[Health schools for patients with chronic cardiac insufficiency (innovative technologies)].

Modern programs for the management of patients with chronic cardiac insufficiency (CCI) envisage systemic preventive measures aimed to extend their information support, stimulate compliance, optimize feedback, modify lifestyle, and facilitate social adaptation. Traditional health schools for CCI patients as a form of secondary prophylaxis helps to achieve the above goals even though efficiency of this work needs to be further improved. Results of regular medical examination of 417 patients with CCI (294 women and 123 men) visiting local outpatient facilities in 2008 were analysed. 207 of them attended health schools, where they were educated and treated with the use of innovative technologies. It was shown that frequency of hospitalization for decompensated CCI was not significantly different in these and control patients. However, the educated patients needed less emergency ambulance care, made fewer unplanned visits to their doctors, and had better results of clinical and functional studies. The efficiency of education was gender related. Men showed significantly lower frequency of hospitalization, improved health scores and results of 6-min walking test.

The effect of mutations in EF-Tu on its affinity for tRNA as measured by two novel and independent methods of general applicability.

Elongation factor Tu is essential for binding and a correct delivery of aminoacyl-tRNA during protein biosynthesis. For a good characterization of its interaction with tRNA in terms of structure-function relationship, determinations of kinetic equilibrium parameters are of great value. We describe two novel methods for that purpose. One method is based on EF-Tu protection of the tRNA 3' acceptor end against RNase A cleavage and yields the Kd value together with the corresponding dissociation and association rate constants from one single set of experiments. The other is a rapid method for screening relative affinities of mutant EF-Tus for tRNA. It is based on competition between EF-Tu species with and without a (His)6 extension for the same aminoacyl-tRNA and yields a relative Kd value. The method can be of general importance for the measuring of ligand affinities of all sorts of His-tagged proteins. Both methods are illustrated by their application in the analysis of mutant EF-Tus with changed interactions with tRNA and antibiotics. Raising the assay temperature from 4 to 37 degrees C causes a 30-fold increase of Kd for EF-Tu x GTP x Phe-tRNA complexes. The mutation K237E leads to rapid inactivation at the latter temperature. A parallel is found between the order of increasing Kd values for EF-Tus with mutation G316D, A375T and Q124K, respectively, and their order of increasing resistance to kirromycin.

Ribosomal decoding processes at codons in the A or P sites depend differently on 2'-OH groups.

The importance of 2'-OH groups of codons for binding of cognate tRNAs to ribosomal P and A sites was analyzed applying the following strategy. An mRNA of 41 nucleotides was synthesized with the structure C16-GAA-UUC-GUC-C16 coding for glutamic acid (E), phenylalanine (F) and valine (V), respectively, in the middle (EFV-mRNA). A second template, the E(dF)V-mRNA, was identical except that it carried a deoxyribo-codon-dUdUdC- for phenylalanine. tRNA binding to the P site is totally insensitive to the presence or absence of the 2'-OH group of the P-site codon, and tRNA binding to the P site is also not affected if the A-site codon lacks the 2'-OH groups. However, binding is impaired if the deoxy-codon is present at the E site. In sharp contrast, the A-site binding of Ac-aminoacyl-tRNA was severely reduced in the presence of the deoxy-codon at the A site as well as at the P site. The results demonstrate that the correctness of base pairing is also "sensed" via a correct sugar structure of the codon, e.g. positioning of the sugar pucker (2'-OH), during the decoding process at the A site (elongation) but not during the decoding at the P site (initiation).

[Analysis of stationary kinetics of translation elongation within the framework stereospecific stabilization hypothesis of codon- anticodon complexes in a ribosome. I. Kinetic schemes of factorless elongation]. / Analiz statsionarnoi kinetiki élongatsii transliatsii v ramkakh gipotezy o stereospetsificheskoi stabilizatsii kodon-antikodonovykh kompleksov na ribosomes. I. Kineticheskie skhemy besfaktornoi élongatsii.

Dependences of steady-state rates of polypeptide elongation on concentrations of substrate (aminoacyl-tRNA) and product (deacylated tRNA) in the absence of elongation factors and GTP are theoretically analyzed in context of stereospecific stabilization of the codon-anticodon complexes at a ribosome. General kinetic scheme and different ribosome isomerization stages are examined. The effect of isomerization stage allows to identify reaction stage experimentally. Regulation of the direct reaction by product and regulation of the reverse reaction by substrate are possible. Under certain conditions elongation system may show kinetic cooperativity.

[Analysis of stationary kinetics of translation elongation within the framework of the stereospecific stabilization hypothesis of codon- anticodon complexes in a ribosome. II. Kinetic schemes in the presence of protein elongation factors and GTP]. / Analiz statsionarnoi kinetiki élongatsii transliatsii v ramkakh gipotezy o stereospetsificheskoi stabilizatsii kodon-antikodonovykh kompleksov na ribosome. II. Kineticheskie skhemy v prisutstvii belkovykh faktorov élongatsii i GTP.

Kinetics of the factor-dependent polypeptide elongation is theoretically studied in context of stereospecific stabilization of the codon-anticodon complexes at a ribosome. Kinetic schemes for the different ribosome isomerization stages are examined. The dependence of steady-state elongation rate on elongation factor concentration for each of the schemes is unique, allowing to identify isomerization stages experimentally.

Synergism between the GTPase activities of EF-Tu.GTP and EF-G.GTP on empty ribosomes. Elongation factors as stimulators of the ribosomal oscillation between two conformations.

A remarkable positive cooperativity between the GTPase activities of EF-Tu and EF-G on empty ribosomes from Escherichia coli has been discovered. This cooperativity implies a decrease of the corresponding apparent KM values of the empty ribosome for either elongation factor: from more than 10 microM to 0.5 microM for EF-Tu.GTP by the addition of 0.25 microM EF-G and from 0.7 microM to 0.5 microM for EF-G.GTP by the addition of 3 microM EF-Tu. In a further analysis of this phenomenon, the effects of various specific antibiotics were studied: thiostrepton, fusidic acid, tetracycline, pulvomycin and kirromycin appeared to inhibit the synergistic effect, whereas streptomycin was found to stimulate it. Even in the present minimal system the ribosomes respond to the above-mentioned antibiotics in a way surprisingly similar to that in the coupled system with mRNA and tRNAs. The cooperativity seems not to be due to a simultaneous binding of the two elongation factors to the ribosome as revealed by studying the effects of fusidic acid and kirromycin, and by band-shift experiments by means of gel electrophoresis under non-denaturing conditions. Our experimental data and the kinetic analysis of alternative models provide evidence that EF-Tu.GTP and EF-G.GTP interact sequentially with empty ribosomes that oscillate between two different conformations, one for each elongation factor. Apparently, ribosomes have an intrinsic property for oscillation as normally observed during protein synthesis with a frequency paced by the events of tRNA binding and translocation.

Effect of E. coli ribosomal protein S1 on the fidelity of the translational elongation step: reading and misreading of poly(U) and poly(dT).

Ribosomal protein S1 was selectively removed from E. coli ribosomes by affinity chromatography and the effect of added S1 on the translation of poly(dT) [which is read as poly(U) in the presence of neomycin] and on the misreading of poly(U) and poly(dT) were examined. S1 enhances the translation of poly(dT) at low template concentration, which is similar to the effect of S1 on poly(U) translation. The misreading of poly(dT) by E. coli ribosomes is at a lower level than is the case with poly(U). This low misreading is the same for "S1-dependent" and "S1-independent" modes of translation. On the other hand, the misreading of poly(U) is significantly reduced when S1 is present. These results thus indicate that S1 not only facilitates the binding of mRNA to the ribosome as already known, but also plays a role in the correct codon-dependent selection of aminoacyl-tRNA.

Cell-free translation systems from different eukaryotes differ in their sensitivity to a template sugar-phosphate backbone.

To study the role of a messenger sugar-phosphate backbone in the ribosomal decoding process, poly(U) and poly(dT) template activity in different eukaryotic systems has been compared. 80S ribosomes from Saccharomyces cerevisiae appeared to be able to translate poly(dT) both in the presence and in the absence of elongation factors, contrary to poly(U). However, ribosomes from higher eukaryotes (wheat germ, rabbit liver) are completely inefficient in poly(dT) translation. Moreover, rabbit liver ribosomes fail to bind effectively phenylalanyl-tRNA in the presence of poly(dT) although the polynucleotide seems to interact with the ribosomal decoding center. It is also of particular interest that hybrid ribosomes formed from the yeast and rabbit liver subunits can translate poly(dT) only when the large ribosomal subunit from yeast is used.

Interaction of yeast tRNA(Phe) anticodon arm with 40S ribosomal subunit of rabbit liver.

The 15-nucleotide analog of yeast tRNA(Phe) anticodon arm binds cooperatively to two sites of poly(U) programmed 40S ribosome like intact tRNA(Phe). The cooperativity coefficients appeared to be about 4 for tRNA(Phe) and 50 for its anticodon arm. Anticodon arm contributes the majority of free energy of tRNA binding to a programmed 40S ribosomal subunit. The correct codon-anticodon pairing seems to play the key role in the cooperativity origin. Contrary to the anticodon arm template independent binding of the whole tRNA to the small ribosomal subunit is revealed.

A codon sugar structure strongly influences tRNA binding to programmed ribosomes.

To study the role of a codon sugar-phosphate backbone in aminoacyl-tRNA selection on the ribosome a comparison of tRNA(Phe) affinity for pdTpdTpdT and prUprUprU in solution, and for correspondingly programmed 30S ribosomal subunits has been performed. In solution the tRNA(Phe) affinity for pdTpdTpdT appeared to be even slightly higher than for prUprUprU, whereas deoxyribocodon was significantly less efficient in the stimulation of Phe-tRNA(Phe) binding to the 30S ribosomal subunit. Some difference in neomycin action in both systems was revealed.

Interaction of ribo- and deoxyriboanalogs of yeast tRNA(Phe) anticodon arm with programmed small ribosomal subunits of Escherichia coli and rabbit liver.

A synthetic ribooligonucleotide, r(CCAGACUGm-AAGAUCUGG), corresponding to the unmodified yeast tRNA(Phe) anticodon arm is shown to bind to poly(U) programmed small ribosomal subunits of both E. coli and rabbit liver with affinity two order less than that of a natural anticodon arm. Its deoxyriboanalogs d(CCAGACTGAAGATCTGG) and d(CCAGA)r(CUGm-AAGA)d(TCTGG), are used to study the influence of sugar-phosphate modification on the interaction of tRNA with programmed small ribosomal subunits. The deoxyribooligonucleotide is shown to adopt a hairpin structure. Nevertheless, as well as oligonucleotide with deoxyriboses in stem region, it is not able to bind to 30S or 40S ribosomal subunits in the presence of ribo-(poly(U] or deoxyribo-(poly (dT) template. The deoxyribooligonucleotide also has no inhibitory effect on tRNA(Phe) binding to 30S ribosomes at 10-fold excess over tRNA. Neomycin does not influence binding of tRNA anticodon arm analogs used. Complete tRNA molecule and natural modifications of anticodon arm are considered to stabilize the arm structure needed for its interaction with a programmed ribosome.

[Status of natural immunity and the ways of its enhancement in patients with goiter]. / Sostoianie estestvennoi rezistentnosti i puti ee povysheniia u bol'nykh zobom.

Disease caused by goiter is marked by changes in cellular and humoral unspecific resistance before and after operation, which at times is of decisive importance in the course of the postoperative period. Natural resistance is inhibited, to a greater measure, in patients with a long history of the disease and in those with diffuse toxic goiter. The work deals with the study of natural resistance in 98 patients with goiter before and after the operation in dynamics. The immunostimulator lysozyme was used for a more favourable course of the postoperative period. This promoted increase of the organism's natural resistance. which had a favourable effect on the course of the postoperative period.

[Factor-free translation of poly(dT) by 80S ribosomes from Saccharomyces cerevisiae]. / Besfaktornaia transliatsiia poli(dT) 80S ribosomami iz drozhzhei Saccharomyces cerevisiae.

The conditions for preparation of 80S ribosomes from S. cerevisiae are suggested. The ribosomes can bind Phe-tRNAPhe in poly(U)-or poly(dT)-directed manner and are shown to be able to translate poly(dT) in the absence of elongation factor and GTP. Effects of different antibiotics on the factor-free translation have been studied.

Correlation between poly(U) misreading and poly(dT) translation efficiency in E coli cell-free systems.

A positive correlation between poly(U) misreading and efficiency of poly(dT) translation has been revealed in cell-free systems from wild-type E coli and streptomycin--resistant mutants with altered ribosomal protein S12. Different factors promoting misreading of poly(U) such as aminoglycoside antibiotics and Mg2+ ions also stimulate poly(dT) translation. The effect of the antibiotics on poly(U) translation efficiency and misreading as well as on poly(dT) decoding is characterised by the same order: neomycin greater than kanamycin greater than streptomycin. S12 mutants ribosomes are less erroneous in poly(U) translation and less efficient in poly(dT) decoding. The data obtained are in good agreement with the hypothesis of stereospecific stabilization of codon-anticodon complexes by the ribosome decoding centre.

The role of a template sugar-phosphate backbone in the ribosomal decoding mechanism. Comparative study of poly(U) and poly(dT) template activity.

To study the role of a template sugar-phosphate backbone in the ribosomal decoding process, poly(U), poly(dT) and poly(dU)-directed cell-free amino acid incorporation was investigated under the influence of neomycin and high concentrations of Mg2+. The specificity of a factor-dependent translation system of Escherichia coli was shown to change according to the principle: "either ribo- or deoxyribopolynucleotide messenger". Poly(dT) is shown to be effectively translated in the absence of elongation factors, both at low (2 degrees C) and high (37 degrees C) temperature. Neomycin inhibits factor-free poly(dT) translation. Little or no poly(U) translation is observed in this system. A chromatographic analysis of the oligophenylalanine residues synthesized seems to show that translocation is the main step responsible for ribosome specificity to the ribo- or deoxyribopolynucleotide template in both factor-dependent and factor-free translation systems.

[The structure of ribosome decoding center]. / O strukture dekodiruiushchego tsentra ribosomy.

To develop the hypothesis of direct interaction between the ribosome and the codon-anticodon sugar-phosphate backbones the model of the ribosome decoding centre structure is proposed. The model is based on the structural complementarity between the protein beta-sheet and the narrow groove of double-stranded RNA. A right handed two-stranded antiparallel beta-sheet is the main structural element of the model. One peptide strand interacts with the codon sugar-phosphate backbone while the second interacts with the anti-codon sugar-phosphate backbone. These stereoregular structures interact between them forming a regular system of H-bonds. The model postulates a specific rule of repetition for some amino acids in both peptide strands. Analysis of known sequences of the E. coli ribosomal proteins indicates such boxes of repeated arginine in proteins S1, S10, S11, S14 and boxes of repeated glycine in proteins S5, S11, (L15. These proteins are discussed as candidates for forming the corresponding "arginine" or "glycine" beta-sheets of the decoding centre and interaction with the codon-anticodon pairs. The properties of such interactions are analysed as well.

[Code correspondence of tRNK Leu 1-6 in the mammary gland of cows]. / Izuchenie kodovogo sootvetstviia tRNK Leu 1-6 molochnoi zhelezy korov.

Preparations of ribosome subunits from the rabbit liver are completely free from the endogenic mRNA admixture and high-active in a system of codon-dependent binding of tRNA. Leucine codons of uridyl-uridyl-adenine (UUA) and uridyl-uridyl-guanine (UUG) are synthesized by the triester method. The correspondence of six leucine isoacceptor tRNA from mammary gland of cows to cytidyl-uridyl-uridine (CUU), UUA and UUG codons is studied. The code response of tRNA is compared with their ability to erroneously translate polyuridylic acid.