RESUMEN

Endolysins are bacteriophage (or phage)-encoded enzymes that catalyse the peptidoglycan breakdown in the bacterial cell wall. The exogenous action of recombinant phage endolysins against Gram-positive organisms has been extensively studied. However, the outer membrane acts as a physical barrier when considering the use of recombinant endolysins to combat Gram-negative bacteria. This study aimed to evaluate the antimicrobial activity of the SAR-endolysin LysKpV475 against Gram-negative bacteria as single or combined therapies, using an outer membrane permeabilizer (polymyxin B) and a phage, free or immobilized in a pullulan matrix. In the first step, the endolysin LysKpV475 in solution, alone and combined with polymyxin B, was tested in vitro and in vivo against ten Gram-negative bacteria, including highly virulent strains and multidrug-resistant isolates. In the second step, the lyophilized LysKpV475 endolysin was combined with the phage phSE-5 and investigated, free or immobilized in a pullulan matrix, against Salmonella enterica subsp. enterica serovar Typhimurium ATCC 13311. The bacteriostatic action of purified LysKpV475 varied between 8.125 µg ml-1 against Pseudomonas aeruginosa ATCC 27853, 16.25 µg ml-1 against S. enterica Typhimurium ATCC 13311, and 32.50 µg ml-1 against Klebsiella pneumoniae ATCC BAA-2146 and Enterobacter cloacae P2224. LysKpV475 showed bactericidal activity only for P. aeruginosa ATCC 27853 (32.50 µg ml-1) and P. aeruginosa P2307 (65.00 µg ml-1) at the tested concentrations. The effect of the LysKpV475 combined with polymyxin B increased against K. pneumoniae ATCC BAA-2146 [fractional inhibitory concentration index (FICI) 0.34; a value lower than 1.0 indicates an additive/combined effect] and S. enterica Typhimurium ATCC 13311 (FICI 0.93). A synergistic effect against S. enterica Typhimurium was also observed when the lyophilized LysKpV475 at ⅔ MIC was combined with the phage phSE-5 (m.o.i. of 100). The lyophilized LysKpV475 immobilized in a pullulan matrix maintained a significant Salmonella reduction of 2 logs after 6 h of treatment. These results demonstrate the potential of SAR-endolysins, alone or in combination with other treatments, in the free form or immobilized in solid matrices, which paves the way for their application in different areas, such as in biocontrol at the food processing stage, biosanitation of food contact surfaces and biopreservation of processed food in active food packing.

Asunto(s)

Antibacterianos , Endopeptidasas , Glucanos , Polimixina B , Fagos de Salmonella , Endopeptidasas/farmacología , Endopeptidasas/química , Endopeptidasas/metabolismo , Polimixina B/farmacología , Antibacterianos/farmacología , Antibacterianos/química , Fagos de Salmonella/genética , Fagos de Salmonella/fisiología , Fagos de Salmonella/química , Glucanos/química , Glucanos/farmacología , Animales , Pruebas de Sensibilidad Microbiana , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Gramnegativas/virología , Ratones , Salmonella typhimurium/virología , Salmonella typhimurium/efectos de los fármacos , Bacteriófagos/fisiología , Bacteriófagos/genética , Proteínas Virales/genética , Proteínas Virales/metabolismo , Proteínas Virales/farmacología , Proteínas Virales/química

RESUMEN

Chlorite dismutase (Cld) catalyzes the reduction of chlorite to chloride and dioxygen. Here, the ligand binding to Cld of Magnetospirillum sp. (MaCld) is investigated with X-ray crystallography and electron paramagnetic resonance (EPR). EPR reveals a large heterogeneity in the structure of wild-type MaCld, showing a variety of low- and high-spin ferric heme forms. Addition of an axial ligand, such as azide or imidazole, removes this heterogeneity almost entirely. This is in line with the two high resolution crystal structures of MaCld obtained in the presence of azide and thiocyanate that show the coordination of the ligands to the heme iron. The crystal structure of the MaCld-azide complex reveals a single well-defined orientation of the azide molecule in the heme pocket. EPR shows, however, a pH-dependent heme structure, probably due to acid-base transitions of the surrounding amino-acid residues stabilizing azide. For the azide and imidazole complex of MaCld, the hyperfine and nuclear quadrupole interactions with the close-by (14)N and (1)H nuclei are determined using pulsed EPR. These values are compared to the corresponding data for the low-spin forms observed in the ferric wild-type MaCld and to existing EPR data on azide and imidazole complexes of other heme proteins.

Asunto(s)

Azidas/química , Imidazoles/química , Magnetospirillum/enzimología , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Tiocianatos/química , Sitios de Unión , Cristalografía por Rayos X , Espectroscopía de Resonancia por Spin del Electrón , Concentración de Iones de Hidrógeno , Ligandos , Modelos Moleculares , Oxidorreductasas/aislamiento & purificación

RESUMEN

Desulfovibrio gigas aldehyde oxidoreductase (DgAOR) is a mononuclear molybdenum-containing enzyme from the xanthine oxidase (XO) family, a group of enzymes capable of catalyzing the oxidative hydroxylation of aldehydes and heterocyclic compounds. The kinetic studies reported in this work showed that DgAOR catalyzes the oxidative hydroxylation of aromatic aldehydes, but not heterocyclic compounds. NMR spectroscopy studies using (13)C-labeled benzaldehyde confirmed that DgAOR catalyzes the conversion of aldehydes to the respective carboxylic acids. Steady-state kinetics in solution showed that high concentrations of the aromatic aldehydes produce substrate inhibition and in the case of 3-phenyl propionaldehyde a suicide substrate behavior. Hydroxyl-substituted aromatic aldehydes present none of these behaviors but the kinetic parameters are largely affected by the position of the OH group. High-resolution crystallographic structures obtained from single crystals of active-DgAOR soaked with benzaldehyde showed that the side chains of Phe425 and Tyr535 are important for the stabilization of the substrate in the active site. On the other hand, the X-ray data of DgAOR soaked with trans-cinnamaldehyde showed a cinnamic acid molecule in the substrate channel. The X-ray data of DgAOR soaked with 3-phenyl propionaldehyde showed clearly how high substrate concentrations inactivate the enzyme by binding covalently at the surface of the enzyme and blocking the substrate channel. The different reactivity of DgAOR versus aldehyde oxidase and XO towards aromatic aldehydes and N-heterocyclic compounds is explained on the basis of the present kinetic and structural data.

Asunto(s)

Aldehído Oxidorreductasas/química , Aldehídos/química , Desulfovibrio gigas/enzimología , Conformación Proteica , Aldehído Oxidorreductasas/metabolismo , Sitios de Unión , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Cinética , Molibdeno/química , Resonancia Magnética Nuclear Biomolecular , Oxidación-Reducción , Unión Proteica , Especificidad por Sustrato

RESUMEN

One of the known complications of gallstones consists in gallbladder perforation with cholecystocutaneous abscesses or external fistulas formation. Nowadays these types of complications are uncommon, having been more frequent. The authors report the case of an 86 year-old woman, bedridden and dependent, referred to our institution by presenting a tumefaction with inflammatory signs in the right upper quadrant. The radiological investigation revealed it to be a subcutaneous abscess associated with a perforated gallbladder, whose histological analysis revealed xanthogranulomatous cholecystitis.

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Uma das complicações conhecidas da litíase vesicular consiste na perfuração da vesícula com consequente formação de abcessos colecisto-cutâneos ou trajectos fistulosos externos. Actualmente este tipo de complicação é pouco frequente na prática diária, tendo sido mais frequente. Os autores relatam o caso de uma senhora de 86 anos, acamada e dependente, referenciada à nossa instituição por apresentar uma tumefacção no quadrante superior direito, com sinais inflamatórios associados. A investigação imagiológica da referida tumefacção revelou tratar-se de um abcesso subcutâneo associado a perfuração da vesicula biliar, cuja análise histológica revelou colecistite xantogranulomatosa.

Asunto(s)

Absceso/etiología , Colecistitis/complicaciones , Enfermedades del Tejido Conjuntivo/etiología , Granuloma/complicaciones , Tejido Subcutáneo , Xantomatosis/complicaciones , Anciano de 80 o más Años , Femenino , Humanos

RESUMEN

OBJECTIVES: To review the most common disorders depicted with conventional videodefecography, and to compare the defecographic abnormalities between symptomatic patients according to their gender and age. METHODS: Conventional videodefecography studies of 300 patients (24 men, 266 women; mean age - 57.7) performed in a 32-month period were reviewed for the following parameters: anorectal angle, movement of the pelvic floor, intussusceptions, incontinence and rectocele. The results were analyzed using the chi-square test. RESULTS: Normal findings were observed in 16.7% men and 7.5% women. In women, the most frequent pathological findings were rectocele (62%), descending perineum syndrome (42.8%), intussusceptions (33.8%), incontinence (10.5%), dyskinetic puborectalis syndrome (9.4%) and rectal prolapse (4.5%); in men the most frequent pathology was the dyskinetic puborectalis syndrome (37.5%). This syndrome is more likely in men than in women (p = 0.01; OR 5.78); descending perineum syndrome (p = 0.027; OR 2.8) is more likely to occur in women. Women with perineal descent younger than 50 years frequently present an increased descent during evacuation (81.8%), while those older than 50 years already have a low pelvic floor during rest (60.3%) (p < 0.001; OR 6.8), with little change in evacuation. CONCLUSION: Videodefecographic findings vary with age and gender.

RESUMEN

Mononuclear Mo-containing enzymes of the xanthine oxidase (XO) family catalyze the oxidative hydroxylation of aldehydes and heterocyclic compounds. The molybdenum active site shows a distorted square-pyramidal geometry in which two ligands, a hydroxyl/water molecule (the catalytic labile site) and a sulfido ligand, have been shown to be essential for catalysis. The XO family member aldehyde oxidoreductase from Desulfovibrio gigas (DgAOR) is an exception as presents in its catalytically competent form an equatorial oxo ligand instead of the sulfido ligand. Despite this structural difference, inactive samples of DgAOR can be activated upon incubation with dithionite plus sulfide, a procedure similar to that used for activation of desulfo-XO. The fact that DgAOR does not need a sulfido ligand for catalysis indicates that the process leading to the activation of inactive DgAOR samples is different to that of desulfo-XO. We now report a combined kinetic and X-ray crystallographic study to unveil the enzyme modification responsible for the inactivation and the chemistry that occurs at the Mo site when DgAOR is activated. In contrast to XO, which is activated by resulfuration of the Mo site, DgAOR activation/inactivation is governed by the oxidation state of the dithiolene moiety of the pyranopterin cofactor, which demonstrates the non-innocent behavior of the pyranopterin in enzyme activity. We also showed that DgAOR incubation with dithionite plus sulfide in the presence of dioxygen produces hydrogen peroxide not associated with the enzyme activation. The peroxide molecule coordinates to molybdenum in a η(2) fashion inhibiting the enzyme activity.

Asunto(s)

Aldehído Oxidorreductasas/química , Aldehído Oxidorreductasas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Desulfovibrio gigas/enzimología , Aldehído Oxidorreductasas/antagonistas & inhibidores , Animales , Proteínas Bacterianas/antagonistas & inhibidores , Bovinos , Cristalografía por Rayos X , Espectroscopía de Resonancia por Spin del Electrón , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Peróxido de Hidrógeno/farmacología , Cinética , Ligandos , Modelos Moleculares , Conformación Proteica , Xantina Oxidasa/antagonistas & inhibidores , Xantina Oxidasa/química , Xantina Oxidasa/metabolismo

RESUMEN

INTRODUCTION: Pulmonary angiography by computed tomography (CT) is the method of choice for the detection of acute pulmonary embolism (PE). Studies have shown that the severity of PE can be estimated by clot burden scores. OBJECTIVE: To evaluate the correlation between an angiographic clot burden score (Qanadli score - QS) and parameters of right ventricular dysfunction (RVD) in patients admitted for PE. METHODS: We performed a retrospective study of 107 patients (60% female) admitted to an intensive care unit for PE (intermediate/high risk) between January 1, 2007 and September 30, 2011. Images from 16-slice multidetector CT angiography were reviewed in 102 patients and the QS calculated. Based on a cut-off of 18 points established by ROC curve analysis, two groups were formed (A<18 points vs. B ≥18 points) and the clinical, laboratory, ECG, echocardiographic and CT angiography parameters were compared. The statistical analysis was performed using SPSS. RESULTS: The overall mean age was 61.4 years. With regard to symptoms at admission, there was a greater prevalence in group B of fatigue, chest pain and syncope (p=0.017), with higher Geneva and Wells scores and shock index. In terms of ECG parameters, heart rate and percentage of right bundle branch block, T-wave inversion (V(1)-V(3)) and S(1)Q(3)T(3) pattern (p=0.034) were higher in group B, as was the ECG score (p=0.009). Laboratory tests revealed that group B had higher troponin and d-dimers, with lower creatinine clearance by the MDRD formula (p=0.020) and PO(2)/FiO(2) ratio. Echocardiography showed higher pulmonary artery systolic pressure in group B, and CT angiography revealed larger right ventricular (RV) diameters and higher RV/LV ratio (p=0.002), and greater superior vena cava, azygos vein and coronary sinus diameters in this group. Pulmonary artery (PA) diameter and the PA/aorta ratio were similar. Interventricular septal bowing and reflux of contrast into the inferior vena cava (p=0.001) were greater in group B, and QS>18 was an independent predictor of RVD (RV/LV ratio>1) (OR: 10.85; p<0.001) (area under the curve on ROC analysis: 0.79; p<0.001). The percentage of patients receiving fibrinolytic treatment was higher in group B (p=0.045), and in-hospital mortality was similar in both groups (overall 4.9%). CONCLUSIONS: QS >18 points proved to be an independent predictor of RVD in PE, and correlated linearly with variables associated with higher morbidity and mortality.

Asunto(s)

Embolia Pulmonar/complicaciones , Embolia Pulmonar/diagnóstico por imagen , Disfunción Ventricular Derecha/etiología , Enfermedad Aguda , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Radiografía , Estudios Retrospectivos , Adulto Joven

RESUMEN

In alkaptonuria, deficiency of homogentisate 1,2-dioxygenase leads to the accumulation of homogentisic acid (HGA) and its metabolites in the body, resulting in ochronosis. Reports of patients with alkaptonuria who have decreased kidney function are rare, but this seems to play an important role in the natural history of the disease. We describe a 68-year-old female with chronic kidney disease (CKD) of unknown etiology who started peritoneal dialysis (PD) after 5 years of follow-up and who was diagnosed with alkaptonuria at this time. Progressive exacerbation of ochronotic manifestations had been noted during these last few years, as kidney function worsened. After PD initiation, the disease continued to progress, and death occurred after one year and a half, due to severe aortic stenosis-related complications. Her 70-year-old sister was evaluated and also diagnosed with alkaptonuria. She had no renal dysfunction. Higher HGA excretion and significantly milder ochronosis than that of her sister were found. We present two alkaptonuric sisters with similar comorbidities except for the presence of CKD, who turned out to have totally different evolutions of their disease. This report confirms that kidney dysfunction may be an important factor in determining the natural history of alkaptonuria.

RESUMEN

A 65-year-old woman with a dual-chamber pacemaker implanted in 2006 for symptomatic carotid sinus hypersensitivity was incidentally found to have loss of ventricular capture on routine pacemaker interrogation. A chest X-ray raised the suspicion of perforation and migration of the right ventricular lead, confirmed by three-dimensional echocardiogram and CT scan. On the basis of this case, we review myocardial lead perforation, including predisposing factors, pathophysiological mechanisms, diagnostic approach and therapeutic options.

Asunto(s)

Falla de Equipo , Migración de Cuerpo Extraño/diagnóstico , Migración de Cuerpo Extraño/etiología , Lesiones Cardíacas/diagnóstico , Lesiones Cardíacas/etiología , Marcapaso Artificial , Anciano , Femenino , Humanos

RESUMEN

Computed tomographic andiography has becoming the main diagnostic imaging method for the evaluation of patients with suspected pulmonary embolism (PE), due to its high sensitivity and specificity and potential cost-effectiveness. The advances in multidetector computed tomography (MDCT) scanners and optimization of scanning protocols via thinner collimation and faster scanning times have greatly improved the ability to detect PE. The diagnosis is made by direct visualization of a low attenuation filling defect (intraluminal thrombus) that partially or completely occludes a contrast filled artery. Differentiate between acute and chronic PE is often possible through the identification of characteristic findings, such as the diameter of the affected artery and the location and relationship of the thrombus to the vessel walls. Awareness of the major error factors in the CT diagnosis of PE can help further increase the sensitivity and specificity of this imaging method.

Asunto(s)

Angiografía/métodos , Embolia Pulmonar/diagnóstico , Tomografía Computarizada por Rayos X/métodos , Enfermedad Aguda , Angiografía/economía , Medios de Contraste , Análisis Costo-Beneficio , Humanos , Tomografía Computarizada Multidetector/economía , Tomografía Computarizada Multidetector/métodos , Embolia Pulmonar/fisiopatología , Sensibilidad y Especificidad , Tomografía Computarizada por Rayos X/economía