RESUMEN
In eukaryotes, RNA polymerase II (pol II) transcribes all protein-coding genes and many noncoding RNAs. Whereas many factors contribute to the regulation of pol II activity, the Mediator complex is required for expression of most, if not all, pol II transcripts. Structural characterization of Mediator is challenging due to its large size (â¼20 subunits in yeast and 26 subunits in humans) and conformational flexibility. However, recent studies have revealed structural details at higher resolution. Here, we summarize recent findings and place in context with previous results, highlighting regions within Mediator that are important for regulating its structure and function.
Asunto(s)
Complejo Mediador/fisiología , ARN Polimerasa II/genética , Animales , Microscopía por Crioelectrón , Cristalografía por Rayos X , Humanos , Complejo Mediador/química , Complejos Multiproteicos/química , Transcripción GenéticaRESUMEN
OXA-239 is a class D carbapenemase isolated from an Acinetobacter baumannii strain found in Mexico. This enzyme is a variant of OXA-23 with three amino acid substitutions in or near the active site. These substitutions cause OXA-239 to hydrolyze late-generation cephalosporins and the monobactam aztreonam with greater efficiency than OXA-23. OXA-239 activity against the carbapenems doripenem and imipenem is reduced â¼3-fold and 20-fold, respectively. Further analysis demonstrated that two of the substitutions (P225S and D222N) are largely responsible for the observed alteration of kinetic parameters, while the third (S109L) may serve to stabilize the protein. Structures of OXA-239 with cefotaxime, doripenem and imipenem bound as acyl-intermediates were determined. These structures reveal that OXA-239 has increased flexibility in a loop that contains P225S and D222N. When carbapenems are bound, the conformation of this loop is essentially identical with that observed previously for OXA-23, with a narrow active site that makes extensive contacts to the ligand. When cefotaxime is bound, the loop can adopt a different conformation that widens the active site to allow binding of that bulky drug. This alternate conformation is made possible by P225S and further stabilized by D222N. Taken together, these results suggest that the three substitutions were selected to expand the substrate specificity profile of OXA-23 to cephalosporins and monobactams. The loss of activity against imipenem, however, suggests that there may be limits to the plasticity of class D enzymes with regard to evolving active sites that can effectively bind multiple classes of ß-lactam drugs.
Asunto(s)
Acinetobacter baumannii/enzimología , Sustitución de Aminoácidos , Proteínas Bacterianas/química , Carbapenémicos/química , Cefotaxima/química , Imipenem/química , beta-Lactamasas/química , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Carbapenémicos/metabolismo , Carbapenémicos/farmacología , Dominio Catalítico , Cefotaxima/metabolismo , Cefotaxima/farmacología , Clonación Molecular , Cristalografía por Rayos X , Doripenem , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Imipenem/metabolismo , Imipenem/farmacología , Cinética , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , beta-Lactamasas/genética , beta-Lactamasas/metabolismoRESUMEN
Background: The goal of this study was to determine if there is an association between glenohumeral synovitis and early post-operative pain after arthroscopic rotator cuff repair. Methods: Fifty patients with symptomatic rotator cuff tears were prospectively enrolled prior to RCR. Baseline ASES score, VAS Pain score, forward elevation, and external rotation were recorded. Intra-operatively, synovitis was graded on a scale of zero to six as based on a previously validated scoring system. VAS Pain scores were obtained from patients post-operatively on days one through 14, week 6, and 3 months. Results: Average intra-operative synovitis score was 2.4 ± 1.6. No significant correlation was found between synovitis score and pre-operative forward elevation (P=0.171), external rotation (P=0.126), VAS Pain (P=0.623), or ASES (P=0.187) scores. No significant correlation was found between synovitis score and post-operative VAS Pain level at any time point. There was a moderate correlation between both pre-operative VAS Pain and ASES scores and post-operative VAS Pain in the first post-operative week. Workers' compensation patients had worse pain at 3 months post-operatively compared to non-workers compensation patients (P=0.038). Conclusion: This study reveals that macroscopically assessed glenohumeral synovitis does not have any significant correlation with pre-operative or post-operative pain in patients undergoing arthroscopic rotator cuff repair; although higher pre-operative pain levels, worse pre-operative ASES scores, and workers compensation status do influence post-operative pain levels in arthroscopic rotator cuff repair.