Search

Resumen Objetivo Identificar los factores asociados con el mal control de la anticoagulacion con antagonistas de la vitamina K (AVK) en pacientes con fibrilacion auricular no valvular (FANV) atendidos en consultas de Neurologia y Medicina Interna de Espana. Metodos Estudio transversal, multicentrico, anidado en el estudio ALADIN, de sujetos con FANV, tratados con AVK, atendidos en consultas de Medicina Interna o Neurologia de Espana. El grado de control de la anticoagulacion se estudio mediante el metodo directo y el de Rosendaal, considerando los 6 meses previos a la inclusion. Resultados De los 1.337 pacientes incluidos en ALADIN, 750 estaban tratados con AVK, con informacion completa sobre el INR de los ultimos 6 meses en 383 pacientes. Las puntuaciones medias del indice de Charlson, CHADS2, CHA2DS2-VASc y HAS-BLED fueron 1,94 ± 1,54; 3,10 ± 1,26; 4,63 ± 1,54 y 2,20 ± 0,90, respectivamente. El 46,2% y el 47,0% de los pacientes presentaban un control adecuado de la anticoagulacion por los metodos directo y Rosendaal, respectivamente. El control inadecuado de la anticoagulacion se asocio por el metodo directo con diabetes (OR: 2,511; IC 95%: 1,144-5,659), antecedentes de INR inestable (OR: 35,371; IC 95%: 15,058-83,083) y la realizacion de > 6 controles en los ultimos 6 meses (OR: 4,747; IC 95%: 2,094-10,759), y por el metodo de Rosendaal, con los antecedentes de INR inestable (p Conclusiones Pese al alto riesgo tromboembolico, solo estaban bien controlados algo mas de la mitad. Los factores asociados al mal control de la anticoagulacion fueron la diabetes, INR inestable, > 6 controles de INR y el HAS-BLED.

Intrapulmonary vascular dilatations (IPVD) are considered a complication of cirrhosis. The technique of choice for their diagnosis is contrast-enhanced echocardiography (CEE). The aim of this study was to determine the usefulness of contrast-enhanced transcranial Doppler (CETD) in the diagnosis of IPVD.We consecutively included patients evaluated for liver transplantation. A cross-sectional study was conducted. The investigator interpreting CETD was blind to the results of the gold standard (CEE). The accuracy of the diagnostic test was evaluated through sensitivity, specificity, positive and negative predictive values, and likelihood ratio.CETD (n=43) showed a right-to-left shunt in 23 patients (62.2%): 4 early, 2 indeterminate and 17 late. Nineteen (51,4%) cases were classified as IPVD. With CEE (n=37), 10 procedures (27%) were negative for shunt, 27 (73%) were positive, and 21 (56.8%) were compatible with IPVD. Patients with and without IPVD showed no differences in age, sex, etiology, severity, or MELD score, independently of the diagnostic test. In the diagnostic validity study (n=37) of CETD versus CEE, the AUC for diagnostic yield was 0.813% (95%CI: 0.666-0.959; P=.001), sensitivity was 76.2% (95%CI: 54.9-89.4) and specificity was 90% (95%CI: 63.9-96.5). The positive likelihood ratio was 6.095.We found a high prevalence of IPVD in candidates for liver transplantation. When a late right-to-left shunt with recirculation is observed, CETD has a high probability of detecting IPVD, with few false-positive results. Because this technique has not previously been described in this indication, similar studies are needed for comparison.

The regulation of the pac gene encoding the penicillin G acylase of Escherichia coli W ATCC 11015 has been investigated by a molecular approach using lacZ as a reporter gene. This analysis revealed that a region of 170 bp located upstream of the pac structural gene contains the regulatory sequences that control its expression. The cAMP receptor protein is involved not only in the catabolite repression of penicillin G acylase production caused by glucose but also in the induction of pac gene expression by phenylacetic acid. Primer extension analyses have demonstrated that the transcription of the pac gene can be initiated from at least three different promoters. Although all these promoters are functional, their relative activity depends on the transcribed gene, the P1 and P3 promoters being more active in the presence of the pac gene, whereas the P2 promoter was stronger when the upstream region of the pac gene was fused to the lacZ reporter. A deletion of the region surrounding the -10 box of the P3 promoter produced a constitutive expression of the fused gene indicating that this sequence is required for phenylacetic acid induction and suggesting that the expression of the pac gene is regulated by a repression mechanism. This work reveals that the regulation of the pac gene is more complex than previously envisioned and provides new clues to investigate further this interesting regulatory system.

In contrast with the general thought that penicillin G acylases (PGAs) were only able to hydrolyse amides or esters of higly hydrophobic acids, we have demonstrated that the PGA from Kluyvera citrophila catalysed the hydrolysis of 4-nitrophenyl esters of acetic, propionic, butyric and valeric acids. Values of kcat. and kcat./Km were greatest for the first compound and less than values for benzylpenicillin by factors of 30 and 7, respectively. 4-Nitrophenyl acetate was hydrolysed faster than 2-nitrophenyl acetate but slower than phenyl acetate. The pH dependence of the reaction was similar to that of benzylpenicillin. Several experiments showed that hydrolysis of 4-nitrophenyl acetate was not catalysed by contaminating esterase activity. The implications for the structural basis of substrate binding are discussed. These substrates open, for the first time, a way to investigate the kinetic parameters of PGA at the presteady-state and provides a new perspective about the role of PGA in nature.

The nucleotide sequence of the 5′-terminal region of the pac gene encoding the penicillin G acylase from Kluyvera citrophila ATCC 21285 has been determined. The transcriptional start site has been identified by primer extension analysis in a different position to that previously found for the homologous pac gene of Escherichia coli W ATCC 11105. Two nucleotide changes in the −35 box appear to be responsible of the promoter displacement in K. citrophila. A putative upstream promoter element (A+T-rich enhancer sequence) and a binding site for the cAMP receptor protein (CRP) were located upstream of the −35 box. Transcriptional lacZ and cat fusions demonstrated that pac expression was subjected to catabolite repression mediated by cAMP and its receptor protein. Remarkably, phenylacetic acid which is a potent inducer of the penicillin G acylase from E. coli, was only able to cause a significant induction of the pac expression in CRP+ cells cultured in the presence of glucose, suggesting that this effect is CRP-dependent.

Abbreviation used: NIPAB, N-(3-carboxy-4-nitrophenyl)-phenylacetamide. PMID: 8687381, The kinetics of release of 4-nitrophenol were followed by stopped-flow spectrophotometry with two 4-nitrophenyl ester substrates of penicillin G acylase from Kluyvera citrophila. With the ester of acetic acid, but not of propionic acid, there was a pre-steady-state exponential phase, the kinetics of which were inhibited by phenylacetic acid (a product of hydrolysis of specific substrates) to the extent predicted from Ki values. This was interpreted as deriving from rapid formation (73 mM-1.s-1) and slow hydrolysis (0.76 s-1) of an acetyl derivative of the side chain of the catalytic-centre residue Ser-290. With the mutant F360V, which differs from the wild-type enzyme in its ability to hydrolyse adipyl-L-leucine and has a kcat for 4-nitrophenyl acetate one-twentieth that of the wild-type enzyme, the corresponding values for the rates of formation and hydrolysis of the acetyl-enzyme were 11.1 mM-1.s-1 and 0.051 s-1 respectively. The ratio of these rate constants was three times that for the wild-type enzyme, suggesting that the mutant is less impaired in the rate of formation of an acetyl-enzyme than in its subsequent hydrolysis., The work at Newcastle upon Tyne was supported by the Biotechnology and Biological Sciences Research Council. A. R. was supported by a pre-doctoral fellowship from the Ministerio de Educación y Ciencia.

Escherichia coli (muT, mutD, Leu-) cells transformed with plasmid pYKD59 harbouring the pac gene encoding penicillin acylase (PA) from Kluyvera citrophila ATCC 21285 were exposed to environmental conditions that made expression of this enzyme essential for growth. Under these conditions, spontaneous mutants were isolated that used adipyl-L-leucine as the sole source of L-leucine. DNA sequencing of the mutant pac genes identified a transversion mutation of thymine to guanine at position 1163. This mutation was located in the beta-subunit of the enzyme and resulted in conversion of Phe-360 to valine. The assignment of this mutation to the shift in substrate specificity was further confirmed by site-directed mutagenesis. Secondary-structure prediction of the region surrounding Phe-360 suggests that this mutation should not produce any significant structural change. The purified mutant acylase was able to hydrolyse adipyl-, glutaryl-, valeryl-, caproyl-, heptanoyl- and phenoxyacetyl-L-leucine at pH 5 with greater efficiency than the wild-type enzyme. However, the mutant enzyme was not able to hydrolyse glutaryl-7-aminocephalosporanic acid and had lost 90% and 50% of activity on penicillin G and phenylacetyl-L-leucine respectively. Nevertheless, mutant PA retained its original activity on 6-nitro-3-phenylacetamidobenzoate and p-nitrophenylphenylacetate, suggesting that the binding specificity of PA by the acyl and amine moieties of the substrate are not independent phenomena. The small differences observed between the c.d. spectra of the mutant enzyme recorded at pH 5 and 8 suggest the existence of different conformational states at the two pH values, but these differences were indistinguishable from those observed in the native enzyme and cannot be correlated with the shift in substrate specificity. Our results demonstrate that it is possible to change the specificity of PA by laboratory evolution and use it to identify the amino acids involved in substrate recognition. However, the synchronous participation of the alpha- and beta-subunits in the complex induced-fit-like mechanism of acylases suggests that, to obtain new enzymes for industrial application, the selection pressure should be specifically designed for the compound of interest.