[Cardiovascular resistance to orthostatic stress in athletes after aerobic exercise].

In the paper cardiovascular resistance to orthostatic stress in the athletes in the two-hour recovery period after prolonged aerobic exercise was investigated. The reaction of the cardiac (stroke volume and cardiac output) and peripheral blood volumes in the lower and upper limbs, abdominal and neck regions in response to the tilt-test before and during two hours after exercise (30 min, heart rate = 156 +/- 8 beats/min) was determined by impedance method: It is found that: (1) at baseline distribution of blood flow in favor of the neck-region in response to the tilt-test, in spite of the decrease in cardiac output, was more efficient in athletes, that was due to a large decrease in blood flow to the lower extremities, and increased blood flow in the neck region; (2) after exercise it was established symptoms of potential orthostatic intolerance: postural hypotension and tachycardia, reduced peripheral pulse blood volume, expressed in a standing position, and reduced effectiveness of the distribution of blood flow in the direction of the neck region; (3) the abilityto effectively distribute blood flow in favor of the neck region in athletes after exercise remained elevated, which was due to a large decrease in blood flow in the abdominal region at the beginning, and in the lower limbs at the end of the recovery period.

[Computer modelling and topographo-anatomic substantiation of TMJ ankylosis surgical treatment].

Results of topographo-anatomic research of lateral and deep area of face with the use of three-dimensional computer modelling was presented. Application of the received data at operations of patients with ankilosis of the temporomandibular joint gave good results. It allows to draw a conclusion of possibility of this technique in a wide clinical practice.

Ca2+/Calmodulin reverses phosphatidylinositol 3,4, 5-trisphosphate-dependent inhibition of regulators of G protein-signaling GTPase-activating protein activity.

Regulators of G protein signaling (RGS proteins) are GTPase-activating proteins (GAPs) for G(i) and/or G(q) class G protein alpha subunits. RGS GAP activity is inhibited by phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) but not by other lipid phosphoinositides or diacylglycerol. Both the negatively charged head group and long chain fatty acids (C16) are required for binding and inhibition of GAP activity. Amino acid substitutions in helix 5 within the RGS domain of RGS4 reduce binding affinity and inhibition by PIP(3) but do not affect inhibition of GAP activity by palmitoylation. Conversely, the GAP activity of a palmitoylation-resistant mutant RGS4 is inhibited by PIP(3). Calmodulin binds all RGS proteins we tested in a Ca(2+)-dependent manner but does not directly affect GAP activity. Indeed, Ca(2+)/calmodulin binds a complex of RGS4 and a transition state analog of Galpha(i1)-GDP-AlF(4)(-). Ca(2+)/calmodulin reverses PIP(3)-mediated but not palmitoylation-mediated inhibition of GAP activity. Ca(2+)/calmodulin competition with PIP(3) may provide an intracellular mechanism for feedback regulation of Ca(2+) signaling evoked by G protein-coupled agonists.

[Thymosin alpha-1 and hybrid proteins consisting of tumor necrosis factor-alpha and thymosin alpha-1 enhance the efficacy of vaccination against the causative agent of plague]. / Timozin al'fa-1 i gibridnye belki, sostoiashchie iz faktora nekroza opukholei al'fa i timozina al'fa-1, uvelichivaiut éffektivnost' vaktsinatsii protiv vozbuditelia chumy.

The influence of the preparation of chemical thymosin alpha 1 (T), recombinant thymosin alpha 1 (rT), tumor necrosis factor (TNF) and hybrid proteins on their basis (T-TNF, TNF-T and T-TNF-T) on the effectiveness of immunization against Y.pestis have been studied. The preparations of T and hybrid proteins exhibit immunostimulating action, enhancing specific immunity when injected at different periods of the vaccinal process against Y.pestis virulent strain 231 in experiments on mice and guinea pigs. The highest effectiveness and reproducibility of results is observed after the use of hybrid protein T-TNF-T. An increase in immunity after the use of the preparations of hybrid proteins is accompanied by the activation of its T-cell element. The influence of rT on the restoration of the immune system of white mice after their exposure to sublethal doses of gamma radiation has been shown.

[Synthesis of an artificial gene coding for thymosin alpha1 and its expression in Escherichia coli as a hybrid with human tumor necrosis factor]. / Sintez iskusstvennogo gena, kodiruiushchego timozin alpha1, i ego ékspressiia v Escherichia coli v sostave gibridov s faktorom nekroza opukholei cheloveka.

Chemical-enzymatic synthesis and cloning in Escherichia coli of an artificial gene encoding the immunoactive peptide thymosin alpha 1 have been carried out. Recombinant plasmids were constructed which contain fusion genes coding for hybrids of human tumour necrosis factor (TNF) and thymosin alpha 1 as N- or C-terminal part of the hybrid protein. In the C-terminal hybrid protein, TNF and thymosin alpha 1 are linked through a methionine residue, thus allowing for thymosin alpha 1 to be cleaved off the rest of the hybrid protein with cyanogen bromide. In case of the N-terminal hybrid protein, the linker between the thymosin alpha 1 and TNF sequences is the acid-labile dipeptide Asp-Pro. Expression of the hybrid genes in E. coli and properties of the recombinant proteins were studied. The N-terminal hybrid protein was secreted into periplasmic space, in contrast with the C-terminal hybrid protein, which formed insoluble aggregates inside bacterial cells. Procedures for the isolation of both hybrid proteins were developed. The N-terminal hybrid protein displayed full biological activity in the cytotoxic assay on the mouse fibroblast L-929 whereas the C-terminal hybrid protein proved to be much less active. Treatment of the hybrid protein TNF-thymosin alpha 1 with cyanogen bromide lead to a mixture of two polypeptides, from which thymosin alpha 1 was purified to homogeneity by simple chromatographic procedures.

[Synthesis, secretion, and proteolytic degradation of diphtheria toxin in Escherichia coli]. / Sintez, sekretsiia i proteoliticheskaia degradatsiia difteriinogo toksina v Escherichia coli.

The recombinant plasmids have been constructed encoding the synthesis of a full-sized diphtheria toxin from its own or PR, PL-promoters of bacteriophage lambda in Escherichia coli cells. The high level constitutive synthesis of toxin results in slow cell growth and plasmid elimination. The toxin was mainly detected in the periplasm, partially in the membrane and to a less extent in the cytoplasm and culturing medium. The dimeric form of toxin was found in the cytoplasm. Participation of toxin B-subunit in secreting of the toxin into culturing medium is discussed. Proteolytic degradation of the synthesized toxin in different Escherichia coli strains was demonstrated. The process takes place in cytoplasm and periplasm mainly. The main enzyme participating in the process is a La-protease. The data on proteolysis obtained by immunoprecipitation immunoblotting, affinity chromatography and in mini-cells of Escherichia coli are summarized.

[The heterogeneity of collection strains of Yersinia pseudotuberculosis and their molecular biological properties]. / Geterogennost' muzeinykh shtammov Yersinia pseudotuberculosis i ikh molekuliarno-biologicheskie svoistva.

The capacity of Y. pseudotuberculosis strains for disassociation with the appearance of S- and P-forms has been studied. Strains 852 and 9547 show high stability in S-forms, their conversion into R-forms occurring at 40-42 degrees C. Strain 6953 shows pronounced polymorphism and instability of its associations at different growth temperatures. Strain 9532 exists in S- and R-forms which retain their stability during numerous subculturings at different growth temperatures and prolonged storage. This strain has plasmids of 130, 72.2, 5.7 kb. All plasmids are retained in S- and R-forms, i. e. the dissociation of the strain is not accompanied by the loss of plasmids. The conversion of the strain from the S-form into the R-form leads to changes in the structure of lipopolysaccharide and the composition of low-molecular (less than 23 kD) proteins in the outer and inner membranes. In tests on guinea pigs the LD50 of the R-form of the strain is tenfold greater than that of its S-form. The dissociants of strain 9532 are transformed by plasmid DNA with equal efficiency and equally inherit them without selective pressure.

[Specific proteolysis by fibrinolysin-coagulase from Yersinia pestis of Yersinia pseudotuberculosis outer membrane proteins coded by the Ca(2+)-dependence plasmid]. / Spetsificheskii proteoliz fibrinolizinokoagulazoi Yersinia pestis belkov vneshnei membrany, kodiruemykh plazmidoi Ca(2+)-zavisimosti v Yersinia pseudotuberculosis.

The pesticinogenicity 9.5 kb plasmid from Yersinia pestis strain EV76 has been marked by the kanamycin phosphotransferase gene inserted into PstI site and designated pP3. The obtained plasmid pP3 determines the synthesis of 45 kd pesticin, alpha and beta-forms of fibrinolysin coagulase (37 and 35 kd) and the 29, 19 and 13 kd proteins in Escherichia coli mini cells. When transferred into Yersinia pseudotuberculosis strain 6933 the plasmid causes the proteolysis of outer membrane proteins. The 150 kd protein is reduced to 138 kd, the 48.5 kd protein is reduced to 45 kd. The proteins secreted into the cultural medium (51 and 38 kd) are also cleaved. The proteolysis of the 150 kd protein was found to occur at the stage of secretion via the inner membrane. The purified fibrinolysin coagulase from Escherichia coli strain JM83 harbouring the plasmid pP3 induces the proteolysis in vitro of the isolated membrane proteins from Yersinia pseudotuberculosis strain 6953 similar to the proteolysis registered in vivo.

[Cloning of Yersinia pseudotuberculosis calcium-dependence plasmid pYV6953 BamHI fragments and their analysis in Escherichia coli mini-cells]. / Klonirovanie BamHI-fragmentov plazmidy kal'tsiizavisimosti pYV 6953 Yersinia pseudotuberculosis i ikh analiz v mini-kletkakh Escherichia coli.

Calcium dependence plasmid pYV6953 (70.4 kb) in Yersinia pseudotuberculosis cells codes for the major quantities synthesis of 150; 48.5; 19.4 Kd outer membrane proteins and the 51, 38, 27 Kd proteins secreted into the culturing medium. These outer membrane and secreted proteins are synthesized in considerable amounts in Yersinia pseudotuberculosis strains 6953 and 9547 at 37 degrees C and in the absence of calcium ions in the culturing medium. BamHI fragments of the plasmid pYV6953 as components of the recombinant plasmids code for the synthesis of 150; 66.6; 51; 48.5; 47; 38 and 21.5 Kd proteins in Escherichia coli mini cells. The synthesis of 150 and 48.5 Kd proteins is determined by the BamHI fragment 9 of the plasmid pYV6953 (3.3 kb). Addition of up to 8% of ethanol inhibiting the protein synthesis eliminates the 150 Kd protein but not the 48.5 Kd synthesis. The 48.5 Kd protein is concluded to be a subunit of the 150 Kd protein. The plasmid pYV6953 is different from the known plasmids pIB1 and pCD1 plasmids as far as the outer membrane and secreted proteins coded by the plasmids are concerned.

[Effect of the structure of oligonucleotide substrates on interaction with methylase Eco dam]. / Vliianie struktury oligonukleotidnykh substratov na vzaimodeistvie s metilazoi Eco dam.

The interaction of Eco dam methylase with various synthetic oligonucleotide substrates was investigated. The "imperfect" duplexes contained a normal GATC recognition sequence in one chain of the enzyme recognition site and had some defects in the complementary chain, i.e., the absence of one or several nucleotide residues or the presence of S-methyl thiophosphate groups at the 3'-termini. The 3'-S-methyl thiophosphate residue has the same effect on the methylation of oligonucleotide complexes as does the absence of internucleotide phosphate in the analogous complexes. The presence of both GA dinucleotides in the recognition site is necessary for a productive enzyme-substrate interaction. The experimental data suggest that Eco dam methylase does form a symmetrical enzyme-substrate complex which is similar to that formed by type II restriction enzymes.

[Chemical synthesis and properties of oligonucleotide substrates for restriction endonuclease BamHI and methyltransferase Eco dam]. / Khimicheskii sintez i svoistva oligonukleotidnykh substratov dlia éndonukleazy restriktsii BamHI i metiltransferazy Eco dam.

Oligodeoxyribonucleotides which form a number of duplexes, containing the recognition sequences for endonuclease BamHI and DNA methylase Eco dam, were synthesised by the phosphotriester approach. Furthermore, synthesis of 3'-phosphorylated oligodeoxyribonucleotides from corresponding S-methyl phosphorothioate triester oligomers is described. The synthetic duplexes are characterized by some defects in the recognition sequences for endonuclease BamHI and methylase Eco dam, viz. nick, absence of an internucleotide phosphate, modifications (including partial single-strandedness) of the recognition site. Interaction of the enzymes with these synthetic substrates was investigated.

[Automated synthesis of oligodeoxyribonucleotides. VI. Properties of segmental supports based on cellulose]. / Avtomaticheskii sintez oligodezoksiribonukleotidov. VI. Issledovanie svoistv segmentnykh nositelei na osnove tselliulozy.

The course of development of the rapid automated synthesis of oligodeoxyribonucleotides, various cellulose supports have been studied. Mild acid or aqueous ammonia treatment of Whatman or Filtrak paper disks is shown to improve characteristics of the supports, increasing the capacity in terms of the first immobilized nucleoside. New segmental supports based on cotton or flax were prepared and used successfully in automated synthesis of oligodeoxyribonucleotides on "Victoriya-2" synthesizer.

[Rapid automated synthesis on paper disks of oligodeoxyribonucleotides constituting the promoter fragment of the vaccinia virus genome]. / Bystryi avtomatizirovannyi sintez na bumazhnykh diskakh oligodeoksiribonukleotidov, sostavliaiushchikh promotornyi fragment genoma virusa ospovaktsiny.

For automation of segmental solid-phase synthesis a simple approach leading to the optimal scheme of synthesis of a large numbers of oligonucleotides in one reaction vessel has been proposed. An advantage of the scheme as compared with synthesis in four reaction vessels is a lower number of condensation steps and increased economy of the process. Sixteen oligodeoxyribonucleotides constituting promoter fragment of the viral genome have been synthesised by the modified segmental method on "Victoriya-2" synthesizer according to the optimal scheme.

[A simplified variant of the Maxam-Gilbert method for determining the primary structure of oligonucleotides and DNA fragments]. / Uproshchennyi variant metoda Maksama-Gilberta dlia opredeleniia pervichnoi struktury oligonukleotidov i fragmentov DNK.

A modification to the Maxam-Gilbert method is proposed that involves precipitation of the nucleotide material with the acetone solution of lithium perchlorate after the completion of chemical reactions to remove the reagents. Modification of cytosine residues is carried out in the presence of lithium chloride. The new mode of precipitation simplifies and speeds up the analysis of oligonucleotides and DNA fragments.

[Effect of Ecodam DNA-methylase on single-stranded sequences and synthetic oligonucleotides]. / Deistvie DNK-metilazy Ecodam na odnonitchatye posledovatel'nosti i sinteticheskie oligonukleotidy.

Interaction of the Ecodam methylase with different substrates were investigated among them the double- and single-stranded DNAs and synthetic oligonucleotides containing some defects in the GATC sequence. These defects were:nick, the absence of one internucleotide phosphate of nucleotide; partially single-stranded form on the recognition site etc. It was demonstrated that the presence of both G . A-dinucleotides in the recognition site is necessary for productive enzyme-substrate interaction. The absence of T and/or C residues is less dramatic for methylase activity. The Ecodam methylase is capable to modify the single-stranded oligonucleotides by forming the double-stranded structure in the symmetric recognition sequences GATC.

Does the DNA methylase Eco dam pair nucleotide sequences to form site-specific duplexes?

The Eco dam methylase is active on denatured DNA and single-stranded synthetic oligonucleotides containing GATC sites. The results suggest that on interaction with single-stranded oligonucleotides the Eco dam methylase is able to form a duplex structure within the GATC site, and that this duplex site is a substrate for enzyme.

[Synthesis of a 33-member polynucleotide containing the "core" att site of phage lambda DNA and its cloning]. / Sintez 33-chlennogo polinukleotida, soderzhashchego "core" att sait DNK bakteriofaga lambda, i ego klonirovanie.

Two polynucleotides containing 33 monomeric units were synthesized by a solid-phase phosphotriester method. These polynucleotides form a duplex with protruding 5'-ends, which allows to clone the duplex in EcoRI site of a cloning vehicle. Each polynucleotide was purified by electrophoresis in polyacrylamide gel, and the duplex obtained was cloned in EcoRI site of pUR 222 plasmid DNA. The structure of the cloned duplex containing the "core" att site of phage lambda was confirmed by sequencing.

Structure subtraction as an approach to investigation of the mechanism of restriction enzyme action.

Endonuclease BamHI cleaves the phosphodiester bonds between the guanine residues within the duplex DNA sequence G decreases GATCC. The substrate characteristics of oligonucleotides, containing some defects in the sequence recognized by endonuclease (nick, absence of some internucleotide phosphate or nucleotide, partially single-stranded form of the recognition site) were investigated. The results suggest that the specificity of synthetic oligonucleotide cleavage is strongly dependent on the ribosophosphate backbone intactness inside the recognition site. BamHI was found not to hydrolyse the phosphodiester bonds outside the double helix. Also BamHI forms a productive complex with the non-symmetrical substrate, having half the recognition sites, of a single strand.

[Complexes of nonionic triether analogs of oligonucleotides with polynucleotides]. / Kompleksy neionnykh triéfirnykh analogov oligonukleotidov s polinukleotidami.

Alkyl triester analogues of oligodeoxynucleotides were synthesized: [Tp(CH3)]8T(Ac), [Tp(C2H5)]8T, [dGp(C2H5)]2G, [dAp(C2H5)]2A. Their binding to complementary polyribo-and polydeoxyribonucleotides was studied by UV-spectroscopy and gel-filtration. The DNA complexes of analogues were shown to be more thermostable than the RNA ones independent on the nucleic base nature and alkyl residue size of the analogue used. Mg-salt increased the thermostability of the oligonucleotide analogue--RNA complexes.