybrid protein cleavage with CNBr, tuftsin has been isolated using ion-exchange and thin-layer chromatography.

[Mutagenesis, induced by phosphotriester analogs of oligonucleotides and directed to the cleavage site of double-spiral DNA]. / Mutagenez, indutsirovannyi fosfotriéfirnymi analogami oligonukleotidov i napravlennyi na mesta razryva dvuspiral'noi DNK.

The mutagenic properties of phosphotriester analogues revealed in course of interaction with linearized plasmid DNA were studied. The plasmid-based model system permitting one to test reliably the induced mutations is proposed. The efficiency of mutagenesis was shown to depend on the length of the oligonucleotide-mutagen and the genotype of the transformed Escherichia coli strain. The possible mechanisms involved in mutagenesis are discussed.

[Characteristics of nucleolar organization and function under condition of complete spatial in situ disassembly of its main structural components]. / Osobennosti organizatsii i funktsionirovaniia iadryshka v usloviiakh polnogo prostranstvennogo razobshcheniia ego osnovnykh strukturnykh komponentov in situ.

It is well known that fibrillar centers (FC) constitute an essential structural component of the active nucleolus in mammalian cells, yet their role in regulation of ribosomal gene transcription still remains an open question. Here, we studied the activity of endogenous RNA polymerase I upon partial and complete unraveling of nucleoli and FCs. The pattern of BrUTP incorporation in nuclei of hypotonically-treated cells was shown to be essentially the same as in the control untreated cells. Moreover, the sites of BrUTP incorporation, which revealed the active PNA polymerase I, were completely coincident with UBF-binding sites. These observations allow to conclude that structural integrity of FCs is not a prerequisite for maintenance of the active RNA polymerase I transcriptional complex. When the action of hypotonic shock was ceased and the cells were transferred to a complete cultural medium, the swollen nucleoli recovered to the control state. Therefore it is possible to conclude that none of the main morphological nucleolar counterparts, such as FCs, dense fibrillar component or the pars granulosa, is responsible for the maintenance of the nucleolar structural and functional integrity. A suggestion is made that this role may be played by the nucleolar matrix associated with the RNA polymerase I transcriptional complex.

[Cytopathological effects of protein synthesis inhibitor emetine on HeLa cells and their nucleoli]. / Tsitopaticheskie éffekty vozdeistviia ingibitora sinteza belka émetina na kletki i iadryshki kul'tury HeLa.

Eukaryotic cell nucleolus is a highly dynamic structure, which is sensitive to all changes within or outside cell borders. Numerous data are available on changes of the nucleolar structure and functions under different treatments. However, almost nothing is known about the action of translation inhibitors on the nucleolus, although these substances, together with TNF-alpha, are commonly used for apoptosis induction, both for scientific and therapeutic purposes. Emetine is one of such inhibitors. We have shown that emetine suppresses cell viability, decreases mitotic index, and induces apoptosis in HeLa cells. Emetine action is irreversible, and it sensitizes cells to unfavourable external conditions. The emetine action causes redistribution of UBF, one of RNA-polymerase I factor, from the nucleolus to nucleoplasm even after a short exposure, i.e. when the morphology of the nucleus and chromatin still keeps its native pattern. It is important that other nucleolar proteins, such as fibrillarin and B23, are not recognized in the nucleoplasm until the very late stages of apoptotic process. A suggestion is made that changes in UBF localization may be associated with the onset of ribosomal repeat cleavage and migration of rDNA-"free" fragments from the nucleolus to nucleoplasm. It looks likely that these changes can serve as an initial morphological indication of apoptosis.

[Isolation and study of recombinant strains of Salmonella typhimurium SL 7207, producing HBcAG and HBcAG-HBs]. / Poluchenie i issledovanie rekombinantnykh shtammov Salmonella typhimurium SL 7207, produtsiruiushchikh HBcAg i HBcAg-HBs.

Recombinant strains producing hepatitis B virus (HBcAg) core protein and chimeric core protein exposing on its surface the major immunogenic epitope of HBsAg (HBcAg-HBs) were constructed on the base of attenuated S. typhimurium SL 7202 strain. The resultant Salmonella strains produced proteins which were capable of self-assembly into virus-like particles and showed antigenic properties of both core and surface hepatitis B proteins. A single rectal immunization with recombinant S. typhimurium induced humoral and cellular immune response to HBcAg and HBsAg. Specific anti-HBcAg were detected in animal sera and intestinal tissues, which indicated the formation of specific mucosal immunity.

[The effect of x-ray contrast agents on the rheological properties of the blood]. / Vliianie rentgenokontrastnykh sredstv na reologicheskie svoistva krovi.

The effects of X-ray contrast agents on human blood rheology were investigated. The main cause of rheological effects of X-ray contrast media is their action on the structure and function of erythrocytes. There was a relationship between the physicochemical properties and the mechanism of action. The major active factor is the high osmolality of X-ray contrast media solutions. Lipid solubility, specific features of molecular structures and the composition of a dosage form played a definite role.

[The biochemical and rheological properties of the blood during the performance of angiography using Ultravist]. / Biokhimicheskie i reologicheskie svoistva krovi pri provedenii angiografii s pomoshch'iu ul'travista.

It is shown that Ultravist makes it possible to receive clear visualization of vessel channel in the zone "of interest" without changing functional conditions of heart vascular system and biochemical blood parameters (level of erythrocytes, bilirubine, urine nitrogen, activity of aspartataminotransferase). In concentration 30 mg/ml in vitro and in vivo Ultravist decreases a viscosity limit not affecting other rheological properties of blood. A mechanism of the found Ultravist effect and prospects of its application in practice for children are considered.