[Treatment of elderly patients with combination of coronary artery disease and aortic valve pathology].

Increasingly, there are patients with concomitant coronary artery disease and aortic valve, especially in elderly patients, who often have severe comorbidities and high surgical risk, which is undoubtedly a certain effect on the choice of method and tactics of treatment. Today, there are several approaches to the treatment of patients in this category, and all of them have certain advantages and disadvantages.

BORIS, a paralogue of the transcription factor, CTCF, is aberrantly expressed in breast tumours.

BORIS (for brother of the regulator of imprinted sites), a paralogue of the transcription factor, CTCF, is a novel member of the cancer-testis antigen family. The aims of the present study were as follows: (1) to investigate BORIS expression in breast cells and tumours using immunohistochemical staining, western and real-time RT-PCR analyses and (2) assess potential correlation between BORIS levels in tumours with clinical/pathological parameters. BORIS was detected in all 18 inspected breast cell lines, but not in a primary normal breast cell culture. In 70.7% (41 of 58 cases) BORIS was observed in breast tumours. High levels of BORIS correlated with high levels of progesterone receptor (PR) and oestrogen receptor (ER). The link between BORIS and PR/ER was further confirmed by the ability of BORIS to activate the promoters of the PR and ER genes in the reporter assays. Detection of BORIS in a high proportion of breast cancer patients implies potential practical applications of BORIS as a molecular biomarker of breast cancer. This may be important for diagnosis of the condition and for the therapeutic use of BORIS. The ability of BORIS to activate promoters of the RP and ER genes points towards possible involvement of BORIS in the establishment, progression and maintenance of breast tumours.

Dual role of DNA methylation inside and outside of CTCF-binding regions in the transcriptional regulation of the telomerase hTERT gene.

Expression of hTERT is the major limiting factor for telomerase activity. We previously showed that methylation of the hTERT promoter is necessary for its transcription and that CTCF can repress hTERT transcription by binding to the first exon. In this study, we used electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) to show that CTCF does not bind the methylated first exon of hTERT. Treatment of telomerase-positive cells with 5-azadC led to a strong demethylation of hTERT 5'-regulatory region, reactivation of CTCF binding and downregulation of hTERT. Although complete hTERT promoter methylation was associated with full transcriptional repression, detailed mapping showed that, in telomerase-positive cells, not all the CpG sites were methylated, especially in the promoter region. Using a methylation cassette assay, selective demethylation of 110 bp within the core promoter significantly increased hTERT transcriptional activity. This study underlines the dual role of DNA methylation in hTERT transcriptional regulation. In our model, hTERT methylation prevents binding of the CTCF repressor, but partial hypomethylation of the core promoter is necessary for hTERT expression.

[Synthetic proteins with antiviral properties of alpha-interferons]. / Iskusstvennye belki, vosproizvodiashchie protivovirusnye svoistva alpha-interferonov.

Consensus sequence 30-36 LKDRHDF of human alpha-interferons is inserted into the C- and N-terminal sequences of the artificial protein albeferon using genetic engineering methods in order to obtain artificial proteins with antiviral activity. Albeferon, obtained by Dolgikh et al. (Protein Eng., 1996, vol. 9, pp. 195-201), already contains the IFN-alpha 2 130-137 fragment and possesses antiproliferative activity comparable with that of IFN-alpha 2. According to CD spectroscopy, both proteins have regular secondary structures similar to that of the precursor protein. They exhibit antiviral activities, and the activity of one of them is comparable with that of IFN-alpha 2. At the same time, their cytotoxic properties are displayed only at relatively high concentrations, which substantially exceed the minimal antiviral concentrations.

[Introduction of biologically active fragments of interferon-alpha2 and insulin into the artificial protein albebetin affects immunogenicity of the final construct]. / Vvedenie biologicheski aktivnykh fragmentov interferona-al'fa2 i insulina v sostav iskusstvennogo belka al'bebetina vliiaet na immunogennost' itogovoi konstruktsii.

The immunogenicity of the artificial protein albebetin and its derivatives with active peptide fragments was investigated. We also studied the influence of the peptides on the immunogenicity of the whole construct and contribution of each component to the immunogenicity. Two of three studied proteins contained active peptides from human IFN-alpha and insulin. Three continuous antigenic sites with different immunogenic potential were recognized in the chimerical proteins. The interferon fragment was the immunodominant site in the albeferon and albeferon-insulin molecules, while the insulin fragment displayed low immunogenic activity. All continuous B-epitopes are located at the boundaries of the secondary structure elements and at the predicted surface-located sites of albebetin molecule. Thus, peptide fragments attached to the artificial protein carrier can influence immunogenicity of the resulting construct.

Structure and stability of recombinant protein depend on the extra N-terminal methionine residue: S6 permutein from direct and fusion expression systems.

Two permuted variants of S6 ribosomal protein were obtained in direct and fusion expression systems, respectively. The product of direct expression contained the extra N-terminal methionine residue. The structural properties and conformational stability of these permuteins were compared using 1-D (1)H-NMR, circular dichroism, intrinsic fluorescence, differential scanning calorimetry and resistance to urea-induced unfolding. A pronounced difference in all the parameters studied has been demonstrated. This means that the structure of recombinant protein can be sensitive to peculiarities of the expression and purification procedures, leading particularly to the presence or absence of the Met at the first position in the target protein sequence.

[Effect of a biologically active interferon fragment on the structure of the synthetic protein carrier]. / Issledovanie vliianiia biologicheski aktivnogo fragmenta interferona na strukturu iskusstvennogo belka-nositelia.

A biologically active de novo protein albeferon was studied by physical techniques including CD spectroscopy in far and near ultraviolet regions and microcalorimetry. Albeferon was obtained by grafting an active octapeptide interferon fragment into a de novo protein albebetin used as a carrier. It was shown that attachment of the octapeptide into its molecule did not weaken albebetin but even slightly improved its structure and stability. The obtained results can be used for de novo protein design and improvement.

Can grafting of an octapeptide improve the structure of a de novo protein?

Structural properties and conformational stability of de novo proteins -- albebetin and albeferon (albebetin with a grafted interferon fragment) -- were studied by means of CD spectroscopy, gel filtration and urea-induced unfolding. The results allow us to conclude that albebetin possesses the properties of the molten globule state. Grafting of the octapeptide to the N-terminus of this de novo protein affects its structure. We show here that albeferon maintains a secondary structure content of albebetin; it becomes more compact and much more stable toward urea-induced unfolding as compared to albebetin and even possesses some weak tertiary structure (at least around Tyr7). This means that the structure of the artificial protein albebetin can be improved by a simple procedure of octapeptide grafting to its N-terminus.

[Expression of mutant horse cytochrome c genes in Escherichia coli]. / Ekspressiia mutantnykh genov tsitokhroma c loshadi v Escherichia coli.

Here we describe genetically engineered constructs for the expression in Escherichia coli of genes for horse cytochrome c mutants. These constructs allow the expression of the cytochrome c genes together with hemeligase, an enzyme which covalently links heme to cytochrome. Careful selection of producer strains and the adjustment of the conditions of expression provided for expression levels of 10-15 mg of protein per liter of culture. This is by an order of magnitude greater than the expression previously achieved in yeast. A series of horse cytochrome c mutants were obtained in this way.

S6 permutein shows that the unusual target topology is not responsible for the absence of rigid tertiary structure in de novo protein albebetin.

Ribosomal protein S6 from Thermus thermophilus was modified to form the unusual unique topology designed earlier for a de novo protein albebetin. The S6 gene was cloned, sequenced and circularly permutated by means of genetic engineering methods. The permutated gene was expressed in Escherichia coli and the permutein was isolated and investigated by means of circular dichroism, fluorescence spectroscopy and scanning microcalorimetry. The permutated protein revealed a pronounced secondary structure close to that of the wild type S6 protein and a rigid tertiary structure possessing cooperative temperature melting. It means that the unusual new topology of albebetin is compatible with a rigid tertiary structure, it may be realized in natural proteins and it is not responsible for the absence of rigid structure in albebetin.