RESUMEN
The RNA world hypothesis regarding the early evolution of life relies on the premise that some RNA sequences can catalyze RNA replication. In support of this conjecture, we describe here an RNA molecule that catalyzes the type of polymerization needed for RNA replication. The ribozyme uses nucleoside triphosphates and the coding information of an RNA template to extend an RNA primer by the successive addition of up to 14 nucleotides-more than a complete turn of an RNA helix. Its polymerization activity is general in terms of the sequence and the length of the primer and template RNAs, provided that the 3' terminus of the primer pairs with the template. Its polymerization is also quite accurate: when primers extended by 11 nucleotides were cloned and sequenced, 1088 of 1100 sequenced nucleotides matched the template.
Asunto(s)
ARN Catalítico/metabolismo , ARN Polimerasa Dependiente del ARN/metabolismo , ARN/biosíntesis , Secuencia de Bases , Secuencia Conservada/genética , Evolución Molecular Dirigida , Datos de Secuencia Molecular , Mutagénesis/genética , Conformación de Ácido Nucleico , ARN/genética , ARN Catalítico/química , ARN Catalítico/genética , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/genética , Análisis de Secuencia de ARN , Especificidad por Sustrato , Moldes GenéticosRESUMEN
The present objective was to determine whether hydrogen peroxide (H(2)O(2)) increases transvascular albumin clearance and lung weight in an isolated rat lung and whether posttreatment with cAMP-enhancing agents can prevent these increases. Transvascular albumin clearance was assessed by (125)I-labeled albumin clearance ((125)I-albumin flux/perfusate concentration of (125)I-albumin) at a given fluid filtration. Nonlinear regression analysis of transvascular albumin clearance vs. fluid filtration yielded values for the permeability-surface area product (PS) and the reflection coefficient (sigma). H(2)O(2) decreased sigma from a control value of 0.93 to 0.38, did not change PS, and increased lung weight. Posttreatment with isoproterenol, a beta(2)-adrenergic-receptor agonist, reduced the H(2)O(2)-induced decrease in sigma to 0.65 and augmented the increase in lung weight. Posttreatment with CP-80633, a phosphodiesterase 4 inhibitor, further reduced the H(2)O(2)-induced decrease in sigma to 0.79 and blocked the rise in lung weight. In the presence of isoproterenol or CP-80633, H(2)O(2) increased PS. Therefore, H(2)O(2) increased the convective and diffusive clearances of albumin across an intact pulmonary vasculature. Furthermore, inhibition of cAMP metabolism more effectively attenuated the H(2)O(2)-induced increases in convective albumin clearance and lung weight as compared with stimulation of cAMP production.
Asunto(s)
Permeabilidad Capilar/efectos de los fármacos , AMP Cíclico/biosíntesis , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiología , Peróxido de Hidrógeno/toxicidad , Inhibidores de Fosfodiesterasa/farmacología , Agonistas Adrenérgicos beta/farmacología , Animales , Quelantes/farmacología , Deferoxamina/farmacología , Técnicas In Vitro , Isoproterenol/farmacología , Pulmón/irrigación sanguínea , Pulmón/efectos de los fármacos , Pulmón/patología , Masculino , Tamaño de los Órganos/efectos de los fármacos , Perfusión , Pirimidinonas/farmacología , Ratas , Ratas Sprague-Dawley , Albúmina Sérica Bovina/metabolismoRESUMEN
The class I RNA ligase ribozyme, isolated previously from random sequences, performs an efficient RNA ligation reaction. It ligates two substrate RNAs, promoting the attack of the 3'-hydroxyl of one substrate upon the 5'-triphosphate of the other substrate with release of pyrophosphate. This ligation reaction has similarities to the reaction catalyzed by RNA polymerases. Using data from steady-state kinetic measurements and pulse-chase/pH-jump experiments, we have constructed minimal kinetic frameworks for two versions of the class I ligase, named 207t and 210t. For both ligases, as well as for the self-ligating parent ribozyme, the rate constant for the chemical step (k(c)) is log-linear with pH in the range 5.7-8.0. At physiological pH, the k(c) is 100 min(-1), a value similar to those reported for the fastest naturally occurring ribozymes. At higher pH, product release is limiting for both 207t and 210t. The 210t ribozyme, with its faster product release, attains multiple-turnover rates (k(cat) = 360 min(-1), pH 9.0) exceeding those of 207t and other reported ribozyme reactions. The kinetic framework for the 210t ribozyme describes the limits of this catalysis and suggests how key steps can be targeted for improvement using design or combinatorial approaches.
Asunto(s)
ARN Ligasa (ATP)/química , ARN Catalítico/química , Sitios de Unión , Catálisis , Simulación por Computador , Difosfatos/química , Difosfatos/metabolismo , Activación Enzimática , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Modelos Químicos , Ácidos Fosfóricos/metabolismo , ARN Ligasa (ATP)/antagonistas & inhibidores , ARN Ligasa (ATP)/metabolismo , ARN Catalítico/metabolismo , Especificidad por SustratoRESUMEN
Of 64 complete iliac obstructions, 50 (78%) were recanalized using the Gruntzig balloon catheter. Life-table analysis of the patency rate over a 4-year period gives a cumulative success rate of 78%. Only three of eight obstructions that involved both the common and external iliac arteries were successfully dilated. The only serious complication was distal embolization, which occurred in two cases (3.1%). Although the procedure is difficult, with a relatively low technical success rate, the high cumulative patency rate should make it an option in the treatment of all patients with totally occluded iliac artery segments.