[3-Dimensional microscopy as a method for volume measurement in cells undergoing apoptosis].

Different patterns of cell volume perturbations are commonly used for modes of cell death: necrosis (cell swelling) and apoptosis (cell shrinkage). In this study we employed recently developed three dimensional microscopy for the measurement of the volume of attached vascular smooth muscle cells transfected with E1A-adenoviral protein. These cells undergo rapid apoptosis in the absence of growth factors or in the presence of staurosporine. In 30-60 min of serum deprivation the volume of these cells is increased by -40% that corresponds to the time point of maximal activation of caspase 3 and chromatin cleavage. In 10-15 min swollen cells exhibit morphological collapse indicated by formation of apoptotic bodies. In contrast to serum-deprived cells, staurosporine leads to attenuation of cell volume by 30%. In this case, apoptotic bodies are detected in -2.5 hr after maximal shrinkage. Thus, our results show that cell shrinkage can not be considered as universal hallmark of apoptosis. The role of stimulus-specific cell volume perturbation in the triggering of the cell death machinery should be examined further.

[Myogenic tone of blood vessels in health and disease: role of purinergic signaling system and Na+, K+ 2Cl+ cotransport].

The narrowing of blood vessels evoked by increasing intraluminal pressure and termed as myogenic response plays a key role in the maintenance of the blood flow rate independently of the variation of systemic arterial pressure. Myogenic response MR is accompanied by decreased electrical membrane potential in the vascular smooth muscle cells (VSMC) that, in turn, leads to activation of voltage-gated Ca2+ channels and elevation of [Ca2+]i. Upstream mechanisms underlying VSMC depolarization remain poorly understood. In this review, we summarized data obtained in our laboratories and by other researchers showing tissue-specific impact of purinergic signaling system and Na+, K+, 2Cl- cotransport in the development of myogenic responses along microcirculation.

[Effect of electrostatic interactions on formation and properties of soluble heteroprotein conjugates based on proteolytic enzymes]. / Vliianie élektrostaticheskikh vazimodeistviii na formirovanie i svoistva rastvorimykh geterobelkovykh kon''iugatov na osnove proteoliticheskikh fermentov.

The regularities of directed polycondensation of proteins with opposite charges by glutaric aldehyde were studied using serum albumin and proteolytic enzymes (urokinase, trypsin, alpha-chymotrypsin) as models. The electrostatic interaction of oppositely charged protein macromonomers in the polycondensation process was shown to facilitate the selective synthesis of soluble heteroprotein conjugates with molecular mass from 2 x 10(5) to 9 x 10(5); these conjugates have controlled component composition, high enzyme content (up to 3 to 4 molecules of an enzyme per one albumin molecule) and completely retain the enzyme catalytic activity. The dependence of rate constants and activation parameters of thermal inactivation of the heteroconjugates upon their composition, molecular mass and the degree of modification of the protein amino groups was investigated. Heteroconjugates of electrically asymmetric proteins, in particular, trypsin and serum albumin, were found to be several hundred times more stable than the starting enzymes at 38-60 degrees C.

[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.

[Immobilized thrombolytic enzymes and their use in ophthalmology]. / Immobilizovannye tromboliticheskie fermenty i ikh ispol'zovanie v oftal'mologii.

Soluble highly active conjugates were obtained after complex formation or covalent binding of trypsin and urokinase with polymers of various chemical structure as well as by means of condensation of the enzyme molecules as a result of which oligo- and heterooligoproteins were produced. Pharmacological activity of the conjugates obtained was studied in the course of treatment of rabbits with experimental hyphemia. All the preparations produced exhibited high therapeutic efficiency: the time of hyphemia resorption was decreased, the effect of immobilized enzymes was prolonged as compared with controls. Dependence on the molecular mass and the dose of enzymatic preparations of the period of hyphemia resorption was studied using heteromers of urokinase and hemoglobin. Maximal activity was noted with the preparations of molecular mass 4.10(5) daltons; they maintained the optimal level of pharmacological activity even after 4-fold decrease in the dose used as compared with other immobilized preparations of trypsin and urokinase. The immobilized preparations were 20-fold more effective as compared with native unmodified enzymes.

[Analysis of the secondary structure of urokinase by the circular dichroism method]. / Issledovanie vtorichnoi struktury urokinazy metodom krugovogo dikhroizma.

The secondary structure of urokinase with molecular weight 33 000 dalton was studied by the circular dichroism method. The secondary structure parameters were calculated based on the protein reference CD spectra resulted in the following secondary structure parameters: approximately 30% aminoacid residues constitute the alpha-helical regions, the same amount forms the beta-structure and an essential fractions contributes the beta-turns. Conformational stability of urokinase to alkaline pH (up to 11.6) and high temperature (up to 80 degrees) in 0.1 M phosphate buffer pH 6.6 was found.

[Isolation and purification of urokinase]. / Vydelenie i ochistka urokinazy.

An improved procedure is developed for isolation of urokinase from human urine by means of ion exchange on cyrboxyl cationite SMT at pH 4.7. with subsequent stepwice elution by phosphate buffer, rH7.0. Depigmentation of the urine was achieved by pretreatment with an anionite FAF. Chromatographically pure preparation of urokinase was obtained as shown by gel filtration through Sephadex G-100 and G-150.

[Preparation and properties of trypsin modified by polysaccharides]. / Poluchenie i svoistva tripsina, modifitsirovannogo polisakharidami.

Trypsin modified by dextran preactivated with 2-amino-4,6-dichloro-1,3,5-triazine or ethyl chloroformate has been prepared. Properties of the resulting conjugates with respect to hydrolysis of synthetic substrates--methyl ester benzoyl L-arginine and n-nitrophenyl ester of n'-guanidine benzoic acid as well as high molecular weight substrates--casein and fibrin--have been studied. Stability of conjugates during autolysis and irreversible heat denaturation has been investigated.