[Association-dissociation of molecules of hemoglobin and polymeric hemoglobin in solutions].

The process of association-dissociation of hemoglobin molecules into dimers of its subunits in water-saline solutions is studied by the method of gel-penetrating chromatography and ultrafiltration. The quantitative assessment of stabilization of quaternary structure of hemoglobin in chemically bound polymer derivative in comparison with native peptide on the basis of building differential concentration curves is conducted for the first time. By the method of atomic-force spectroscopy, the morphology of nanoparticles of hemoglobin and its modified polymeric derivative is studied.

[Modification of hemoglobin by glutaric aldehyde and electrochemical properties of the conjugates]. / Modifikatsiia gemoglobina glutarovym al'degidom i issledovanie élektrokhimicheskikh svoistv poluchennykh kon''iugatov.

Electrochemical properties of macromolecules of modified haemoglobin obtained by polycondensation with glutaric aldehyde have been investigated by means of potentiometric titration, PAG-electrophoresis, ion-exchange chromatography, and (for evaluation of isoelectric point) distribution between two aqueous polymeric phases. Introduction of additional functional groups into the macromolecule is possible by using various agents blocking polycondensation, which makes it possible to change the resulting charge and the isoelectric point.

[Soluble heteroprotein conjugates based on recombinant pro-urokinase for ophthalmology]. / Rastvorimye geterobelkovye kon''iugaty na osnove rekombinantnoi prourokinazy dlia oftal'mologii.

A procedure is developed for precise synthesis of soluble highly active heteroprotein conjugates enriched in the enzyme with prearranged molecular mass and composition, containing blood serum albumin and recombinant prourokinase. Correlation between the pharmacological activity of the heroconjugates produced and their molecular mass from 2.2 x 10(5) to 5 x 10(5) was studied in experimental vitreous body hemorrhage (hemophthalmia). Heteroconjugates with molecular mass 5 x 10(5) and more exhibited distinctly higher activity due to prolonged fibrinolytic effect. The best medicinal efficiency of the immobilized prourokinase with molecular mass 9 x 10(5) proved to be also in 50-fold decrease of its dose administered into the vitreous body as compared with native enzyme effect.

[Effect of chemical modification of hemoglobin on its biological activity]. / Vliianie khimicheskoi modifikatsii gemoglobina na ego biologicheskuiuaktivnost'.

Oligomeric hemoglobins were produced after polycondensation with glutaric aldehyde (OHb) as well as after modification with pyridoxal-5-phosphate (OHb-PLP). OHb exhibited high affinity to oxygen (P50 = 17 torr) as well as a decreased rate of the subunits cooperative interaction (n = 1.5-1.6); OHb-PLP possessed P50 = 27 torr, n = 2.2 (for pO2 greater than P50), Bore effect -0.4. The P50 pattern was not distinctly altered in presence of approximately 15% Met hemoglobin, while already 8% of the Met form affected the "n" parameter. Biological activity of OHb and OHb-PLP was studied.

[Study of oligohemoglobin solutions in vitro and in vivo]. / Issledovanie rastvorov oligogemoglobina in vitro i in vivo.

Solutions of oligohemoglobins (OHb) with middle molecular mass 100 x 10(3) = 400 x 10(3), obtained after polycondensation of hemoglobin with glutaric aldehyde as well as oligohemoglobin modified with pyridoxal-5-phosphate, were studied in vivo and in vitro (plethoric administration, isovolemic metabolic substitution, hemorrhagic shock; at a dose of 0.5-1.8 g/kg of body mass; dogs, rabbits, rats). With increase of the OHb molecular mass period of its circulation in blood was elevated; blood plasma protected OHb from autooxidation; half-life of OHb was about 12-18 hrs in isovolemic metabolic substitution and in hemorrhagic shock and about 5 hrs--in plethoric administration. During circulation molecular mass selection of OHb occurred, where low molecular fraction decreased and high molecular fraction--accumulated; sign and value of human erythrocyte charge were similar both in OHb solutions with concentration up to 5% and in physiological solution.

[The prevalence of secondary resistance to peroral hypoglycemic agents among patients with type-2 diabetes mellitus]. / Rasprostranennost' vtorichnoi rezistentnosti k peroral'nym sakharosnizhaiushchim sredstvam sredi bol'nykh sakharnym diabetom II tipa.

Prevalence of secondary resistance to peroral sugar-reducing agents (PSRA) among patients with type II diabetes mellitus was studied. The majority of patients (81%) who were allocated to insulin treatment for the first time had received peroral sugar-reducing preparation prior to that treatment. Mean duration of illness in these patients was 9.94 +/- 0.6 years. Females predominated among those persons showing resistance to PSRA; they all presented with obesity.

Cell-free and cell-bound circulating DNA in breast tumours: DNA quantification and analysis of tumour-related gene methylation.

Tumour development is characterised by the increased circulating DNA (cirDNA) concentration and by tumour-related changes in blood plasma DNA. Concentration of cirDNA and methylation of RARbeta2, RASSF1A and HIC-1 gene promoters were investigated in cell-free and cell-surface-bound fractions from healthy donors, patients with breast cancer, and patients with breast fibroadenoma. Tumour development was shown to lead to significant changes in the distribution of cirDNA between cell-free and cell-surface-bound fractions. Analysis of RARbeta2 and RASSF1A methylation in the total cirDNA provides 95% diagnostic coverage in breast cancer patients, 60% in patients with benign lesions, and is without false-positive results in healthy women. Results of the study indicate that methylation-specific PCR of RARbeta2 and RASSF1A genes based on the total cirDNA combined with the quantitative analysis of cirDNA distribution between cell-bound and cell-free fractions in blood provide the sensitive and accurate detection and discrimination of malignant and benign breast tumours.

Plasma content of extracellular nucleic acids in donors and patients with mammary tumors.

The concentrations of extracellular DNA and RNA were measured in the plasma of donors and patients with fibroadenoma and breast cancer. The content of extracellular DNA surpassed the normal in 80% plasma samples from patients with mammary tumors. Extracellular RNA was detected in 30% plasma samples from donors and patients with breast tumors. No correlations were found between plasma concentration of extracellular DNA and size and stage of tumor growth. Hence, measurement of extracellular DNA in the plasma of patients can be used only as an accessory test for tumor diagnosis.

Microdisperse form of immobilized hemoglobin modelling the erythrocytes.

Disperse systems based on hemoglobin sorption immobilization in reticular carboxylic polyelectrolytes are proposed as model of erythrocytes. The efficiency of oxygen transport of these systems is much higher than that of native hemoglobin and is comparable with the gas transport of erythrocytes. This is believed to be due to highly selective sorption of hemoglobin in microdisperse forms of permeable polyelectrolytes. Microparticles of immobilized hemoglobin exhibit high local concentration of hemoglobin: the protein mass being one order of magnitude than that of polymer-carrier. Besides, it is presumed that structure of the carboxylic polyelectrolyte matrix is a polymer analog of 2,3-diphosphoglycerate. Microdispersion of immobilized hemoglobin exhibits a aggregative resistance and have identity of surface charge with erythrocytes. Studies in vivo have revealed biocompatibility of immobilized hemoglobin.

[Increase in oxygen-transporting efficiency of hemoglobin when chemically modified]. / Povyshenie kislorodtransportnoi éffektivnosti gemoglobina pri ego khimicheskoi modifikatsii.

Polycondensation of human hemoglobin with glutaraldehyde premodified with reagents bearing acid groups (glutamic acid or sodium bisulfite), has been carried out. The resulting modified hemoglobin displays better oxygen transport characteristics in comparison with native hemoglobin.

Modelling of gas transport function of erythrocytes in haemoglobin sorption immobilization.

Colloid-disperse systems based on haemoglobin sorption immobilization in reticular polyelectrolytes are proposed and investigated. The efficiency of oxygen transport of these systems is much higher than that of native haemoglobin and is comparable with the efficacy of gas transport of erythrocytes. This is believed to be due to highly selective sorption immobilization of haemoglobin in microdisperse forms of permeable carboxylic reticular polyelectrolytes. Microparticles exhibit high local concentration of haemoglobin, the protein mass being by one order of magnitude higher than that of the polymer carrier.

Synthesis, physico-chemical and biological properties of crosslinked modified hemoglobin.

Polymer aspects of polycondensation of pyridoxylated hemoglobin with glutaraldehyde have been considered. On the basis of the investigation of reaction kinetics, the mechanism of chemical crosslinking of hemoglobin molecules into oligohemoglobin is proposed. Owing to the statistical character of the reaction, the resulting macromolecules are polydisperse with respect to the degree of modification of hemoglobin amino groups, and size of oligohemoglobin molecules. The formation of hemoglobin oligomers was studied by varying the following reaction conditions: pH, the components ratio, and their concentrations. It is shown that the net electric charge of the oligohemoglobin molecule depends on the terminating agents. However, these agents have no effect on the electrophoretic mobility of erythrocytes in oligohemoglobin solutions. The efficiency of oxygen transport of these solutions is close to that of human blood erythrocytes. Oligohemoglobin circulation in the blood of animals after intravenous infusion leads to rapid removal of low molecular weight fractions from blood and to the accumulation of high molecular weight fractions in plasma. The period of half-release of oligohemoglobin from the organism is 14-16 h.