[Protective action figurations for superoxide dismutase - chondroitin sulfate - catalase bienzyme conjugate after its medicative administration in endotoxin shock]. / Pokazateli zashchitnogo deistviia bifermentnogo kon"iugata superoksiddismutaza-khondroitinsul'fat-katalaza posle ego lechebnogo vvedeniia pri éndotoksicheskom shoke.

Previously it found that the bienzymatic conjugate superoxide dismutase-chondroitin sulfate, catalase (SOD-CHS-CAT) increased the survival rate of rats with endotoxic shock caused by the administration of lipopolysaccharide (LPS). This effect was observed both in preventive (before LPS) and therapeutic conjugate administration (after the administration of LPS). This study shows that the development of endotoxic shock is accompanied by increased levels of NO in the liver, lungs, kidneys, heart; administration of the SOD-CHS-CAT conjugate insignificantly influenced this parameter. At the same time, the changes in blood urea and creatinine suggest the protective effect of the conjugate on renal function, while diverse changes in biochemical parameters studied complicate the formation of the agreed conclusions on the state of other organs.

Stratification of chondroitin sulfate binding sites in 3D-model of bovine testicular hyaluronidase and effective size of glycosaminoglycan coat of the modified protein.

A 3D-model of bovine testicular hyaluronidase (BTH) was constructed based on established tertiary structure of human hyaluronidase Hyal1 using a molecular homological modeling method in silico. The analysis of the BTH 3D-model demonstrated lysine residue stratification during enzyme modification. The 3D-model of chondroitin sulfate (CHS)-modified hyaluronidase (BTH-CHS) was obtained by modeling covalent binding of lysine residues with benzoquinone-activated CHS. The degree of enzyme modification and the length of CHS chains were varied during 3D modeling. The importance of deep BTH modification degree for the formation of active and stable enzyme derivatives was shown, as determined earlier experimentally. The effective size of the CHS coat for productive BTH modification was confirmed. It is theoretically achieved at the increase in molecular mass of BTH-CHS derivative to approximately 140-180 kDa and can be practically obtained, according to experimental data, using CHS of different molecular mass (30-50 as well as 120-140 kDa).

Specific effects of chondroitin sulfate-modified hyaluronidase on the function of progenitor cells.

We have demonstrated the possibility of stimulation of the function of various types of precursor cells with hyaluronidase modified with chondroitin sulfate. Parenteral administration of modified hyaluronidase increased the number of fibroblast, granulomonocyte, and erythroid CFU in the hemopoietic tissue. The changes in the pool of mesenchymal progenitor cells were more pronounced in comparison with those induced by native enzyme.

[Thrombolysis with plasminogen activators: use and research of serine proteinases, promise and actuality].

Physiological plasminogen activators of tissue and urokinase type (serine proteinases) are effective thrombolytic agents. Research and development of their novel forms contributed to progress of thrombolytic therapy, advanced treatment of acute coronary syndrome and a marked decrease in the lethal index.

[Endothelial glycocalyx of blood circulation. I. Finding, components, structure organization].

In normal state, a complex multicomponent system called glycocalyx is present on the surface of endothelial vascular system. The structure of the glycocalyx is determined by a group ofproteoglycans, glycoproteins and glycosaminoglycans, originating from endothelial cells and blood flow. Due to its complexity and location on the border of the system of blood circulation, glycocalyx participates in a number of functions supporting the metabolism of the vascular wall. Complete or partial loss of this structure in pathologicalconditions leads to inconsistencies in the vascular wall and changes in its functions. The first part of this review considers the history of detection and determination of endothelial glycocalyx structure, utilized methods and approaches. The molecular composition of the glycocalyx, properties of its components and glycocalyx structure organization are described. The English version of the paper: Russian Journal of Bioorganic Chemistry, see also http://www.maik.ru.

[Endothelial glycocalyx of blood circulation. II. Biological functions, state at norm and pathology, bioengineering application].

In normal state, a complex multicomponent system called glycocalyx is present on the surface of endothelial vascular system. Due to complexity of its composition and location on the border between vessel wall and blood circulation, glycocalyx participates in a number of functions supporting the metabolism of the vascular wall. In pathological conditions undergo complete or partial loss of this structure, which leads to inconsistencies in the vascular wall and change its functions. The functions of endothelial glycocalyx are its involvement in the regulation of vascular permeability, transduction and transformation by the shear stress of blood flow on endothelium, the molecular regulation of glycocalyx microenvironment and its interaction with circulating blood cells. Also briefly be considered participation of glycocalyx in the implementation of cardiovascular diseases, their correction, bioengineering application of glycocalyx and its components.

Cardiological biopharmaceuticals in the conception of drug targeting delivery: practical results and research perspectives.

The results of the clinical use of thrombolytic and antithrombotic preparations developed on the basis of protein conjugates obtained within the framework of the conception of drug targeting delivery in the organism are considered. A decrease has been noted in the number of biomedical projects focused on these derivatives as a result of various factors: the significant depletion of financial and organizational funds, the saturation of the pharmaceutical market with preparations of this kind, and the appearance of original means for interventional procedures. Factors that actively facilitate the conspicuous potentiation of the efficacy of bioconjugates were revealed: the biomedical testing of protein domains and their selected combinations, the optimization of molecular sizes for the bioconjugates obtained, the density of target localization, the application of cell adhesion molecules as targets, and the application of connected enzyme activities. Enzyme antioxidants and the opportunity for further elaboration of the drug delivery conception via the elucidation and formation of therapeutic targets for effective drug reactions by means of pharmacological pre- and postconditioning of myocardium arouse significant interest.

[Antioxidant and antiaggregant effects of covalent bienzyme superoxide dismutase-chondroitin sulfate-catalase conjugate in platelet interactions].

A covalent bienzyme superoxide dismutase-chondroitin sulfate-catalase conjugate (SOD-CHS-CAT) demonstrated the dose-dependent inhibitory action on induced aggregation of platelets in the presence of hydrogen peroxide. Antioxidant activity of SOD-CHS-CAT appeared at much lower doses than for other CAT derivatives. We detected the antiaggregation effect of SOD-CHS-CAT for platelet aggregation induced by ADP, serotonin, TRAP (with their different mechanism of action). Novel properties of SOD-CHS-CAT confirmed with its action agains spread-eagle platelets on glass surface. The new characteristics of SOD-CHS-CAT conjugate underline the prospects of its biopharmaceutical development for antioxidant therapy.

Effect of hydrogen peroxide and catalase derivatives on functional activity of platelets.

Effects of H(2)O(2) on platelet aggregation were estimated in vitro in the presence and absence of inductors (ADP, serotonin, TRAP) and native and modified catalase. Dose-dependent effect of H(2)O(2) (50 µM or more) was investigated in a pathophysiological concentration of 300 µM inducing platelet aggregation. H(2)O(2) modulated aggregation induced by ADP, serotonin, and TRAP significantly increasing the initial platelet aggregation followed by disaggregation, which was always more pronounced than in control. Catalase derivatives (native and modified forms) dose-dependently reduced the effect of H(2)O(2) and completely abolished it in a dose of 9000 U catalase activity per 1 ml of solution for native catalase and 1200 U/ml for modified one. Modified catalase, in contrast to native one, produced an independent inhibitory effect on induced platelet aggregation. Components of modified catalase (individual substance and simple mixture) had no antiaggregant effect.

[Do electrostatic interactions determine glycation of hyaluronidase derivatives with N-acethylhexosamines?].

Glycation of native hyaluronidase and its chondroitin sulfate modified form was studied with N-acethylglucosamine, N-acethylgalactosamine and their mixture, as well as hyaluronan fragments (n = 0-4) and their mixture. The modified form of hyaluronidase exhibited higher inactivation than native enzyme. The chondroitin sulfate modification of hyaluronidase altered its surface electrostatic potential, but this effect was not crucial for inactivation of hyaluronidase derivatives. The observed picture of the glycation action on hyaluronidase derivatives was opposite for glycation with mono- and di-saccharides. Such results give us the informative enzyme test for in vivo system in order to determine the dominant type of glycation agents in bloodstream and its origin.

Biopharmacology of enzyme conjugates: vasoprotective activity of supramolecular superoxide dismutase-chondroitin sulfate-catalase derivative.

Bienzyme conjugate was obtained by the covalent connection of superoxide dismutase with catalase through endothelial glycocalyx glycosaminoglycan - chondroitin sulfate (SOD-CHS-CAT). This SOD-CHS-CAT conjugate has vasoprotective activity in respect to platelet interactions, tonus of the ring arterial fragment of a rat blood vessel, as well as normalization of hemodynamic parameters in rats and rabbits in conditions of oxidative stress caused by the administration of hydrogen peroxide. The SOD-CHS-CAT conjugate had antiplatelet potential due to its antiaggregation action manifested through the combination of enzyme activities and an acquired supramolecular structure. The influence on arterial fragment tonus was equivalent for SOD and CAT in native and conjugated form. Blood pressure and heart rate were significant and effectively normalized with SOD-CHS-CAT conjugate in rats and rabbits (after hydrogen peroxide administration as a perturbance stimulus). We have discovered the possibility of using the antioxidant bienzyme conjugate in chronic prophylaxis. It is important for a real development of the oral form of the SOD-CHS-CAT conjugate. These results indicate that the development of enzyme conjugates can be medically significant, as a promising approach for the creation of new drugs.

Role of the glycosaminoglycan microenvironment of hyaluronidase in regulation of its endoglycosidase activity.

The glycosaminoglycan microenvironment of testicular hyaluronidase was simulated by multipoint covalent attachment of the enzyme to glycans as a result of benzoquinone activation. The efficiency of their binding was assessed using gel chromatography, ultrafiltration, titration of surface amino groups of the enzyme, electrophoresis, as well as judging by the value of residual endoglycosidase activity and its inhibition with heparin. Copolymer glycosaminoglycans, such as dermatan sulfate and heparin, inactivated the endoglycosidase activity as a result the C-5 epimerization of hexuronic acid. It was shown that glucuronic acid and, to a lesser extent, N-acetylglucosamine determine the specificity of hyaluronidase. The chondroitin-sulfate microenvironment made the enzyme resistant to heparin inhibition because the equatorial orientation of the OH groups is similar to that in hyaluronic acid. Model experiments with dextran and dextran sulfate showed that sulfation of the glycan chain increased its rigidity, thus hampering the stabilizing effect on hyaluronidase. The effect of chondroitin sulfate on the endoglycosidase activity of hyaluronidase had additive character and did not directly affect the small fragment of the active site of the enzyme located at the bottom of a groove. The glycosaminoglycan microenvironment of hyaluronidase, containing an iduronic acid residue, the alpha1-3 and alpha1-4 glycosidic bond, inactivated the hyaluronidase activity of the enzyme, whereas simple polymers (such as gluco- and galactoaminoglycans) potentiated it due to a similar way of linking--beta(1e-4e) and beta(1e-3e). To understand the nature of these interactions in detail, the effect of oligomeric glycosaminoglycan fragments and their derivatives on hyaluronidase should be studied.

Influence of DNA aptamer structure on the specificity of binding to Taq DNA polymerase.

The secondary structure of DNA aptamer to Taq DNA polymerase was established as a hairpin. Both stem and loop structures of DNA ligand were shown to be involved in the interaction with Taq DNA polymerase. Moreover, the structure and sequence of DNA aptamer that was the most effective inhibitor of DNA polymerase activity were established. This crucial structure was evaluated as a GC-rich stem longer than 17 bp, and a loop consisting of 12 bases with strictly determined nucleotide sequence. It was demonstrated that nucleotide in position 23 counting from the 5;-end of DNA ligand was involved in direct contact with Taq DNA polymerase. The ability of optimized DNA aptamer TQ21-11 to form a complex with the enzyme was increased 5-fold in comparison to the initial aptamer.

Resistance of dextran-modified hyaluronidase to inhibition by heparin.

Properties of native and aldehyde dextran-modified hyaluronidase (with surface amino group modification about 98%) were investigated. Optimal endoglycosidase activity of the native enzyme was observed at 0.15 M NaCl and pH 5.5 and electrostatic interactions influenced the enzyme activity. The inhibitory effect of heparin on hyaluronidase activity slightly differed at pH 5.5 (1.5-fold inhibition) and 7.5 (1.2-fold inhibition). Ionic strength of the reaction medium only slightly influenced the effect of heparin. Modification of hyaluronidase with dextran increased hydrophobic interactions and steric hindrance. Conjugation with dextran increased the resistance of hyaluronidase activity to denaturing agents (urea, guanidinium hydrobromide) and extended the optimal conditions for maximal endoglycosidase activity (pH 4.5-6.5, the range of NaCl concentration from 0.1 to 0.3 M). The conjugation also reduced electrostatic effects on the active site of hyaluronidase and efficacy of heparin inhibition. At pH 7.5 the enzyme was almost insensitive to heparin. The resistance of dextran-modified hyaluronidase to heparin points to approaches for subsequent studies of the heparin-binding site of this enzyme and biomedical trial of the stabilized enzyme for the treatment of acute cardiovascular lesions.

Chemical modification of hyaluronidase regulates its inhibition by heparin.

Chemical modification of surface amino groups of bovine testicular hyaluronidase with aldehyde dextran was conducted. It was found that with the increase of modification degree of hyaluronidase amino groups the value of residual enzymatic catalytic activity is decreased rather monotonously. It turned out that the value of inhibition of enzyme activity by heparin considerably depends on modification degree of enzyme. This dependence is of a threshold character. Sharp conformational changes in the enzyme occurring at 70-90% degree of its modification considerably lowers heparin inhibition. The higher the degree of hyaluronidase modification, the weaker its inhibition by heparin. More completely/deeply modified derivatives of hyaluronidase (modification degree 96-100%) are practically not inhibited by heparin. Thus, chemical conjugation of hyaluronidase with aldehyde dextran regulates the value of enzyme inhibition by heparin. Hyaluronidase modification becomes an informative tool to study the mechanism of inhibition of its enzyme activity and an efficient means for the development of new therapeutic preparations improving tissue permeability during cardiovascular injuries.

[Regulation of hyaluronidase inhibition through its chemical modification]. / Reguliatsiia ingibirovaniia geparinom gialuronidazy posredstvom ee khimicheskoi modifikatsii.

The modification of hyaluronidase by aldehydodextran regulates inhibition of the enzyme by heparin. A 70-90% modification of the surface amino groups of hyaluronidase results in sharp conformational changes and a substantial decrease of its inhibition by heparin, whereas hyaluronidase derivatives with a modification degree of 96-100% are practically uninhibited.

Targeting of single-stranded DNA and RNA containing adjacent pyrimidine and purine tracts by triple helix formation with circular and clamp oligonucleotides.

The aim of this work was to construct an anti-messenger targeted to the pim-1 oncogene transcript, based on circular or clamp oligodeoxyribonucleotides. The formation of bimolecular triplexes by clamp or circular oligonucleotides was investigated using single-stranded targets of both DNA (5'-CCCTCCTTTGAAGAA-3') and RNA type (5'-CCCUCCUUUGAAGAA-3'). The third, 'Hoogsteen' strand of the triplex was represented by G,T-rich sequences. The secondary structures of the complexes were determined by thermal denaturation, circular dichroism and gel mobility shift experiments and shown to depend on the nature of the target strand. With DNA as target, the sequence of a clamp (or circular) oligonucleotide that formed the triple helix was 3'-GGGAGGAAACTTCTTTT-TTGTTGTTT-TT-GGTGGG-5', where the first TT dinucleotide (in italics) is a linker and the second TT (bold) represents the bridge through which the 'Hoogsteen' strand switches from one strand of the Watson-Crick duplex to the other, once the duplex is formed by the corresponding portion of the anti-messenger (underlined). The portion of the 'Hoogsteen' sequence of the triplex between the two TT dinucleotides binds to the 3' extremity of the target strand and runs parallel to it. The portion situated at the 5' end of the oligonucleotide switches to the purine tract of the complementary strand of the duplex and is antiparallel to it. In contrast, with RNA as target, for a branched clamp oligonucleotide that formed a triple helix over its entire length (5'-TTCTTCAAAGGAGGG-3' 3'-GGGTGGTTT-T-GTTGTT-5') the portion of the 'Hoogsteen' sequence that bound to the 3' extremity of the target strand had to be antiparallel to it.

[Third generation of plasminogen activators. New directions in research]. / Aktivatory plazminogena tret'ego pokoleniia. Novoe napravlenie izucheniia.

Recent information on the development of new plasminogen activators (PA) is reviewed. The results of studies of PA mutant derivatives synthesized by recombinant DNA techniques are discussed. Data on chimeric (hybrid) forms of PAs and their chemically synthesized conjugates are presented. The trends in the search for PAs are analyzed. A new direction in the study of third-generation PAs for combined plasminogen activation and in the further development of the methods of thrombolytic therapy is outlined.

Physico-chemical and biological properties of antisense phosphodiester oligonucleotides with various secondary structures.

The influence of the secondary structure of oligonucleotides having a natural phosphodiester backbone on their ability to interact with DNA and RNA targets and on their resistance to the nucleolytic digestion is investigated. Oligonucleotides having hairpin, looped and snail-like structure are found to be much more stable to nuclease degradation in different biological media and inside cells than the linear ones. The structured oligonucleotides can also hybridise with their DNA and RNA targets.

New thrombolytic strategy: bolus administration of tPA and urokinase-fibrinogen conjugate.

Increased efficacy of thrombolytic therapy requires a comprehensive search for new and novel therapeutic strategies. Many new modified forms of plasminogen activators have been obtained by means of chemical and biological synthesis. However, clinical findings demonstrate that the reperfusion level achieved during thrombolysis remains the same for various thrombolytic agents, irrespective of an extensive search for an "ideal" thrombolytic. Thrombolytic therapy may be complicated by treatment delays, cumbersome schemes of preparation and administration, and hemorrhagic and rethrombotic events. These limitations may be overcome, at least in part, by applying combined thrombolysis with plasminogen activators exhibiting complementary actions and different pharmacokinetic profiles. The combined action of native thrombolytics allows the use of lower doses and simplified schemes of administration, yielding encouraging results in experimental models. Long-acting forms of plasminogen activators are being developed and tested in combination with tissue-type plasminogen activator as a trigger of thrombolysis. The combination of short- and long-acting plasminogen activators appears promising and potentially eligible for bolus administration to patients. On the basis of our own experimental results and data in the literature, we suggest a new thrombolytic strategy connected with the single injection of a combination of complementary and pharmacokinetically different plasminogen activators.