[Stimulation of DNA molecules association with amphiphilic derivatives of 1,3-diazaadamantane containing hydrophobic side chanins].

Earlier, a new class of compounds--amphiphilic derivatives of 1,3-diazaadamantanes, capable of facilitating the strand exchange in the system of short oligonucleotides was revealed. Longer hydrophobic side chains of 1,3-diazaadamantanes promoted stronger acceleration of the reaction. In this study, interaction with DNA of two 1,3-diazaadamantane derivatives containing different side chains was investigated by use of optical methods. Concentration of the investigated 1,3-diazaadamantans micelles formation were determined by the means of monitoring fluorescence intensity enhancement of 1-anilinonaphtalene-8-sulphonate probe; as well as the ranges of concentrations where the compounds/water mixtures existed as true solutions. 1,3-diazaadamantanes affinity to DNA was determined with Fluorescent Intercalator Displacement (FID) approach. Significant increase in hydrodynamic volume of short DNA hairpins in the complexes with 1,3-diazaadamantanes was revealed by estimation of the fluorescence polarization of ethidium bromide probe bound to the hairpins. Intermolecular association of DNA hairpins upon binding with 1,3-diazaadamantans was confirmed by Förster resonance energy transfer in system of an equimolar mixture of fluorescently labeled with Cy-3 and Cy-5 hairpins. In this study, the number of positive charges at 1,3-diazaadamantane derivatives containing side chains of different lengths was demonstrated to affect their affinity to DNA, whereas longer length of the hydrophobic side chains ensured more efficient interaction between the DNA duplexes that may facilitate, in particular, DNA strand exchange.

Comparative genomics of the eukaryotes.

A comparative analysis of the genomes of Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae-and the proteins they are predicted to encode-was undertaken in the context of cellular, developmental, and evolutionary processes. The nonredundant protein sets of flies and worms are similar in size and are only twice that of yeast, but different gene families are expanded in each genome, and the multidomain proteins and signaling pathways of the fly and worm are far more complex than those of yeast. The fly has orthologs to 177 of the 289 human disease genes examined and provides the foundation for rapid analysis of some of the basic processes involved in human disease.

Correlation of intrinsic DNA curvature with DNA property periodicity.

Twelve di- and trinucleotide parameter sets representing various structural, thermodynamic or bendability-related properties of DNA were tested in the prediction of DNA curvature applying Fourier analysis on curved and straight, A/T-type or G/C-type DNA sequence motifs. The best predictions were obtained with a new consensus bendability scale created by combining a nucleosome-based and a deoxyribonuclease I-based parameter set. Geometry calculations on the same sequences showed that the helical parameters derived from NMR structures can correctly predict curvature, as distinct from the parameters derived from X-ray crystallographic analysis.

Distribution of sequence-dependent curvature in genomic DNA sequences.

The distribution of inherent, sequence-dependent curvature was calculated for a number of prokaryotic (M. genitalium, H. influenzae, M. jannaschii), viral (adenovirus 2, equine herpes virus 1), phage (M13, lambda), eukaryotic (S. cerevisiae) and mitochondrial genomes as well as E. coli and human genomic fragments. The genomic averages are in the range of 6-8 degrees/helical turn and only about 20% of DNA is curved less than 3 degrees/helical turn. The prokaryotes and phages appear to have a consistently higher frequency of curved DNA in their genomes than the other genomes tested. Long, highly curved segments, similar to artificially designed curved DNA, are apparently absent from the genomes. Short, curved segments, differing in G+C content may provide environmentally modulated conformational signals for gene regulation. A WWW-server was constructed for the prediction of curved sites from DNA sequences (http://icgeb.trieste.it/dna/curve_it.html/)..

Distribution of bending propensity in DNA sequences.

Local bending propensity and curvature of DNA can be characterized using a vector description of DNA bendability, based on a set of parameters derived from deoxyribonuclease I (DNase I) cleavage experiments. Two characteristics-arithmetic and vector averages of bendability-were successfully used to predict experimentally known bendable, rigid and curved segments in DNA. A characteristic distribution of bendability is conserved in evolutionarily related kinetoplast sequences. An analysis of the M. genitalium and H. influenzae genomes as well as fragments of human and yeast genomes shows, on the other hand, that highly curved segments--similar to artificially designed curved oligonucleotides--are extremely rare in natural DNA.

Residues responsible for distinct biological functions are characterized by different statistical features of sequential and spatial neighborhoods: a thermolysin example.

An investigation of the functional topography of thermolysin was carried out using frequency analysis of its primary and tertiary structures. The statistical validity of this approach was estimated for the enzyme active site, the substrate-binding pocket, the inter-domain interface and calcium-binding sites' predictions. We showed that frequency analysis of primary structure could be employed to predict the localization of contiguous parts of the inter-domain interface. The same approach appears to be unsuitable to a search for conformation-dependent enzyme active sites and substrate-binding pockets. In contrast, frequency analysis of the spatial neighborhood is not effective for predicting the inter-domain interface as distinct from the active site, substrate-binding pocket and calcium-binding sites. These differences should be taken into account when investigating and understanding protein structure-function relationships.

Synthetic peptides in the determination of hepatitis A virus T-cell epitopes.

Computer search for probable T-epitopes of hepatitis A virus capsid proteins was performed using an integrated set of programs. Eight segments of the VP1, VP2, VP3 and VP4 proteins were chosen and synthesised. Five peptides previously examined as probable B-epitopes were used as well. All the peptides were tested for their ability to stimulate proliferation of lymph node T-cells primed with synthetic peptides. Almost all predicted T-epitopes affected the T-cell proliferation. None of the peptides had mitogenic activity. We demonstrated that regions 17-33 and 276-298 of VP1 are possible immunodominant promiscuous sites activating lymphocytes of all mouse haplotypes.

Identification of the transcription initiation site of the asexually expressed rRNA genes of the malaria parasite Plasmodium berghei.

The start site of the A-type ribosomal RNA transcription units of the rodent malaria parasite, Plasmodium berghei, has been identified. The two A-type units cannot be distinguished within the transcription unit, yet exist as single copies on different chromosomes. Gene transcription initiates 820 bp upstream of the A-type small subunit (SSU) ribosomal gene and two major processing sites were mapped 610 and 611 nucleotides upstream of the SSU in the external transcribed spacer region. Surprisingly the nucleotide sequence of the DNA region containing the putative ribosomal promoter lacked repetitive DNA sequences typical of ribosomal promoters. This region was further analysed by computer using programs designed to reveal sequence-dependent structural features. Comparison of DNA curvature, duplex stability and pattern of twist angle variation revealed a striking degree of conservation between the ribosomal promoters from Plasmodium and other eukaryotes.

Protein engineering of de novo protein with predesigned structure and activity.

The de novo protein albebetin has been engineered (J. Mol. Biol. 1992, 225, 927-931) to form a predesigned tertiary fold that has not yet been observed in natural proteins. Analysis of albebetin expressed in a cell-free system and in Escherichia coli revealed its compactness, relative stability, and the secondary structure close to the predesigned one. The blast-transforming biological activity of human interferon was grafted to albebetin by attachment of an eight amino acid interferon fragment to the N-terminus of albebetin next to its first methionine residue. The chimeric protein was expressed in a wheat germ cell-free translation system and tested for its structural properties, receptor binding, and biological activity. According to the tests, albebetin incorporating the active interferon fragment has a compact and relatively stable structure, and binds the murine thymocyte receptor effectively. It activates the blast transformation reaction of thymocyte cells even more efficiently than human interferon at low concentrations.

[Use of frequency analysis for localization of functionally important regions of thermolysin]. / Ispol'zovanie chastotnogo analiza dlia lokalizatsii funktsional'no znachimykh oblastei termolizina.

A theoretical investigation of the functional topography of thermolysin molecule was carried out using frequency analysis of its primary and tertiary structures. The statistical validity of predictions was estimated for the enzyme active site, substrate-binding pocket, interdomain interface, and calcium-binding sites. It was shown that frequency analysis of primary structure could be employed to predict the localization of contiguous parts of the interdomain interface. Primary structure analysis cannot be used to search for the conformation-dependent enzyme active site and substrate-binding pocket. On the contrary, frequency analysis of interresidues contacts is not so effective for prediction of the interdomain interface as compared with active site, substrate-binding pocket, and calcium-binding sites. The set of original algorithms proposed could be used in searching for functional sites in various proteins.

[Theoretical analysis of the amino acid sequence of receptors for growth hormones and prolactins. Prediction of ligand-binding segments]. / Teoreticheskii analiz aminokislotnykh posledovatel'nostei retseptorovgormonov rosta i prolaktinov. Predskazanie ligand-sviazyvaiushchikh uchastkov.

At present the growth hormone and prolactin receptors were cloned along with their variant forms from human, rat, mouse, rabbit, bovine and sheep tissues. The functional topography of receptors is practically unknown. Because of the high price and difficulty of protein's total mutagenesis, it is reasonable to carry on a theoretical analysis of structures of receptors to predict the most probable ligand-binding sites. We studied the primary structures of known prolactin and growth hormone receptors using theoretical methods proved to be powerful in earlier structure--activity relationship investigations. We analyzed the secondary structure, conservative positions, hydrophilicity profiles of the growth hormone and prolactin receptors, and used the original method based on information theory to predict the sites which are promising for mutagenesis or peptide synthesis as probable ligand-binding sites. Three segments corresponding to the main conservative, hydrophilic and rare sites were predicted to form the ligand-binding determinant.

[A method of searching for amphipathic structures in protein sequences]. / Metod poiska amfipatichnykh struktur v belkovykh posledovatel'nostiakh

A simple method for searching amphipathic helices based on estimation of correlation between hydrophobicity distribution and periodic function is proposed. The method was examined in a series of proteins with known T-cell epitopes, which are mostly amphipathic helices. The predictive power of the method is discussed.

[Search for T-epitopes of the hepatitis A virus using synthetic peptides]. / Poisk T-épitopov virusa gepatita A s pomoshch'iu sinteticheskikh peptidov.

Computer search for probable T-epitopes of the hepatitis A virus capsid proteins was performed using developed integrated set of programmes. The following peptides were chosen and synthesised by solid phase technique: 75-92 VP1, 115-139 VP1, 209-221 VP1, 69-99 VP2, 80-99 VP2, 45-57 VP3, 137-150 VP3 and 1-23 VP4. Peptides 1-17 VP1, 10-33 VP1, 11-25 VP1, 75-85 VP1 and 276-298 VP1 previously examined as probable B-epitopes were used as well. All the peptides were tested for their ability to stimulate proliferation of lymph node T-cells primed with synthetic peptides. Almost all the predicted T-epitopes did affected the T-cell proliferation. 10-33 VP1 and 276-298 VP1 stimulated lymph node proliferation of all tested mouse strains. 107-126 VP1 and 115-126 VP1 did not influence proliferation of lymphocytes of mice primed with these peptides but stimulated proliferation of T-cells of F1 (CBA x C57Bl6) mice primed with 115-139 VP1.

[Modeling antigenic determinants of the hepatitis A virus using synthetic peptides]. / Modelirovanie antigennykh determinant virusa gepatita A s pomoshch'iu sinteticheskikh peptidov.

Monoclonal and polyclonal anti-hepatitis A (HAV) antibodies were used to search for peptides mimicking the antigenic determinants of HAV. Synthetic peptides VP1 115-139, VP1 117-139, VP1 126-139, VP2 69-99, VP2 80-99, VP3 45-57, VP3 137-150, were shown to bind the anti-HAV antibodies in ELISA. Peptides VP1 115-139, VP1 117-139, VP2 69-99 were utilized to produce the antipeptide antibodies. Mice were immunized with the free peptides or with their conjugates with ovalbumin. Only the free VP2 69-99 caused formation of HAV binding antibodies.

[Conformational transitions of DNA in concentrated neutral salt solutions]. / Konformatsionnye perekhody DNK v kontsentrirovannykh rastvorakh neitral'nykh solei.

Salt anions can be arranged in lyotropic series of the action of conformational stability of DNA. This effect is universal for proteins and DNA. It is explained by the salt changes of transfer free energy of macromolecular inner groups to the solvent. Effect mechanism is the combination of anion direct interaction with the exposed inner groups and in indirect way through the changes of water structure. Individual features of the salt effect on DNA are the induced transitions in DNA duplex and the changes of differential stability of AT- and GC-base pairs. An important role in these phenomena plays the dehydration of DNA in the reduction of water activity in concentrated salt solutions. Local changes in medium polarity, hydration level of DNA molecule at different stages of their functioning can explain the regulatory role in intracellular processes, as well as selectivity and specificity of the action of individual ions in the cell.

[Solubility of adenosine in concentrated salt solutions]. / Rastvorimost' adenozina v kontsentrirovannykh solevykh rastvorakh.

The solubility of adenosine has been measured in concentrated neutral salt solutions of NaClO4 (salt-destabilizer) and Na2SO4 (salt-stabilizer) as a function of salt concentration and temperature. The thermodynamic functions of adenosine transfer from water to salt solutions have been estimated. The values of delta Ftr, delta Htr, delta Str adenosine transfer in NaClO4 solutions are negative, in Na2SO4--positive. The results are considered in connection with the mechanism of high concentrations of salt action on DNA in the water solutions.

[Artificial proteins with a given spatial structure and biological activity]. / Iskusstvennyi belok s zadannoi prostranstvennoi strukturoi i biologicheskoi aktivnost'iu.

Biologically active fragment 131-138 of human interferon alpha 2 carrying blast-transforming activity of the protein was attached to the N-terminus of the de novo protein albebetin with predetermined tertiary structure by means of genetic engineering. The chimeric protein was expressed in a wheat germ cell-free translation system and tested for compactness, stability and biological activity. According to the tests used albebetin with interferon fragment has a compact and relatively stable structure. It binds murine thymocyte receptor with high affinity and activates efficiently thymocyte blast transformation at a concentration of 10(-11) M.

[The synthetic peptide 62-75 VP3 of the hepatitis A virus induces virus-binding antibodies]. / Sinteticheskii peptid 62-75 VP3 virusa gepatitia A indutsiruet virussviazyvaiushchie antitela.

Peptide (P6) representing amino acids 62-75 of HAV capsid protein VP3 was synthesized. Pure P6, octamer form of P6, and P6 conjugated to KLH were injected into rabbits. The sera against these preparations reacted with denaturized VP3 in immunoblotting tests; however, only anti-P6-KLH serum was capable of binding the native HAV.

[The effect of the tempi of rehabilitation on the psychological status and quality of life in myocardial infarct patients]. / Vliianie tempov reabilitatsii na psikhologicheskii status i kachestvo zhizni bol'nykh infarktom miokarda.

104 patients 3 to 12 months after myocardial infarction were evaluated psychologically and by quality of life. Faster hospital rehabilitation contributed to better quality of life than in patients on conventional rehabilitation. The absence of sanatorium rehabilitation was associated with anxiety, hypochondria, depression registered 12 months after MI onset and thus with lowering of life quality.

[A means for the hemodynamic correction of coronary insufficiency in the basin of the right coronary artery]. / Sposob gemodinamicheskoi korrektsii koronarnoi nedostatochnosti v basseine pravoi venechnoi arterii.

To synchronize the bloodstream along the shunt and the bloodflow along coronary channel hemodynamically protected aortocoronary shunt was elaborated and applied in 1 patient. The monocusp was formed, dipped in the aorta lumen by means of V-figurative cut in the ascending aorta wall. An aperture in the aorta wall, disposed behind the monocusp in direction of bloodstream, was closed with the autovena patch, in centre of which the section was made and the autovenous transplant was sewn along its edge.