RESUMEN
Serine proteases and its inhibitors are involved in physiological process and its deregulation lead to various diseases like Chronic Obstructive Pulmonary Disease (COPD), pulmonary emphysema, skin diseases, atherosclerosis, coagulation diseases, cancer, inflammatory diseases, neuronal disorders and other diseases. Serine protease inhibitors have been described in many species, as well as in plants, including cowpea beans (Vigna unguiculata (L.) Walp). Here, we purified and characterized a protease inhibitor, named VuEI (Vigna unguiculata elastase inhibitor), from Vigna unguiculata, with inhibitory activity against HNE (human neutrophil elastase) and chymotrypsin but has no inhibitory activity against trypsin and thrombin. VuEI was obtained by alkaline protein extraction followed by three different chromatographic steps in sequence. First, an ion exchange chromatography using Hitrap Q column was employed, followed by two reversed-phase chromatography using Source15RPC and ACE18 columns. The molecular mass of VuEI was estimated in 10.99 kDa by MALDI-TOF mass spectrometry. The dissociation constant (Ki) to HNE was 9 pM. These data indicate that VuEI is a potent inhibitor of human neutrophil elastase, besides to inhibit chymotrypsin.
Asunto(s)
Elastasa de Leucocito/aislamiento & purificación , Semillas/química , Inhibidores de Serina Proteinasa/aislamiento & purificación , Vigna/química , Animales , Bioensayo , Bovinos , Extractos Vegetales/químicaRESUMEN
Pulmonary inflammatory diseases are a major burden worldwide. They have in common an influx of neutrophils. Neutrophils secrete unchecked proteases at inflammation sites consequently leading to a protease/inhibitor imbalance. Among these proteases, neutrophil elastase is responsible for the degradation of the lung structure via elastin fragmentation. Therefore, monitoring the protease/inhibitor status in lungs non-invasively would be an important diagnostic tool. Herein we present the synthesis of a MeO-Suc-(Ala)2-Pro-Val-nitroxide, a line-shifting elastase activity probe suitable for Electron Paramagnetic Resonance spectroscopy (EPR) and Overhauser-enhanced Magnetic Resonance Imaging (OMRI). It is a fast and sensitive neutrophil elastase substrate with Km =â¯15⯱â¯2.9⯵M, kcat/Km =â¯930,000â¯s-1 M-1 and Km =â¯25⯱â¯5.4⯵M, kcat/Km =â¯640,000â¯s-1 M-1 for the R and S isomers, respectively. These properties are suitable to detect accurately concentrations of neutrophil elastase as low as 1â¯nM. The substrate was assessed with broncho-alveolar lavages samples derived from a mouse model of Pseudomonas pneumonia. Using EPR spectroscopy we observed a clear-cut difference between wild type animals and animals deficient in neutrophil elastase or deprived of neutrophil Elastase, Cathepsin G and Proteinase 3 or non-infected animals. These results provide new preclinical ex vivo and in vivo diagnostic methods. They can lead to clinical methods to promote in time lung protection.
Asunto(s)
Elastina/química , Elastasa de Leucocito/química , Pulmón/enzimología , Neumonía/enzimología , Animales , Líquido del Lavado Bronquioalveolar/química , Catepsina G/química , Elastina/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Humanos , Elastasa de Leucocito/aislamiento & purificación , Pulmón/efectos de los fármacos , Pulmón/patología , Imagen por Resonancia Magnética , Ratones , Mieloblastina/química , Neutrófilos/enzimología , Oligopéptidos/síntesis química , Oligopéptidos/química , Oligopéptidos/farmacología , Neumonía/metabolismo , Neumonía/patología , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Especificidad por SustratoRESUMEN
The growing incidence of chronic wounds in the world population has prompted increased interest in chronic wound dressings with protease-modulating activity and protease point of care sensors to treat and enable monitoring of elevated protease-based wound pathology. However, the overall design features needed for the combination of a chronic wound dressing that lowers protease activity along with protease detection capability as a single platform for semi-occlusive dressings has scarcely been addressed. The interface of dressing and sensor specific properties (porosity, permeability, moisture uptake properties, specific surface area, surface charge, and detection) relative to sensor bioactivity and protease sequestrant performance is explored here. Measurement of the material's zeta potential demonstrated a correlation between negative charge and the ability of materials to bind positively charged Human Neutrophil Elastase. Peptide-cellulose conjugates as protease substrates prepared on a nanocellulosic aerogel were assessed for their compatibility with chronic wound dressing design. The porosity, wettability and absorption capacity of the nanocellulosic aerogel were consistent with values observed for semi-occlusive chronic wound dressing designs. The relationship of properties that effect dressing functionality and performance as well as impact sensor sensitivity are discussed in the context of the enzyme kinetics. The sensor sensitivity of the aerogel-based sensor is contrasted with current clinical studies on elastase. Taken together, comparative analysis of the influence of molecular features on the physical properties of three forms of cellulosic transducer surfaces provides a meaningful assessment of the interface compatibility of cellulose-based sensors and corresponding protease sequestrant materials for potential use in chronic wound sensor/dressing design platforms.
Asunto(s)
Vendajes , Celulosa/metabolismo , Fibra de Algodón , Elastasa de Leucocito/aislamiento & purificación , Elastasa de Leucocito/metabolismo , Péptidos/metabolismo , Absorción Fisicoquímica , Humanos , Permeabilidad , Porosidad , HumectabilidadRESUMEN
The neutrophil enzyme, myeloperoxidase, by converting hydrogen peroxide (H2O2) and chloride to hypochlorous acid (HOCl), provides important defense against ingested micro-organisms. However, there is debate about how efficiently HOCl is produced within the phagosome and whether its reactions with phagosomal constituents influence the killing mechanism. The phagosome is a small space surrounding the ingested organism, into which superoxide, H2O2 and high concentrations of proteins from cytoplasmic granules are released. Previous studies imply that HOCl is produced in the phagosome, but a large proportion should react with proteins before reaching the microbe. To mimic these conditions, we subjected neutrophil granule extract to sequential doses of H2O2. Myeloperoxidase in the extract converted all the H2O2 to HOCl, which reacted with the granule proteins. 3-Chlorotyrosine, protein carbonyls and large amounts of chloramines were produced. At higher doses of H2O2, the extract developed potent bactericidal activity against Staphylococcus aureus. This activity was due to ammonia monochloramine, formed as a secondary product from protein chloramines and dichloramines. Isolated myeloperoxidase and elastase also became bactericidal when modified with HOCl and antibacterial activity was seen with a range of species. Comparison of levels of protein modification in the extract and in phagosomes implies that a relatively low proportion of phagosomal H2O2 would be converted to HOCl, but there should be sufficient for substantial protein chloramine formation and some breakdown to ammonia monochloramine. It is possible that HOCl could kill ingested bacteria by an indirect mechanism involving protein oxidation and monochloramine formation.
Asunto(s)
Antibacterianos/metabolismo , Cloraminas/metabolismo , Gránulos Citoplasmáticos/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Ácido Hipocloroso/metabolismo , Elastasa de Leucocito/metabolismo , Peroxidasa/metabolismo , Antibacterianos/farmacología , Burkholderia cepacia/efectos de los fármacos , Burkholderia cepacia/crecimiento & desarrollo , Cloraminas/farmacología , Mezclas Complejas/química , Gránulos Citoplasmáticos/química , Enterococcus faecalis/efectos de los fármacos , Enterococcus faecalis/crecimiento & desarrollo , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Humanos , Ácido Hipocloroso/farmacología , Cinética , Elastasa de Leucocito/aislamiento & purificación , Viabilidad Microbiana/efectos de los fármacos , Neutrófilos/química , Peroxidasa/aislamiento & purificación , Cultivo Primario de Células , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/crecimiento & desarrollo , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/crecimiento & desarrollo , Streptococcus/efectos de los fármacos , Streptococcus/crecimiento & desarrolloRESUMEN
Ultrafiltration with a hemoconcentrator may remove excess fluid load and alleviate tissue edema and has been universally adopted in extracorporeal circulation protocols during pediatric cardiac surgery. Balanced ultrafiltration is advocated to remove inflammatory mediators generated during surgery. However, whether balanced ultrafiltration can remove all or a portion of the inflammatory mediator load remains unclear. The inflammatory mediator removal capacity of zero-balanced ultrafiltration was measured during pediatric extracorporeal circulation in vitro. Extracorporeal circulation was composed of cardiotomy reservoir, D902 Lilliput 2 membrane oxygenator, and Capiox AF02 pediatric arterial line filter. The Hemoconcentrator BC 20 plus was placed between arterial purge line and oxygenator venous reservoir. Fresh donor human whole blood was added into the circuit and mixed with Ringer's solution to obtain a final hematocrit of 24-28%. After 2 h of extracorporeal circulation, zero-balanced ultrafiltration was initiated and arterial line pressure was maintained at approximately 100 mmHg with Hoffman clamp. The rate of ultrafiltration (12 mL/min) was controlled by ultrafiltrate outlet pressure. Identical volume of plasmaslyte A was dripped into the circuit to maintain stable hematocrit during the 45 min of the experiment. Plasma and ultrafiltrate samples were drawn every 5 min, and concentrations of inflammatory mediators including interleukin-1ß (IL-1ß), IL-6, IL-10, neutrophil elastase (NE), and tumor necrosis factor-α (TNF-α) were measured. All assayed inflammatory mediators were detected in the ultrafiltrate, demonstrating that the ultrafiltrator may remove inflammatory mediators. However, dynamic observations suggested that the concentration of NE was highest among the five inflammatory mediators in both plasma and ultrafiltrate (P < 0.001). IL-1ß had the lowest concentration in plasma, whereas the concentration of TNF-α was the lowest in ultrafiltrate (P < 0.001). Concentrations of all inflammatory mediators in the ultrafiltrate did not increase linearly compared with those in plasma. The respective ultrafiltrate to plasma concentration and amount ratios indicated that the total removal effect of hemoconcentrator on the inflammatory mediators was 4.17 ± 2.68% for IL-1ß, 0.64 ± 0.69% for IL-6, 0.24 ± 0.18% for IL-10, 2.84 ± 1.65% for NE, and 0.51 ± 0.81% for TNF-α, respectively. Balanced ultrafiltration may selectively remove inflammatory mediators from serum. Respective ratios of inflammatory mediators in ultrafiltrate compared with plasma, as well as total amount of inflammatory mediators in the ultrafiltrate suggest that balanced ultrafiltration removes a limited portion of the total inflammatory mediator load.
Asunto(s)
Circulación Extracorporea/instrumentación , Mediadores de Inflamación/sangre , Mediadores de Inflamación/aislamiento & purificación , Ultrafiltración/instrumentación , Análisis Químico de la Sangre , Niño , Diseño de Equipo , Hemodinámica , Humanos , Interleucinas/sangre , Interleucinas/aislamiento & purificación , Elastasa de Leucocito/sangre , Elastasa de Leucocito/aislamiento & purificación , Factor de Necrosis Tumoral alfa/sangre , Factor de Necrosis Tumoral alfa/aislamiento & purificaciónRESUMEN
BACKGROUND: Pulmonary ischemia/reperfusion (I/R) injury is associated with degradation of structural proteins. Preconditioning by short-term inhalation of nitric oxide (NO) ameliorates some of the severe consequences of an I/R cycle. The aim of this study was to evaluate the effects of NO preconditioning on I/R-induced changes of matrix metalloproteinase (MMP) activity. MATERIALS AND METHODS: Left lung in situ ischemia in rats was maintained for 1 h, followed by reperfusion for 30 min or 4 h. In the NO group, animals inhaled NO (15 ppm) for 10 min directly before ischemia. Changes of expression or activity of MMPs (MMP-2, MMP-7, MMP-9, MMP-14) and of neutrophil elastase (NE) in bronchoalveolar lavage fluid (BALF), lung tissue, and arterial plasma were analyzed by zymography and Western blotting. Western blotting was also used to detect tissue inhibitors of matrix proteases, the extracellular metalloproteinase inducer (EMMPRIN or CD147), and endostatin, a proteolytic collagen fragment. RESULTS: Ischemia resulted in an increase of lavagable MMP activity (12.3-fold MMP-2, 8.1-fold MMP-7) at 30 min reperfusion. The activity of MMP-9 and NE in lung tissue progressively increased with time, whereas MMP-14 and MMP-2 were constant. Inhalation of NO prevented the early increase of MMP-2 and MMP-7 in BALF, but the level of MMP-9 and NE in tissue was not affected. The expression of tissue inhibitors of matrix proteases and EMMPRIN did not respond to any treatment. The release of endostatin proceeded in parallel to the level of MMPs in BALF. Significant correlations between MMP-9 and myeloperoxidase in lung tissue and between MMP-2/MMP-7 and plasma protein extravasation were found. CONCLUSIONS: The early rise of MMP-2 and MMP-7 in BALF resulted from plasma protein extravasation, whereas MMP-9 and NE were imported into lung tissue via leukocyte invasion. The effect of NO inhalation on lavagable MMPs was secondary to the sealing of the permeability barrier.
Asunto(s)
Precondicionamiento Isquémico/métodos , Pulmón/enzimología , Metaloproteinasas de la Matriz/metabolismo , Óxido Nítrico/farmacología , Daño por Reperfusión/enzimología , Daño por Reperfusión/prevención & control , Animales , Líquido del Lavado Bronquioalveolar , Gelatinasas/sangre , Gelatinasas/aislamiento & purificación , Gelatinasas/metabolismo , Elastasa de Leucocito/aislamiento & purificación , Elastasa de Leucocito/metabolismo , Pulmón/efectos de los fármacos , Masculino , Metaloproteinasas de la Matriz/aislamiento & purificación , Elastasa Pancreática/aislamiento & purificación , Elastasa Pancreática/metabolismo , Ratas , Ratas Sprague-Dawley , Posición Supina , Toracotomía/efectos adversos , Toracotomía/métodosRESUMEN
OBJECTIVE: To purify neutrophil elastase (NE) from dog blood and develop and validate an ELISA for the measurement of canine NE (cNE) in canine serum as a marker for gastrointestinal tract inflammation. SAMPLE POPULATION: Neutrophils from 6 dogs immediately after they were euthanatized and serum from 54 healthy dogs. PROCEDURES: cNE was purified from blood by use of dextran sedimentation, repeated cycles of freezing-thawing and sonication, cation-exchange chromatography, and continuous elution electrophoresis. Antibodies against cNE were generated in rabbits, and an ELISA was developed and validated by determination of sensitivity, dilutional parallelism, spiking recovery, intra-assay variability, and interassay variability. A reference range was established by assaying serum samples from the 54 healthy dogs and by use of the lower 97.5th percentile. RESULTS: cNE was successfully purified from blood, and antibodies were successfully generated in rabbits. An ELISA was developed with a sensitivity of 1,100 mug/L. The reference range was established as < 2,239 mug/L. Ratios of observed-to-expected results for dilutional parallelism for 4 serum samples ranged from 85.4% to 123.1%. Accuracy, as determined by spiking recovery, ranged from 27.1% to 114.0%. Coefficient of variation for 4 serum samples was 14.2%, 16.0%, 16.8%, and 13.4%, respectively, for intra-assay variability and 15.4%, 15.0%, 10.5%, and 14.6%, respectively, for interassay variability. CONCLUSIONS AND CLINICAL RELEVANCE: The purification protocol used here resulted in rapid and reproducible purification of cNE with a high yield. The novel ELISA yielded linear results and was accurate and precise. Additional studies are needed to evaluate the clinical usefulness of this assay.
Asunto(s)
Perros/sangre , Ensayo de Inmunoadsorción Enzimática/veterinaria , Elastasa de Leucocito/aislamiento & purificación , Elastasa de Leucocito/metabolismo , Secuencia de Aminoácidos , Animales , Ensayo de Inmunoadsorción Enzimática/métodos , Elastasa de Leucocito/química , Datos de Secuencia Molecular , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Annexin 1 has been shown to participate through its unique N-terminal domain in the recruitment and activation of leukocytes at sites of inflammation. Peptides derived from this domain are true mimetics of the annexin 1 action in all inflammation models tested and most likely serve as the active entities generated at sites of inflammation. To elucidate mechanisms underlying peptide generation we used isolated blood leukocytes and endothelial cell monolayers. We show that following endothelial adhesion, annexin 1 was externalized from leukocytes and rapidly cleaved. Addition of purified annexin 1 to degranulating leukocytes resulted in the truncation of annexin 1, which seemed to depend on the proteolytic activity of human leukocyte elastase (HLE). The capacity of elastase to proteolytically cleave annexin 1 was confirmed using both purified annexin 1 and HLE. The identification of annexin 1 as a substrate for HLE supports the model in which annexin 1 participates in regulating leukocyte emigration into inflamed tissue through N-terminal peptides generated at inflammatory sites.
Asunto(s)
Anexina A1/química , Anexina A1/metabolismo , Elastasa de Leucocito/química , Adhesión Celular , Células Endoteliales/metabolismo , Células Endoteliales/fisiología , Humanos , Elastasa de Leucocito/aislamiento & purificación , Elastasa de Leucocito/metabolismo , Leucocitos Mononucleares/enzimología , Transporte de Proteínas , Especificidad por SustratoRESUMEN
The urokinase receptor (CD87) participates to the pericellular proteolytic potential of migrating cells and to the recruitment of leukocytes during inflammation. It consists of three structurally homologous domains, with the C-terminal domain D3 attached to cell membranes through a GPI anchor. CD87 is susceptible to an endoproteolytic processing removing the N-terminal domain D1 and generating truncated D2D3 membrane species, thus modulating CD87-associated functions. Full-length or truncated CD87 can be also released from cells via juxtamembrane cleavage by phospholipases and/or by yet unidentified proteinases. Using a recombinant CD87 and the CD87-positive monocytic U937 cell line and isolated blood monocytes, we show by protein immunoblotting and flow immunocytometry that the human neutrophil serine-proteinases elastase and cathepsin G cleave CD87 within the D1-D2 linker sequence, while in addition cathepsin G is highly efficient in cleaving the C terminus of D3. The combination of cathepsin G and elastase provided by degranulated neutrophils results in enzymatic cooperation leading to the release from monocytic cells of a truncated D2D3 species resembling that previously described in pathological body fluids. Using mass spectrometry analysis, the proteolytic fragmentation of synthetic peptides mapping the D1-D2 linker and D3 C-terminal domains identifies potential cleavage sites for each enzyme and suggests the existence of a mechanism regulating the CD87(D1-D2)-associated chemotactic activity. Finally, isolated or combined elastase and cathepsin G drastically reduce the capacity of cells to bind urokinase. Secretable leukocyte serine-proteinases are thus endowed with high potential for the regulation of CD87 expression and function on inflammatory cells.
Asunto(s)
Catepsinas/química , Elastasa de Leucocito/química , Monocitos/enzimología , Neutrófilos/enzimología , Receptores de Superficie Celular/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Secuencia de Aminoácidos , Unión Competitiva , Catepsina G , Catepsinas/metabolismo , Membrana Celular/enzimología , Sinergismo Farmacológico , Humanos , Hidrólisis , Immunoblotting , Elastasa de Leucocito/aislamiento & purificación , Elastasa de Leucocito/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Monocitos/metabolismo , Neutrófilos/metabolismo , Fragmentos de Péptidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Receptores de Superficie Celular/química , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Serina Endopeptidasas , Solubilidad , Células U937RESUMEN
The fusion protein PML-RARalpha, generated by the t(15;17)(q22;q11.2) translocation associated with acute promyelocytic leukemia (APL), initiates APL when expressed in the early myeloid compartment of transgenic mice. PML-RARalpha is cleaved in several positions by a neutral serine protease in a human myeloid cell line; purification revealed that the protease is neutrophil elastase (NE). Immunofluorescence localization studies suggested that the cleavage of PML-RARalpha must occur within the cell, and perhaps, within the nucleus. The functional importance of NE for APL development was assessed in NE deficient mice. Greater than 90% of bone marrow PML-RARalpha cleaving activity was lost in the absence of NE, and NE (but not Cathepsin G) deficient animals were protected from APL development. Primary mouse and human APL cells also contain NE-dependent PML-RARalpha cleaving activity. Since NE is maximally produced in promyelocytes, this protease may play a role in APL pathogenesis by facilitating the leukemogenic potential of PML-RARalpha.
Asunto(s)
Leucemia Promielocítica Aguda/enzimología , Elastasa de Leucocito/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Animales , Médula Ósea/enzimología , Extractos Celulares , Línea Celular Tumoral , Eliminación de Gen , Predisposición Genética a la Enfermedad , Humanos , Células K562 , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/patología , Elastasa de Leucocito/química , Elastasa de Leucocito/deficiencia , Elastasa de Leucocito/aislamiento & purificación , Ratones , Ratones Noqueados , Peso Molecular , Proteínas de Neoplasias/genética , Proteínas de Fusión Oncogénica/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células U937RESUMEN
Introducción. Se ha demostrado que los polimorfonucleares aislados de pacientes diabéticos presentan un mayor grado de activación que los de los pacientes no diabéticos. Además, la diabetes, tanto de tipo 1 como de tipo 2, se asocia con un incremento de la peroxidación lipídica y valores elevados de moléculas de adhesión circulantes. La administración de estreptozotocina (STZ) en ratas neonatales conduce en las ratas adultas a una ligera deficiencia de insulina, con valores de glucemia normales, y son aceptadas como modelo de diabetes tipo 2.Objetivo. En este estudio hemos investigado posibles diferencias en plasma y tejidos de algunos mediadores de la inflamación entre ratas normales y ratas con diabetes tipo 2, después de isquemiareperfusión (I-R) intestinal. Material y métodos. Se utilizaron ratas Wistar a las que se les administró STZ (0 o 30 mg/kg) el día de su nacimiento. Dos meses después, tanto las ratas control como las que tenían diabetes tipo 2 (normoglucémicas) fueron asignadas aleatoriamente a dos grupos. El grupo I fue sometido a un período de 60 min de isquemia intestinal por pinzamiento de la arteria mesentérica superior. Cinco minutos después de la reperfusión, las ratas fueron sacrificadas y se obtuvieron muestras de vena porta (VP), cava infrahepática (CIH), cava suprahepática (CSH), páncreas e intestino. En el grupo control los animales se manipularon de igual forma, pero sin ser sometidos a IR. El óxido nítrico (NO) se midió como NO-2 + NO-3, por la reacción de Griess. Los hidroperóxidos lipídicos (LPO) fueron determinados espectrofotométricamente usando un kit comercial. Los receptores para factor de necrosis tumoral (TNF) de 60 kDa (TNF-R1) y 75 kDa (TNF-R2), y el ICAM-1 se determinaron por el método Elisa. Resultados. Tras la I-R, las ratas diabéticas evidenciaron un aumento de las concentraciones plasmáticas de LPO, NO, ICAM-1 (0,514 + 0,083 frente a 0,046 + 0,011 CIH; 0,574 + 0,075 frente a 0,037 + 0,009 CSH, y 0,528 + 0,067 frente a 0,033 + 0,009 VP; ng/ml; n = 10; p < 0,01), TNF (42,4 + 5,7 CIH, 248,4 + 28,2 CSH y 33,6 + 4,0 VP, pg/ml, en ratas diabéticas frente a no detectable en ratas control; n = 10), TNF-R1 (0,179 + 0,024 frente a 0,023 + 0,011 CIH; 0,233 + 0,032 frente a 0,033 + 0,005 CSH; 0,206 + 0,034 frente a 0,039 + 0,023 VP; ng/ml; n = 10; p < 0,001; p < 0,05 todas) mientras que no se encontraron diferencias en los valores de TNF-R2 entre ambos grupos. Tras I-R, los valores plasmáticos de TNF y NO fueron más elevados en CSH que en CIH y VP, lo que sugiere que el hígado es una importante fuente de ambos mediadores. Hemos observado que tras I-R en ratas diabéticas en el tejido intestinal se produce un aumento en los valores de TNF, interleucina (IL) 1, IL-6 (no significativo) e IL-10, mientras que en el tejido pancreático hay una disminución de TNF- e IL-10 y un aumento de IL-1 e IL-6.Conclusión. La diabetes tipo 2 intensifica la respuesta inflamatoria a la I-R intestinal (AU)
Asunto(s)
Animales , Masculino , Ratas , Mediadores de Inflamación/uso terapéutico , Mediadores de Inflamación/administración & dosificación , Isquemia/diagnóstico , Isquemia/terapia , Neutrófilos , Elastasa de Leucocito/aislamiento & purificación , Peroxidación de Lípido , Estreptozocina/administración & dosificación , Ácido Peracético/administración & dosificación , Espectrofotometría/métodos , Biomarcadores/análisis , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/terapia , Ratas Wistar , Espectrofotometría/clasificación , Espectrofotometría/instrumentación , Espectrofotometría/tendencias , EspectrofotometríaRESUMEN
Carboxypeptidase R (EC 3.4.17.20; CPR) is an unstable basic carboxypeptidase found in fresh serum in addition to carboxypeptidase N (CPN) which is a stable enzyme. CPR in fresh serum is generated from its zymogen (proCPR) during coagulation by trypsin-like enzymes such as thrombin and thrombin/thrombomodulin complexes. Since removal of the C-terminal arginine abrogates the anaphylatoxin activity of C3a and C5a, CPR and CPN are regarded as anaphylatoxin inactivators. We report here that the culture supernatant of activated human neutrophils converts proCPR to CPR. Addition of an elastase specific inhibitor, N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone (MSAAPVCK) to the supernatant of stimulated neutrophils completely inhibited activation of proCPR. On the other hand, a thrombin specific inhibitor, p-Nitrophenyl-p'-amidinophenyl-methanesulfonate hydrochloride (pNP-pAPMS) inhibited only 16% of proCPR activation by the neutrophil supernatant. Furthermore, purified elastase converted proCPR to CPR. Therefore, elastase can activate proCPR directly, or indirectly through activation of some proteases, which have been contaminating in reagents. Release of CPR generating enzymes from neutrophils should play an important role in regulation of excess inflammation.
Asunto(s)
Carboxipeptidasa B2/metabolismo , Carboxipeptidasas/metabolismo , Precursores Enzimáticos/metabolismo , Elastasa de Leucocito/metabolismo , Clorometilcetonas de Aminoácidos/farmacología , Células Cultivadas , Medios de Cultivo , Activación Enzimática , Humanos , Elastasa de Leucocito/antagonistas & inhibidores , Elastasa de Leucocito/aislamiento & purificación , Neutrófilos/enzimología , Inhibidores de Serina Proteinasa/farmacología , Trombina/antagonistas & inhibidores , Trombomodulina/metabolismoRESUMEN
As described previously (Yamashita S. et al., Biol. Pharm. Bull., 23, 519-522 (2000)), high levels of a truncated actin with an N-terminus of Met-44 were detected in neutrophils of patients with Behcet's disease. Since the increase of the truncated actin in neutrophils of patients may be important for understanding the pathology of Behçet's disease, the mechanism of the truncated actin formation was studied. First, to investigate the presence of a specific protease, which cleaves the actin at the site between Val-43 and Met-44, a peptide with a partial amino acid sequence of actin from the N-terminal Pro-38 to Asp-51 was synthesized as the protease substrate. The synthesized peptide was digested with cytosolic fractions of neutrophils from patients and healthy volunteers, and digestion products were analyzed by C18-reverse phase HPLC. The chromatograms of these samples showed that an endoprotease, which cleaved the peptide at a specific site, was present in cytosolic fractions of neutrophils from patients with Behçet's disease. Then, the effects of various kinds of protease inhibitors on the digestion of the peptide were investigated in order to identify the responsible endoprotease. The digestion of the peptide was suppressed by 4-(2-aminoethyl) benzenesulfonylfluoride (AEBSF, a serine protease inhibitor) and N-methoxysuccinyl-Ala-Ala-Pro-Val chloromethylketone (CMK, a polymorphonuclear (PMN)-elastase inhibitor) in the presence of EDTA. Furthermore, PMN-elastase was found to cleave the substrate peptide and actin at the site between Val-43 and Met-44. These results lead to the conclusion that the PMN-elastase is responsible for cleavage of actin at the N-terminal site between Val-43 and Met-44 in neutrophils from patients with Behçet's disease.
Asunto(s)
Actinas/metabolismo , Síndrome de Behçet/sangre , Elastasa de Leucocito/sangre , Neutrófilos/enzimología , Actinas/química , Clorometilcetonas de Aminoácidos/farmacología , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Inhibidores Enzimáticos/farmacología , Humanos , Elastasa de Leucocito/antagonistas & inhibidores , Elastasa de Leucocito/aislamiento & purificación , Datos de Secuencia Molecular , Neutrófilos/metabolismo , Sulfonas/farmacologíaRESUMEN
Human neutrophil elastase (HNE, IEC 3. 4. 21. 37) is a causative factor of inflammatory diseases, including emphysema and rheumatoid arthritis. Enzymatic characterization is important for the development of new drugs involved in the regulation of this enzyme. In this study, we investigated the enzymatic and biochemical properties of five different elastolytic enzymes, with a molecular mass between 24 kDa and 72 kDa. Three elastases, molecular masses of 27, 29, 31 kDa, might be elastase isozymes that have the same NH2-terminal amino acid sequences of Ile-Val-Gly-Gly-Arg-Arg-Ala. The 24-kDa enzyme, which showed the identical NH2-terminal amino acid sequences to elastase, was a degraded fragment of native elastase. The elastolytic activity was conserved at the 6/7 domain of the NH2-terminal region. The inhibitory characteristics of PMSF, DipF were the same as those of native elastases. The 72-kDa molecule, which showed elastolytic activity, might be a trimer formed between native elastases (31 kDa and 29 kDa) and a cathepsin G-like enzyme, which did not show elastolytic activity but enhanced the elastolytic activity of neutrophil elastase. Although this cathepsin G-like enzyme showed weak cathepsin G activity, it has distinguishable NH2-terminal sequences of Ile-Val-Gly-Gly-Ser-Arg-Ala- from those of elastase or cathepsin G. The potentiation of elastolytic activity could be a result of the trimerization of native elastase with a cathepsin G-like enzyme, and was then weakly inhibited by serine protease inhibitors, such as PMSF, DipF. Therefore, we suggest the cathepsin G-like enzyme to be a novel enzyme, which has an important role in the development of inflammation.
Asunto(s)
Catepsinas/sangre , Elastasa de Leucocito/sangre , Neutrófilos/enzimología , Secuencia de Aminoácidos , Catepsina G , Catepsinas/química , Catepsinas/aislamiento & purificación , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Cristalografía por Rayos X , Humanos , Isoenzimas/sangre , Isoenzimas/química , Isoenzimas/aislamiento & purificación , Cinética , Elastasa de Leucocito/química , Elastasa de Leucocito/aislamiento & purificación , Modelos Moleculares , Peso Molecular , Fragmentos de Péptidos/química , Estructura Terciaria de Proteína , Serina EndopeptidasasRESUMEN
Nonsteroidal antiinflammatory drugs(NSAIDs) are known as clinically effective agents for treatment of inflammatory diseases. Inhibition of cyclooxygenase has been thought to be a major facet of the pharmacological mechanism of NSAIDs. However, it is difficult to ascribe the antiinflammatory effects of NSAIDs solely to the inhibition of prostaglandin synthesis. Human neutrophil elastase (HNElastase; HNE, EC 3.4.21.37) has been known as a causative factor in inflammatory diseases. To investigate the specific relationship between HNElastase inhibition and specificity of molecular structure of several NSAIDs, HNElastase was purified by Ultrogel AcA54 gel filtration, CM-Sephadex ion exchange, and HPLC (with TSK 250 column) chromatography. HNElastase was inhibited by aspirin and salicylate in a competitive manner and by naproxen, ketoprofen, phenylbutazone, and oxyphenbutazone in a partial competative manner, but not by ibuprofen and tolmetin. HNElastase-phenylbutazone-complex showed strong Raman shifts at 200, 440, 1124, 1194, 1384, 1506, and 1768 cm(-1). The Raman bands 1194, 1384, and 1768 cm(-1) may represent evidences of the conformational change at -N=N-phi radical, pyrazol ring, and -C=O radical of the elastase-drug complex, respectively. Phenylbutazone might be bound to HNElastase by ionic and hydrophobic interaction, and masked the active site. Inhibition of HNElastase could be another mechanism of action of NSAIDs besides cyclooxygenase inhibition in the treatment of inflammatory diseases. Different inhibition characteristics of HNE-lastase by NSAIDs such as aspirin, phenylbutazone-like drugs and ineffective drugs could be important points for drawing the criteria for appropriate drugs in clinical application.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Elastasa de Leucocito/antagonistas & inhibidores , Cromatografía de Afinidad , Simulación por Computador , Inhibidores Enzimáticos/farmacología , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/aislamiento & purificación , Cetoprofeno/farmacología , Elastasa de Leucocito/aislamiento & purificación , Modelos Moleculares , Naproxeno/farmacología , Fenilbutazona/análogos & derivados , Salicilatos/farmacología , Espectrometría RamanRESUMEN
Nonsteroidal antiinflammatory drugs(NSAIDs) are known as clinically effective agents for treatment of inflammatory diseases. Inhibition of cyclooxygenase has been thought to be a major facet of the pharmacological mechanism of NSAIDs. However, it is difficult to ascribe the antiinflammatory effects of NSAIDs solely to the inhibition of prostaglandin synthesis. Human neutrophil elastase (HNElastase; HNE, EC 3.4.21.37) has been known as a causative factor in inflammatory diseases. To investigate the specific relationship between HNElastase inhibition and specificity of molecular structure of several NSAIDs, HNElastase was purified by Ultrogel AcA54 gel filtration, CM-Sephadex ion exchange, and HPLC (with TSK 250 column) chromatography. HNElastase was inhibited by aspirin and salicylate in a competitive manner and by naproxen, ketoprofen, phenylbutazone, and oxyphenbutazone in a partial competative manner, but not by ibuprofen and tolmetin. HNElastase-phenylbutazone-complex showed strong Raman shifts at 200, 440, 1124, 1194, 1384, 1506, and 1768 cm(-1). The Raman bands 1194, 1384, and 1768 cm(-1) may represent evidences of the conformational change at -N=N-phi radical, pyrazol ring, and -C=O radical of the elastase-drug complex, respectively. Phenylbutazone might be bound to HNElastase by ionic and hydrophobic interaction, and masked the active site. Inhibition of HNElastase could be another mechanism of action of NSAIDs besides cyclooxygenase inhibition in the treatment of inflammatory diseases. Different inhibition characteristics of HNE-lastase by NSAIDs such as aspirin, phenylbutazone-like drugs and ineffective drugs could be important points for drawing the criteria for appropriate drugs in clinical application.
Asunto(s)
Humanos , Antiinflamatorios no Esteroideos/farmacología , Cromatografía de Afinidad , Simulación por Computador , Inhibidores Enzimáticos/farmacología , Isoenzimas/aislamiento & purificación , Isoenzimas/antagonistas & inhibidores , Cetoprofeno/farmacología , Elastasa de Leucocito/aislamiento & purificación , Elastasa de Leucocito/antagonistas & inhibidores , Modelos Moleculares , Naproxeno/farmacología , Fenilbutazona/análogos & derivados , Salicilatos/farmacología , Espectrometría RamanRESUMEN
Cardiopulmonary bypass has been implicated in triggering a multisystem inflammatory response caused by blood contact with the artificial surfaces of the circuit. This leads to increased morbidity levels because of cytotoxic enzymes released from activated neutrophils. Recently, it was discovered that certain inflammatory mediators are permeable to the membrane of the hemoconcentrator. As a result, this study was undertaken to quantitatively characterize the nature of this movement by deriving a sieving coefficient (S) for four inflammatory mediators: myeloperoxidase, elastase, interleukin-6, and lactoferrin. The results show no permeability through the hemoconcentrator for the two neutrophil derived enzymes myeloperoxidase and elastase (S = 0, p > 0.05). Conversely, although larger than the pore size of the hemoconcentrator, lactoferrin sieves through unrestricted (S = 1.030 +/- 0.037, p < 0.0001). Interleukin-6 is removed in concentrations greater than those found in the blood, which yields a sieving coefficient significantly greater than 1.0 (S = 1.246 +/- 0.042, p < 0.0001). In addition to sieving coefficients, this study offers theories as to why these mediators acted as such. One conclusion is that certain mediators are efficaciously removed by the hemoconcentrator and, with additional study, may result in an attenuated inflammatory response.
Asunto(s)
Puente Cardiopulmonar/efectos adversos , Hemofiltración/métodos , Mediadores de Inflamación/sangre , Mediadores de Inflamación/aislamiento & purificación , Anciano , Humanos , Inflamación/etiología , Inflamación/prevención & control , Mediadores de Inflamación/química , Interleucina-6/sangre , Interleucina-6/aislamiento & purificación , Lactoferrina/sangre , Lactoferrina/aislamiento & purificación , Elastasa de Leucocito/sangre , Elastasa de Leucocito/aislamiento & purificación , Masculino , Persona de Mediana Edad , Peso Molecular , Neutrófilos/enzimología , Peroxidasa/sangre , Peroxidasa/aislamiento & purificaciónRESUMEN
Integrin CR3 (CD11b/CD18, Mac-1, alpha M beta 2) mediates the transient adhesion of polymorphonuclear leukocytes (PMN) to surfaces coated with fibrinogen, C3bi, ICAM-1, and other ligands. Recent studies (Cai, T.-Q., and S.D. Wright 1995. J. Biol. Chem. 270:14358) suggest that adhesion may be favored by stimulus-dependent changes in the kinetics of ligand binding by CR3. Cell detachment, on the other hand, must occur by a different mechanism because binding kinetics cannot affect cell adhesion after binding of ligand has occurred. We have sought a mechanism that would reverse binding of ligand to CR3 and report here that lysates of PMN contain an endogenous ligand that binds CR3 and competes the binding of C3bi. Purification and sequence analysis identified the structurally homologous azurophilic granule proteins, elastase, protease 3, and azurocidin as candidates. Studies with purified elastase and azurocidin showed that each bound specifically to purified, immobilized CR3. Elastase may play a role in modulating integrin-mediated cell adhesion because it is expressed at the cell surface, and the expression level is inversely proportional to cell adhesivity. Furthermore, a monoclonal antibody against elastase prevented detachment of PMN from fibrinogen-coated surfaces and blocked chemotaxis, confirming a role for this protein in regulating integrin-mediated adhesion. These studies suggest a model for release of integrin-mediated cell adhesion in which endogenous ligands such as elastase may release adhesion by "'eluting" substrate-bound ligand. A role for the proteolytic activity of elastase appears likely but is not demonstrated in this study.
Asunto(s)
Antígenos CD18/metabolismo , Proteínas Portadoras , Adhesión Celular/fisiología , Elastasa de Leucocito/metabolismo , Antígeno de Macrófago-1/metabolismo , Neutrófilos/fisiología , Secuencia de Aminoácidos , Péptidos Catiónicos Antimicrobianos , Unión Competitiva , Proteínas Sanguíneas/metabolismo , Quimiotaxis de Leucocito/efectos de los fármacos , Complemento C3b/metabolismo , Fibrinógeno/metabolismo , Humanos , Cinética , Elastasa de Leucocito/aislamiento & purificación , Elastasa de Leucocito/farmacología , Ligandos , Datos de Secuencia Molecular , Neutrófilos/enzimología , Inhibidores de Proteasas/farmacología , Unión Proteica/efectos de los fármacosRESUMEN
Human myeloblastin (leukocyte proteinase 3) shares many biochemical properties with leukocyte elastase, but rapidly loses enzymatic activity when raising the pH and/or the ionic strength of an acidic solution or when handled in glass vessels. This poses limits to kinetic experiments requiring long incubation times. After purification, myeloblastin was conveniently stored in a glycine/HCl buffer at pH 3.2, while assays were performed in sodium/potassium phosphate buffer at pH 7.0, ionic strength 0.11, in the presence of 0.05% w/v Triton X-100 and taking care to avoid any contact with glass surfaces. The kinetic parameters of leukocyte elastase and myeloblastin with peptide substrates, irreversible inactivators and glycosaminoglycans were compared under these conditions. MeO-succinyl-Lys(2-picolinoyl)Ala-Pro-Val-4-nitroanilide, an excellent substrate for leukocyte elastase, also proved to be a good substrate for myeloblastin (Km = 16 microM, kcat/Km = 30,600 M(-1)s(-1)). Inactivation of myeloblastin by 3,4-dichloroisocoumarin (ki/Ki = 6,389 M(-1)s(-1)) and MeO-Suc-Ala-Ala-Pro-Val-chloromethane (ki/Ki = 579 M(-1) S(-1)) occurred via a two-step, irreversible complexing mechanism with potencies one-half and one-fifth that of leukocyte elastase, respectively. Glycosaminoglycans such as chondroitin sulfate, dermatan sulfate and a chondroitin polysulfate, interacted with myeloblastin as non-essential activators in the presence of peptide substrates (activation up to a 6.7-fold factor) and as partial inhibitors (about 50% inhibition at saturation) in the presence of elastin. This property distinguishes myeloblastin from leukocyte elastase, which is always inhibited by glycosaminoglycans, independently of the substrate.