[Epidemiology of chronic critical limb ischemia].

Peripheral artery diseases amount to more than 20% of all types of cardiovascular diseases, which corresponds to 2-3% of the total population. 15 to 30% of the population of Russian Federation over 65 years have signs of obliterating arteriopathy. Advance of arterial insufficiency in some cases leads to development of special state - chronic critical limb ischemia (CLI). This article presents a review of statistical data from different sources, showing high prevalence of this severe issue, as well as data of amputations rate, performed in different countries due to CLI, reflecting the extreme relevance of the search for new methods of CLI treatment.

[Cardiosynchronized sequential antegrade pneumocompression in treatment of patients with multifocal atherosclerosis].

The work was aimed at assessing efficacy and safety of indications for and contraindications to a promising method of treatment for multifocal atherosclerosis - enforced external counterpulsation, suggesting a new noninvasive method of treatment of chronic obliterating diseases of lower-limb arteries - cardiosynchronized sequential antegrade pneumocompression.

[The study of the creatine kinase radiation stability in 25Mg-enzyme enrichment experiment].

The radiation inactivation technique was applied to conduct the study on catalytic activity changes of creatine kinase species isolated from the Mid-Asian Death Adder venom. Both intact and the 25Mg-enriched enzyme samples were separately tested. Some differences in radiation stability levels were found to be indicating to a shift in conformational condition (a so called "conformational distribution") of the native enzyme that had happen in the presence of the magnetic nuclei excess.

[Angiotensin-converting enzyme stability in the presence of zinc-ions different concentrations].

Stability of angiotensin-converting enzyme was studied as a dependence on the zinc-ions concentrations brining in the apo-enzyme. Our data were discussed in the terms of a set of initial permissible conformation conditions of a protein (conformation distribution). Apo-enzyme was shown to be able to the radiation activation that is disappearing in the presence of even 10(-6 )mol/l of the zinc-ions.

The porphyrin-fullerene nanoparticles to promote the ATP overproduction in myocardium: 25Mg2+-magnetic isotope effect.

This is a first case ever reported on the fullerene-based low toxic nanocationite particles (porphyrin adducts of cyclohexyl fullerene-C(60)) designed for targeted delivery of the paramagnetic magnesium stable isotope to the heart muscle providing a sharp clinical effect close to about 80% recovery of the tissue hypoxia symptoms in less than 24 h after a single injection (0.03-0.1 LD(50)). A whole principle of this therapy is novel: (25)Mg(2+)-magnetic isotope effect selectively stimulates the ATP overproduction in the oxygen-depleted cells due to (25)Mg(2+) released by the nanoparticles. Being membranotropic cationites, these "smart nanoparticles" release the overactivating paramagnetic cations only in response to the metabolic acidic shift. The resulting positive changes in the heart cell energy metabolism may help to prevent and/or treat the local myocardial hypoxic disorders and, hence, protect the heart muscle from a serious damage in a vast variety of the hypoxia-caused clinical situations including both doxorubicin and 1-methylnicotineamide cardiotoxic side effects. Both pharmacokinetics and pharmacodynamics of the drug proposed make it suitable for safe and efficient administration in either single or multi-injection (acute or chronic) therapeutic schemes.

[The effect of magnesium pool isotopy on reactivation of mitochondrial ATP synthesis suppressed by 1-methyl-nicotine amide].

The ATP-generating activity of both rat myocardial mitochondria and intramitochondrial creatine phosphokinase (CPK) was examined as a function of the incubation medium magnesium pool isotopy. The in vitro systems tested were prepared from the hearts of animals treated with single injection of 1-methyl-nicotine amide (MNA) suppressing the NAD(P)-dependent reactions in vivo. The presense of the 25Mg paramagnetic cations leads to essential compensation of intramitochondrial ATP deficiency caused by the MNA induced blockade of oxidative phosphorylation. This effect is merely unreachable in those systems where the magnesium pool consists of isotopes with a zero nuclear spin (24Mg, 26Mg). The reactivation of mitochondrial ATP synthesis described here involves CPK activity which does not depends on MNA. In this case, a high efficiency of this reactivation seems to be a spin selective phenomenon which requires, predominantly, 25Mg2+ cations.

[Stable isotopes of Mg2+ as activators of the suppressed ATP-generating function of mitochondria].

The ATP-generating activity of rat myocardial mitochondria and intramitochondrial creatine kinase was examined as a function of the isotopy of the incubation medium magnesium pool. The study was performed using in vitro systems prepared from the hearts of animals injected with 1-methylnicotine amide, which suppresses the NAD (NADP)-dependent reactions in vivo. It was shown that the presence of the 25Mg paramagnetic cations essential by compensates for the intramitochondrial ATP deficiency caused by the 1-methyl-nicotine amide-induced blockade of oxidative phosphorylation. This effect is hardey achievable in systems where the magnesium pool consists of isotopes with a zero nuclear spin (24Mg, 26Mg). The restoration of mitochondrial ATP synthesis involves the participation of creatine kinase since the activity of the latter does not depend on 1-methyl-nicotine amide. In this case, the high efficiency of this restaration seems to be a spin-selective phenomenon which requires predominantly 25Mg2+ cations. A possible meaning of the data for further studies on the mechanisms of enzymatic catalysis regulation is discussed.

[Analysis of conditions for different types of enzyme dose response]. / Analiz uslovii polucheniia razlichnykh tipov dozovogo otveta fermenta.

Within the previously described concept designed for a radiation-induced activation/inactivation of enzyme tested, an equation for further analysis of the dose response changing mechanism has been proposed. This mechanism includes both rate and probability of the appearance of different conformation forms of enzyme, as well as with accumulation of the water radiolysis active products.

Conformational differences between native and recombinant horseradish peroxidase revealed by tritium planigraphy.

Significant conformational differences between native and recombinant horseradish peroxidase have been shown by tritium planigraphy, which includes a method of thermal activation of tritium followed by amino acid analysis of the protein preparation. Comparison of radioactivity distribution among the amino acid residues with the theoretical (calculated) accessibility shows that the recombinant enzyme is characterized by high hydrophobicity and compactness of folding. The protective role of oligosaccharides in native enzyme has been confirmed. An unexpected result of the study is a finding on high accessibility of a catalytic histidine residue in solution. An effect of low dose (3 Gy) of irradiation on the accessibility of amino acid residues has been unequivocally demonstrated. The data can be interpreted as swelling of the compact folding and increase in the surface hydrophilicity of the recombinant enzyme. In the case of native enzyme, irradiation does not cause remarkable changes in the accessibility of amino acid residues indicating the possible extensive radical modification of the native enzyme in the life-course of the cell. The catalytic histidine is an exception. It becomes inaccessible after the enzyme irradiation, while its accessibility in the recombinant enzyme increases. An additional observation of a 5-fold decrease in the rate constant towards hydrogen peroxide points to the destructive effect of irradiation on the hydrogen bond network in the distal domain of the native enzyme molecule and partial collapse of the active site pocket.

[Radioenzymatic investigation of membrane and soluble forms of angiotensin-converting enzyme in acetate-phosphate buffer]. / Radioénzimologicheskoe izuchenie membrannoi i rastvorimoi form angiotenzinprevrashchaiushchego fermenta v atsetatno-fosfatnom bufere.

The dose response of soluble and membrane forms of angiotensin-converting enzyme to gamma-irradiation is investigated at different pH values of the medium and at different concentrations of acetate-phosphate buffer. Membrane form of the enzyme is more stable shows principally other conformational equilibrium than the soluble form. "Splitted" activation peaks on the curves of the enzyme dose response are observed.

[Structure-functional features of homologous domains of angiotensin-converting enzyme]. / Strukturno-funktsional'nye osobennosti gomologichnykh domenov angiotenzinprevrashchaiushchego fermenta.

Somatic angiotensin-converting enzyme (ACE) consists of two homologous domains, each of them containing an active site. Differences in substrate specificities and affinity to inhibitors of the active sites of the two domains of bovine ACE are described. The ACE domains demonstrate different thermostability, and the reasons for this difference are analyzed. A structural model of the ACE domains is suggested, which allows us to reveal the structural subdomain important for the protein stability and localize the hydrophobic and the carbohydrate-binding sites.

[ATP-generating creatine kinase: a new compound isolated from Viperidae venom]. / ATP-generiruiushchaia kreatinakinaza: vpervye obnaruzhennyi komponent iada Viperidae.

This is a first report on the presence of creatine kinase, ATP-generating enzyme, in the venom of Vipera xanthia representing the Viperidae family of poisoning snakes. Kinetic and catalytic properties of 40 kD active monomer has been described with a following discussion on the issue of potential of the data listed for further research in either toxic coagulopathy molecular mode or enzyme application dealing with "thrombin-thrombin receptor" interaction studies. An enzyme purification procedure includes hydrophobic (phenyl-Sepharose), anion exchange (MonoQ) and affinity (ADP-Sepharose) chromatographic steps.

A hydrophobic site on the surface of the angiotensin-converting enzyme molecule.

Using the hydrophobic fluorescent dye 8-anilino-1-naphthalenesulfonic acid (8-ANS), a hydrophobic site on the surface of the protein globule of angiotensin-converting enzyme (ACE) from bovine lung was found. The dissociation constant of the ACE-8-ANS complex was estimated as 1.5 +/- 0.2 microM. This hydrophobic site is far from the ACE catalytic sites because the binding of the hydrophobic dye does not influence ACE activity. Shielding of the ACE hydrophobic site due to the complex formation with 8-ANS or Triton X-100 resulted in pronounced stabilization of the enzyme against the action of water radiolysis products during gamma-irradiation of dilute solutions of ACE.

Tryptophanless recombinant horseradish peroxidase: stability and catalytic properties.

The tryptophanless mutant of horseradish peroxidase, W117F, has been constructed and expressed in Escherichia coli. The mutation affects enzyme folding and stability. The optimum composition of the refolding medium requires the presence of ammonium sulfate. The yield of mutant is ca. 8000 U per liter of the optimized refolding medium with the specific activity of 1100-1500 U/mg (compared to 25, 000 U per liter and 2000 U/mg for the recombinant wild-type enzyme). The mutant is more stable in acid media, in the reaction course and toward irradiation. The effect of hydrogen peroxide pretreatment on radiation-induced inactivation of the wild-type and mutant enzyme indirectly indicates participation of Trp-117 in electron transfer pathways through the enzyme molecule. This is in agreement with the steady-state kinetic data interpreted in terms of Trp-117 participation in electron transfer within the Michaelis complex.

[Radiation activation of angiotensin-converting enzyme during low-dose irradiation]. / Radiatsionnaia aktivatsiia angiotenzin-prevrashchaiushchego fermenta pri malykh dozakh oblucheniia.

Radiation activation of angiotensin-converting enzyme (respect to Cbz-Phe-His-Leu as substrate) was obtained at the gamma (137Cs, t(irr) = 10s-2h, D approximately 3 Gy)- and X (plasma foces source, t(irr) = 10(-9)s, Cu-filter, D approximately 2 x 10(-5) Gy)-irradiation. The inactivation of the horseradish peroxidase at the same X-irradiation dose (2 x 10(-5) Gy) took place. Based on the experimental data and on the mathematical model proposed by us we made a conclusion that the special points exist on the dose response curves. Besides, the deduction that an activation is a common process at radiation changes of the different enzymes follows from our model. The activation of tobacco peroxidase (in presence of Ca(2+)-cations and without them) and of recombinant horseradish peroxidase (in presence of H2O2) by gamma-irradiation was really observed (respect to guaiacol as substrate).

Wild-type and mutant forms of recombinant horseradish peroxidase C expressed in Escherichia coli. Substrate specificity and stability under irradiation.

Two horseradish peroxidase C (HRPC) mutants with substitutions in the active center, i.e., Phe41-->His and Phe143-->Glu, were compared to the wild-type recombinant enzyme expressed in Escherichia coli in terms of the enzymatic activity and stability under irradiation. Both mutations caused a significant decrease in activity, but it was still possible to follow the effect of mutations on the key steps of the reaction mechanism. Phe41 can be considered a nonpolar barrier separating histidine residues in the active center and providing a firm noncovalent binding with the highly hydrophobic porphyrin ring. The replacement of Phe41 with the ionizable His residue destabilizes the enzyme. The Phe143-->Glu replacement creates a negative charge at the entrance of the heme-binding pocket, and protects the latter from both donor substrates and free radicals. The radiolytic inactivation of the wild-type and mutant forms of recombinant HRP suggested different binding sites for iodide, 2,2'-bis(3-ethylbenzothiasoline-6-sulfonate (ABTS), guaiacol, and O-phenylene diamine. The study of kinetics and inactivation is in agreement with the direct binding of iodide to the heme porphyrin ring. The results also suggest that the ABTS binding site is less accessible than that for O-phenylene diamine.

[Evaluation of short-term hospitalization in the improvement of specialized aid to children with bronchial asthma]. / Rol'statsionara kratkovremennogo prebyvania v sovershenstvovanii spetsializirovannoi pomoshchi detiam s bronkhial'noi astmoi.

The programmes of allergological examination and individual bronchospasmolytic therapy in children with severe asthma are discussed. These programmes are being realized at specialized departments of allergology where patients stay for two or three days during day-time. The catamnestic efficacy of such programmes is illustrated.