RESUMEN
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.
Asunto(s)
Amputación Quirúrgica , Arteriopatías Oclusivas , Isquemia , Recuperación del Miembro , Adulto , Anciano , Amputación Quirúrgica/métodos , Amputación Quirúrgica/estadística & datos numéricos , Arteriopatías Oclusivas/complicaciones , Arteriopatías Oclusivas/epidemiología , Arteriopatías Oclusivas/cirugía , Arterias/patología , Enfermedad Crónica , Femenino , Humanos , Isquemia/clasificación , Isquemia/epidemiología , Isquemia/fisiopatología , Isquemia/cirugía , Recuperación del Miembro/métodos , Recuperación del Miembro/estadística & datos numéricos , Extremidad Inferior/irrigación sanguínea , Extremidad Inferior/fisiopatología , Masculino , Persona de Mediana Edad , Pronóstico , Medición de Riesgo , Factores de Riesgo , Federación de Rusia/epidemiología , Índice de Severidad de la EnfermedadRESUMEN
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.
Asunto(s)
Arteriosclerosis Obliterante/terapia , Terapias Complementarias , Aparatos de Compresión Neumática Intermitente/tendencias , Arteriosclerosis Obliterante/fisiopatología , Ensayos Clínicos como Asunto , Terapias Complementarias/instrumentación , Terapias Complementarias/métodos , Terapias Complementarias/tendencias , Humanos , Extremidad Inferior/irrigación sanguínea , Evaluación de Resultado en la Atención de Salud , Flujo Sanguíneo Regional , Terapias en Investigación/instrumentación , Terapias en Investigación/métodosRESUMEN
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.
Asunto(s)
Creatina Quinasa/química , Creatina Quinasa/efectos de la radiación , Magnesio/química , Venenos de Víboras/enzimología , Animales , Catálisis/efectos de la radiación , Conformación Proteica/efectos de la radiación , Radioisótopos/químicaRESUMEN
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.
Asunto(s)
Peptidil-Dipeptidasa A/química , Peptidil-Dipeptidasa A/efectos de la radiación , Zinc/química , Apoenzimas/química , Apoenzimas/efectos de la radiación , Cationes Bivalentes/química , Radioisótopos de Cesio , Estabilidad de Enzimas/efectos de la radiaciónRESUMEN
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.
Asunto(s)
Adenosina Trifosfato/biosíntesis , Fulerenos/farmacología , Magnesio/química , Magnetismo , Miocardio/metabolismo , Nanopartículas/química , Porfirinas/química , Animales , Descubrimiento de Drogas , Fulerenos/química , Fulerenos/metabolismo , Fulerenos/farmacocinética , Corazón/efectos de los fármacos , Concentración de Iones de Hidrógeno , Hipoxia/inducido químicamente , Hipoxia/metabolismo , Isótopos/química , Magnesio/metabolismo , Masculino , Microscopía de Fuerza Atómica , Microscopía Electrónica de Transmisión , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Miocardio/ultraestructura , Difracción de Neutrones , Oxígeno/metabolismo , Ratas , Ratas Wistar , Dispersión del Ángulo PequeñoRESUMEN
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.
Asunto(s)
Adenosina Trifosfato/biosíntesis , Magnesio/metabolismo , Mitocondrias Cardíacas/metabolismo , Niacinamida/análogos & derivados , Adenosina Difosfato/metabolismo , Animales , Cationes Bivalentes , Creatina Quinasa/antagonistas & inhibidores , Isótopos , Cinética , Masculino , Mitocondrias Cardíacas/enzimología , Niacinamida/farmacología , Oxidación-Reducción , Fosforilación Oxidativa , Ratas , Ratas Endogámicas SHRRESUMEN
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.
Asunto(s)
Adenosina Trifosfato/biosíntesis , Magnesio/farmacología , Mitocondrias Cardíacas/efectos de los fármacos , Animales , Creatina Quinasa/metabolismo , Masculino , Mitocondrias Cardíacas/enzimología , Mitocondrias Cardíacas/metabolismo , NAD/metabolismo , Ratas , Ratas Endogámicas SHRRESUMEN
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.
Asunto(s)
Enzimas/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Activación Enzimática , Enzimas/química , Enzimas/metabolismo , Hidrólisis , Conformación Proteica , Agua/químicaAsunto(s)
Adenosina Trifosfato/biosíntesis , Magnesio/química , Magnesio/farmacología , Magnetismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Adenosina Difosfato/metabolismo , Animales , Creatina Quinasa/metabolismo , Isótopos/química , Isótopos/farmacología , Mitocondrias/enzimología , Miocardio/citología , Fosforilación Oxidativa/efectos de los fármacos , RatasRESUMEN
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.
Asunto(s)
Peroxidasa de Rábano Silvestre/química , Proteínas Recombinantes/química , Aminoácidos/análisis , Estabilidad de Enzimas/efectos de la radiación , Rayos gamma , Peroxidasa de Rábano Silvestre/genética , Peroxidasa de Rábano Silvestre/aislamiento & purificación , Peroxidasa de Rábano Silvestre/efectos de la radiación , Conformación Proteica/efectos de la radiación , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/efectos de la radiación , TritioRESUMEN
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.
Asunto(s)
Membranas/efectos de la radiación , Peptidil-Dipeptidasa A/efectos de la radiación , Acetatos , Tampones (Química) , Relación Dosis-Respuesta en la Radiación , Estabilidad de Enzimas/efectos de la radiación , Rayos gamma , Concentración de Iones de Hidrógeno , Membranas/enzimología , Peptidil-Dipeptidasa A/química , FosfatosRESUMEN
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.
Asunto(s)
Peptidil-Dipeptidasa A/metabolismo , Secuencia de Aminoácidos , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Peptidil-Dipeptidasa A/química , Conformación Proteica , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Adenosina Trifosfato/biosíntesis , Creatina Quinasa/aislamiento & purificación , Venenos de Víboras/enzimología , Catálisis , Cromatografía de Afinidad , Cromatografía por Intercambio Iónico , Creatina Quinasa/metabolismo , CinéticaRESUMEN
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.
Asunto(s)
Interacciones Hidrofóbicas e Hidrofílicas , Peptidil-Dipeptidasa A/química , Peptidil-Dipeptidasa A/metabolismo , Naftalenosulfonatos de Anilina , Animales , Sitios de Unión , Bovinos , Colorantes Fluorescentes , Rayos gamma , Pulmón/enzimología , Octoxinol , Conformación Proteica/efectos de la radiación , Propiedades de Superficie , TermodinámicaRESUMEN
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.
Asunto(s)
Sustitución de Aminoácidos , Peroxidasa de Rábano Silvestre/metabolismo , Triptófano/química , Secuencia de Aminoácidos , Catálisis , Electrones , Activación Enzimática/efectos de la radiación , Estabilidad de Enzimas/efectos de la radiación , Escherichia coli/genética , Peroxidasa de Rábano Silvestre/química , Peroxidasa de Rábano Silvestre/genética , Peroxidasa de Rábano Silvestre/aislamiento & purificación , Peróxido de Hidrógeno/metabolismo , Concentración de Iones de Hidrógeno , Cuerpos de Inclusión , Mediciones Luminiscentes , Modelos Moleculares , Datos de Secuencia Molecular , Oxidación-Reducción , Plantas/enzimología , Pliegue de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Solubilidad , Triptófano/genéticaRESUMEN
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).
Asunto(s)
Peptidil-Dipeptidasa A/metabolismo , Animales , Bovinos , Activación Enzimática , Rayos gamma , Peroxidasa de Rábano Silvestre/metabolismo , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Peptidil-Dipeptidasa A/efectos de la radiación , Dosis de Radiación , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Peroxidasa de Rábano Silvestre/genética , Isoenzimas/genética , Sitios de Unión , Electroforesis en Gel de Poliacrilamida , Escherichia coli , Modelos Químicos , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/genética , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
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.