ABSTRACT
The availability of enzymes with a high promiscuity/specificity relationship permits the hydrolysis of several substrates with a view to obtaining a certain product or using one enzyme for several productive lines. N-Carbamoyl-ß-alanine amidohydrolase from Agrobacterium tumefaciens (Atßcar) has shown high versatility to hydrolyze different N-carbamoyl-, N-acetyl- and N-formyl-amino acids to produce different α, ß, γ and δ amino acids. We have calculated the promiscuity index for the enzyme, obtaining a value of 0.54, which indicates that it is a modestly promiscuous enzyme. Atßcar presented the highest probability of hydrolysis for N-carbamoyl-amino acids, being the enzyme more efficient for the production of α-amino acids. We have also demonstrated by mutagenesis, modelling, kinetic and binding experiments that W218 and A359 indirectly influence the plasticity of the enzyme due to interaction with the environment of R291, the key residue for catalytic activity.
Subject(s)
Agrobacterium tumefaciens/enzymology , Amidohydrolases/chemistry , Amidohydrolases/metabolism , Amino Acids/biosynthesis , beta-Alanine/metabolism , Agrobacterium tumefaciens/genetics , Chromatography, High Pressure Liquid , Circular Dichroism , Elasticity , Escherichia coli/genetics , Escherichia coli/metabolism , Kinetics , Models, Molecular , Mutagenesis, Site-Directed , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Stereoisomerism , Substrate Specificity , Thermodynamics , beta-Alanine/chemistryABSTRACT
The angiotensin I-converting enzyme (ACE; EC.3.4.15.1) is a dipeptidyl carboxypeptidase that plays a central role in blood pressure regulation. The somatic form of the enzyme is composed of two highly similar domains, usually referred to as N and C domains, each containing one active site. Nevertheless, a 1:1 stoichiometry for the binding of lisinopril, captopril or enalaprilat to somatic pig lung ACE is shown by isothermal titration calorimetry (ITC) and enzymatic assays. The binding of the three inhibitors at neutral pH is very tight and the enthalpy changes are positive, indicating that the binding is entropically driven. The origin of this thermodynamic signature is discussed under the new structural information available.
Subject(s)
Angiotensin-Converting Enzyme Inhibitors/metabolism , Captopril/metabolism , Enalaprilat/metabolism , Lisinopril/metabolism , Lung/enzymology , Peptidyl-Dipeptidase A/metabolism , Angiotensin-Converting Enzyme Inhibitors/pharmacology , Animals , Calorimetry , Captopril/pharmacology , Enalaprilat/pharmacology , Lisinopril/pharmacology , Swine , ThermodynamicsABSTRACT
Angiotensin I converting enzyme (ACE) plays a major role in blood pressure regulation, catalyzing the conversion of angiotensin I to the vasoconstrictor angiotensin II. In this report we describe a two-step affinity chromatography method for preparative purification of ACE from pig lung using Concanavalin-A Sepharose 4B and affinity chromatography on Lisinopril Sepharose 6B. The same purification scheme was used to obtain Cobalt-ACE, where zinc ion located at the active site is replaced by cobalt. Cobalt-ACE visible spectrum shows a characteristic broad peak from 500 to 600 nm. The shape and maximum absorptivity of this peak changes in presence of ACE inhibitors that bind at the catalytic site.
Subject(s)
Chromatography, Affinity/methods , Chromatography, Gel/methods , Lung/enzymology , Peptidyl-Dipeptidase A/isolation & purification , Animals , Electrophoresis, Polyacrylamide Gel , Molecular Weight , Peptidyl-Dipeptidase A/chemistry , Spectrophotometry, Ultraviolet , SwineABSTRACT
Objetivo/métodos: Presentamos un caso de hemangiopericitoma orbitario en un muchacho de 14 años. Se detallan los hallazgos radiológicos de la tomografía axial computerizada (TAC), resonancia magnética nuclear (RMN), y ultrasonografía.Resultados/conclusiones: El diagnóstico por imagen mostró una masa sólida bien circunscrita de localización intraconal. Macroscópicamente el tumor era de coloración púrpura, muy vascularizado y encapsulado. El examen histológico reveló un hemangiopericitoma de grado intermedio, con hipercelularidad y moderado número de mitosis. Destacamos la rareza del hemangiopericitoma orbitario, especialmente cuando se localiza dentro del cono muscular (AU)
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Subject(s)
Adolescent , Male , Humans , Hemangiopericytoma , Orbital NeoplasmsABSTRACT
Se presenta un estudio inmunohistoquímico de expresión de glicoproteína P en 44 tumores óseos malignos (32 osteosarcomas y 12 condrosarcomas) con el propósito de analizar la inmunorreactividad por estirpe histológico y su valor pronóstico potencial. El principio del procedimiento es la determinación de la molécula de glicoproteína P mediante un método inmunohistoquímico que emplea un cóctel de anticuerpos monoclonales específicos (JSB1 y MRK16) prediluidos y el procedimiento estándar de la fosfatasa-antifosfatasa alcalina, con incubación doble de anticuerpo secundario y ese complejo. Las muestras de tejido fueron obtenidas antes de la quimioterapia. La inmunoexpresión se clasificó en seis grados según una escala semicuantitativa de positividad celular.Hay que destacar la expresión intensa y constante de glicoproteína P en la membrana celular de los condrosarcomas (positividad del 100 por ciento y puntuación semicuantitativa de 3,83ñ 1,03). En los osteosarcomas la tinción fue menos constante (positividad del 59,37 por ciento y puntuación semicuantitativa de 2,79 ñ 1,81). Los pacientes con osteosarcomas localizados de alto grado (IIB) y que expresaban glicoproteína P tenían una menor supervivencia (log rank; p < 0,05) e intervalo libre de enfermedad (log rank; p < 0,001). Los hallazgos sustentan a la expresión de glicoproteína P como factor pronóstico en el osteosarcoma (AU)
Subject(s)
Female , Male , Humans , Osteosarcoma/diagnosis , ATP Binding Cassette Transporter, Subfamily B/analysis , Immunohistochemistry/methods , Chondrosarcoma, Mesenchymal/diagnosis , Biomarkers, Tumor/isolation & purification , Osteosarcoma , Chondrosarcoma, Mesenchymal , Disease-Free Survival , Neoplasm Metastasis , Neoplasm Staging/methodsABSTRACT
Inflammatory pseudotumour of lymph nodes is a benign type of lymphadenopathy recently described as a peculiar nodal reaction, which displays distinctive clinical and histological features. The origin of the disease is unknown although an inflammatory etiology has been suggested. We study a new case of inflammatory pseudotumour of lymph nodes which had an unusual clinical presentation, with lymphadenopathy on both sides of the diaphragm and enlargement of liver and spleen. In addition, focal fibrinoid vascular necrosis was observed on histological examination, an unusual finding which makes difficult the differential diagnosis with other lesions, particularly vasculitis.
Subject(s)
Fibroma/diagnosis , Lymphatic Diseases/diagnosis , Vasculitis/diagnosis , Abdomen , Adolescent , Diagnosis, Differential , Humans , Male , Necrosis , SplenomegalyABSTRACT
Ten cases of xanthogranulomatous cholecystitis are presented, 5 women and 5 men, from a total of 439 cholecystectomies (2.2%). In 50% of cases the clinical course was consistent with acute cholecystitis; in 30%, gallbladder cancer was suspected preoperatively; and in 70% of cases cancer was suspected during surgery but intraoperative biopsies showed no malignancy. Definitive pathological findings included early carcinoma of the gallbladder in two patients, and a cholecystocolic fistula in one patient. A perforated gallbladder was found in one patient. The incidence of postoperative septic complications was 18.1%, a figure that doubles that of elective biliary surgery in our hospital.
