RESUMEN
Two recombinant protease inhibitors from Bauhinia bauhinioides, rBbKI (kallikrein inhibitor) and rBbCI (cruzipain inhibitor) were evaluated for insecticidal activity against workers and soldiers of Nasutitermes corniger (order: Isoptera; family: Termitidae) through the inhibitors' effect on the insect's gut enzymes. The inhibitor rBbKI was more effective than rBbCI in inhibiting the termite's gut enzymes. The kallikrein inhibitor showed termiticidal activity in workers with an LC50 of 0.9â¯mgâ¯mL-1 after 4 days. Conversely, rBbKI did not affect the survival of soldiers and rBbCI did not show termiticidal activity against N. corniger. The two inhibitors showed different specificity towards the termite's gut enzymes, representing interesting tools to characterize N. corniger enzymes. The different effects of rBbKI and rBbCI on the termite's enzymes and survival may be linked to slight structural differences between these inhibitors.
Asunto(s)
Bauhinia/química , Insecticidas/farmacología , Isópteros/enzimología , Inhibidores de Proteasas/farmacología , Animales , Cisteína Endopeptidasas , Humanos , Calicreínas/antagonistas & inhibidores , Proteínas Protozoarias/antagonistas & inhibidores , Especificidad por SustratoRESUMEN
En la mujer adulta declina la función ovárica y la producción de estrógenos, con sintomatología física y psíquica, alteración en la regulación del apetito y tendencia a la ingesta de hidratos de carbono. Los psicofármacos empleados para el manejo del estado anímico podrían provocar efectos secundarios, como modificación de la ingesta y peso corporal y efectos neuroendocrinos como regulación de la serotonina, entre otros. Objetivos: Identificar la asociación entre el consumo de psicofármacos y la compulsión glucídica en mujeres adultas. Materiales y Método: Se realizó un estudio analítico de corte transversal sobre 114 mujeres mayores de 40 años que asistieron al consultorio de climaterio del Hospital General de Agudos Bernandino Rivadavia. Se analizaron variables biológicas, antropométricas, conducta alimentaria y consumo de psicofármacos, recabadas mediante encuestas estructuradas, voluntarias y anónimas, y mediciones antropométricas. Para el análisis estadístico se utilizó SPSS 15.0, calculando medidas de tendencia central,comparación de medias y Odds Ratio (OR) con intervalo de confianza (IC) del 95%, test chi cuadrado (x2) y correlación de Pearson con nivel de significación p< 0,05. Resultados: El 67,5% de la muestra presentó sobrepeso u obesidad y el 48,2% circunferencia de cintura (CC) aumentada. El 69,3% refirió compulsión glucídica, siendo los panificados y el chocolate los alimentos de mayor elección en estos episodios. No se observó significación estadística entre las mujeres que consumían psicofármacos para mejorar la calidad de vida, y la presencia de compulsión o estado nutricional alterado. Conclusión: Las mujeres que consumieron psicofármacos no presentaron diferencias en la prevalencia de compulsión glucídica o el estado nutricional alterado...
Asunto(s)
Humanos , Menopausia , Obesidad , Tranquilizantes , MujeresRESUMEN
Introducción: . Objetivos: Estudiar si los valores de TSH influyen sobre el descenso del peso corporal en mujeres adultas con sobrepeso u obesidad. Metodología: Diseño de intervención sobre muestra no probabilística de mujeres >30 años y con IMC ≥25 Kg/m2, que asistieron a un Centro Endocrinológico de la Ciudad Autónoma de Buenos Aires. Resultados: Se estudiaron 105 mujeres (50.8±12.2 años) con un IMC medio de 30.0±4.9 Kg/m2. El 57.1% eran hipotiroideas bajo tratamiento con LT4. Según valor de TSH: Grupo A (27.6%) ≥2.5 mUI/L y Grupo B (72.4%) <2.5 mUI/L. A partir del programa de intervención, la mayoría descendió menos del 1% semanal con respecto a su peso inicial (51.7% grupo A y 56.6% Grupo B), con un grado de adherencia al tratamiento entre el 60 y 70% (51.7% grupo A y 50.1% Grupo B) sin observarse diferencias significativas entre los grupos. No se encontró asociación entre la pérdida de peso semanal con la función tiroidea (OR:0.64; IC:0.29-1.39; p:0.32) ni con valor de TSH (OR:1.21; IC:0.51-2.86; p:0.66). Se encontró correlación directa entre la pérdida de peso semanal y el grado de adherencia al tratamiento (r:0.464; p: 0.000) e inversa con el tiempo transcurrido hasta el primer control (r:-0.30; p: 0.002) y rangos del IMC (r: -0.282; p: 0.004), independientemente de la edad, la función tiroidea o el valor de TSH que presentaban las pacientes. Conclusiones: Los valores de TSH y la función tiroidea no influyeron sobre la pérdida de peso semanal. El descenso de peso se relacionó con la adherencia al tratamiento en forma directa independientemente de la edad, la función tiroidea o el valor de TSH.
Asunto(s)
Humanos , Femenino , Hipotiroidismo , Obesidad , Sobrepeso , MujeresRESUMEN
Few chronic food protein models have described the relationship between allergenicity and the molecular structure of food protein after physical processing. The effect of γ-radiation on the structure of food protein was measured by fluorescence, circular dichroism and microcalorimetry. BALB/c mice were intraperitoneally sensitized and then given non-irradiated and irradiated Con-A by daily gavage for 28days. The tendency to form insoluble amorphous aggregates and partially unfolded species was observed after irradiation. The administration of non-irradiated and irradiated samples at low-dose significantly increased weight loss as well as plasma levels of eotaxin in animals repeatedly exposed to Con-A. Significant lymphocytic infiltrate filling completely the stroma of microvilli and tubular glands was observed in the small intestinal of the group given Con-A irradiated at a low dose. This phenotype was not observed in animals treated with Con-A irradiated at a high dose.
Asunto(s)
Concanavalina A/química , Concanavalina A/inmunología , Concanavalina A/efectos de la radiación , Hipersensibilidad a los Alimentos/etiología , Administración Oral , Animales , Rastreo Diferencial de Calorimetría , Dicroismo Circular , Concanavalina A/administración & dosificación , Modelos Animales de Enfermedad , Relación Dosis-Respuesta en la Radiación , Femenino , Hipersensibilidad a los Alimentos/patología , Rayos gamma , Intestino Delgado/inmunología , Intestino Delgado/patología , Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Microvellosidades/inmunología , Microvellosidades/patología , Conformación Proteica , Pérdida de PesoRESUMEN
AIMS: The aim of this work was to analyse the coagulant and antibacterial activities of lectin isolated from Moringa oleifera seeds that are used for water treatment. METHODS AND RESULTS: The water-soluble M. oleifera lectin (WSMoL) was separated from nonhemagglutinating components (NHC) by chitin chromatography. WSMoL fluorescence spectrum was not altered in the presence of ions that are often present in high concentrations in polluted waters. Seed extract, NHC and WSMoL showed coagulant activity on a turbid water model. Both NHC and WSMoL reduced the growth of Staphylococcus aureus, but only WSMoL caused a reduction in Escherichia coli. WSMoL was also more effective in reducing the growth of ambient lake water bacteria. CONCLUSIONS: Data obtained from this study indicate that WSMoL is a potential natural biocoagulant for water, reducing turbidity, suspended solids and bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Moringa oleifera seeds are a material effective in the treatment of water.
Asunto(s)
Antibacterianos/farmacología , Lectinas/farmacología , Moringa oleifera/metabolismo , Semillas/metabolismo , Antibacterianos/metabolismo , Floculación , Lectinas/química , Lectinas/metabolismo , Eliminación de Residuos Líquidos/métodos , Contaminantes del Agua/química , Purificación del Agua/métodosRESUMEN
BACKGROUND AND PURPOSE: The serine and cysteine peptidase inhibitor, BbCI, isolated from Bauhinia bauhinioides seeds, is similar to the classical plant Kunitz inhibitor, STI, but lacks disulphide bridges and methionine residues. BbCI blocks activity of the serine peptidases, elastase (K(iapp) 5.3 nM) and cathepsin G (K(iapp) 160.0 nM), and the cysteine peptidase cathepsin L (K(iapp) 0.2 nM). These three peptidases play important roles in the inflammatory process. EXPERIMENTAL APPROACH: We measured the effects of BbCI on paw oedema and on leucocyte accumulation in pleurisy, both induced by carrageenan. Leucocyte-endothelial cell interactions in scrotal microvasculature in Wistar rats were investigated using intravital microscopy. Cytokine levels in pleural exudate and serum were measured by elisa. KEY RESULTS: Pretreatment of the animals with BbCI (2.5 mg·kg(-1)), 30 min before carrageenan-induced inflammation, effectively reduced paw oedema and bradykinin release, neutrophil migration into the pleural cavity. The number of rolling, adhered and migrated leucocytes at the spermatic fascia microcirculation following carrageenan injection into the scrotum were reduced by BbCI pretreatment. Furthermore, levels of the rat chemokine cytokine-induced neutrophil chemo-attractant-1 were significantly reduced in both pleural exudates and serum from animals pretreated with BbCI. Levels of interleukin-1ß or tumour necrosis factor-α, however, did not change. CONCLUSIONS AND IMPLICATIONS: Taken together, our data suggest that the anti-inflammatory properties of BbCI may be useful in investigations of other pathological processes in which human neutrophil elastase, cathepsin G and cathepsin L play important roles.
Asunto(s)
Antiinflamatorios/farmacología , Inflamación/tratamiento farmacológico , Proteínas de Plantas/farmacología , Animales , Antiinflamatorios/aislamiento & purificación , Bauhinia/química , Carragenina , Catepsina G/antagonistas & inhibidores , Catepsina G/metabolismo , Catepsina L/antagonistas & inhibidores , Catepsina L/metabolismo , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Citocinas/metabolismo , Modelos Animales de Enfermedad , Edema/tratamiento farmacológico , Edema/fisiopatología , Ensayo de Inmunoadsorción Enzimática , Humanos , Inflamación/fisiopatología , Elastasa de Leucocito/antagonistas & inhibidores , Elastasa de Leucocito/metabolismo , Leucocitos/efectos de los fármacos , Leucocitos/metabolismo , Masculino , Microscopía/métodos , Proteínas de Plantas/aislamiento & purificación , Ratas , Ratas Wistar , SemillasRESUMEN
The serine and cysteine peptidase inhibitor, BbCI, isolated from Bauhinia bauhinioides seeds, is similar to the classical plant Kunitz inhibitor, STI, but lacks disulphide bridges and methionine residues. BbCI blocks activity of the serine peptidases, elastase (Kiapp 5.3 nM) and cathepsin G (Kiapp 160.0 nM), and the cysteine peptidase cathepsin L (Kiapp 0.2 nM). These three peptidases play important roles in the inflammatory process. We measured the effects of BbCI on paw oedema and on leucocyte accumulation in pleurisy, both induced by carrageenan. Leucocyteendothelial cell interactions in scrotal microvasculature in Wistar rats were investigated using intravital microscopy. Cytokine levels in pleural exudate and serum were measured by elisa.Pretreatment of the animals with BbCI (2.5 mg·kg−1), 30 min before carrageenan-induced inflammation, effectively reduced paw oedema and bradykinin release, neutrophil migration into the pleural cavity. The number of rolling, adhered and migrated leucocytes at the spermatic fascia microcirculation following carrageenan injection into the scrotum were reduced by BbCI pretreatment. Furthermore, levels of the rat chemokine cytokine-induced neutrophil chemo-attractant-1 were significantly reduced in both pleural exudates and serum from animals pretreated with BbCI. Levels of interleukin-1â or tumour necrosis factor-á, however, did not change.Taken together, our data suggest that the anti-inflammatory properties of BbCI may be useful in investigations of other pathological processes in which human neutrophil elastase, cathepsin G and cathepsin L play important roles.
Asunto(s)
Animales , Ratas , Bauhinia/microbiología , Bradiquinina , Citocinas , Plantas/inmunología , Preparaciones de Plantas/antagonistas & inhibidores , Elastasa Pancreática , PleuresiaRESUMEN
Objetivo: Describir el perfil lipídico de las mujeres pre y postmenopáusicas y estimar el riesgo cardiovascular (RCV) por el Score de Framingham (SF) y circunferencia de la cintura (CC) en ambas etapas biológicas. (pre y postmenopausicas). Metodología: Muestra por conveniencia de mujeres de 40 a 65 años que concurrieron por primera vez a la consulta nutricional, de enero de 2005 a junio de 2006. Variables: Perfil lipídico (COLT>200, HDL<50, LDL>100 y TG>150), Indice aterogénico (IA)>4,5, Categorías de RCV según Score de Framingham (I, II y III). Se realizó medición directa de Peso, Talla y CC (>80 cm y >88 cm), según técnicas estandarizadas. El análisis estadístico se realizó con el programa Epi 6.04d, con intervalo de confianza (IC) del 95%. Se calculó la significación estadística por Chi2 para las variables correspondientes con valor p<0,05. Resultados: Se incluyeron 216 mujeres, 34,7% premenopáusicas (PM) con edad promedio de 47 ± 4,6 años y 65,3 % postmenopáusicas (PoM) con edad promedio de 57 ± 4,7 años. La edad promedio de menopausia fue de 48 ± 5 años. El 34,6% de PM (IC: 24,5-45,9) presentó IMC = 27,3 y el 70,6% (IC: 59,6-80,1) una CC >80 cm. El 56% de PoM (IC: 47,7-64,0) presentó IMC = 27,3 y 80,8 % (IC: 73,7- 86,7) CC > 80cm. El 100% de la muestra se encontró en Categoría III de RCV según SF. La mayor prevalencia de parámetros lipídicos alterados se observó en el Colesterol total y LDL-C, sin diferencias significativas al comparar ambas etapas biológicas. Conclusiones: Mientras por medición de CC la mayoría de la muestra presenta riesgo aumentado, por determinación del SF, la totalidad presentó bajo riesgo cardiovascular proyectado a 10 años.(AU)
Asunto(s)
Femenino , Menopausia , Premenopausia , Posmenopausia , Riesgo , Enfermedades Cardiovasculares , Hipercolesterolemia , Circunferencia Abdominal , ArgentinaRESUMEN
Objetivo: Describir el perfil lipídico de las mujeres pre y postmenopáusicas y estimar el riesgo cardiovascular (RCV) por el Score de Framingham (SF) y circunferencia de la cintura (CC) en ambas etapas biológicas. (pre y postmenopausicas). Metodología: Muestra por conveniencia de mujeres de 40 a 65 años que concurrieron por primera vez a la consulta nutricional, de enero de 2005 a junio de 2006. Variables: Perfil lipídico (COLT>200, HDL<50, LDL>100 y TG>150), Índice aterogénico (IA)>4,5, Categorías de RCV según Score de Framingham (I, II y III). Se realizó medición directa de Peso, Talla y CC (>80 cm y >88 cm), según técnicas estandarizadas. El análisis estadístico se realizó con el programa Epi 6.04d, con intervalo de confianza (IC) del 95%. Se calculó la significación estadística por Chi2 para las variables correspondientes con valor p<0,05. Resultados: Se incluyeron 216 mujeres, 34,7% premenopáusicas (PM) con edad promedio de 47 ± 4,6 años y 65,3 % postmenopáusicas (PoM) con edad promedio de 57 ± 4,7 años. La edad promedio de menopausia fue de 48 ± 5 años. El 34,6% de PM (IC: 24,5-45,9) presentó IMC = 27,3 y el 70,6% (IC: 59,6-80,1) una CC >80 cm. El 56% de PoM (IC: 47,7-64,0) presentó IMC = 27,3 y 80,8 % (IC: 73,7- 86,7) CC > 80cm. El 100% de la muestra se encontró en Categoría III de RCV según SF. La mayor prevalencia de parámetros lipídicos alterados se observó en el Colesterol total y LDL-C, sin diferencias significativas al comparar ambas etapas biológicas. Conclusiones: Mientras por medición de CC la mayoría de la muestra presenta riesgo aumentado, por determinación del SF, la totalidad presentó bajo riesgo cardiovascular proyectado a 10 años.
Asunto(s)
Femenino , Circunferencia Abdominal , Enfermedades Cardiovasculares , Hipercolesterolemia , Menopausia , Posmenopausia , Premenopausia , Riesgo , ArgentinaRESUMEN
Two Bowman-Birk type trypsin inhibitors (CmTI(1) and CmTI(2)) were purified from Cratylia mollis seeds by acetone precipitation, ion exchange, gel filtration and reverse-phase chromatography. CmTI(1) and CmTI(2), with 77 and 78 amino acid residues, respectively, were sequenced in their entirety and show a high structural similarity to Bowman-Birk inhibitors from other Leguminosae. The putative reactive sites of CmTI(1) are a lysine residue at position 22 and a tyrosine residue at position 49. Different reactive sites, as identified by their alignment with related inhibitors, were found for CmTI(2): lysine at position 22 and leucine at position 49. The dissociation constant K(i) of the complex with trypsin is 1.4 nM. The apparent molecular mass is 17 kDa without DDT and 11 kDa with reducing agent and heating.
Asunto(s)
Fabaceae/química , Semillas/química , Inhibidores de Tripsina/química , Inhibidores de Tripsina/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Bovinos , Concentración de Iones de Hidrógeno , Espectrometría de Masas , Datos de Secuencia Molecular , Desnaturalización Proteica , Homología de Secuencia de Aminoácido , Temperatura , Inhibidores de Tripsina/clasificaciónRESUMEN
The first low-molecular-mass metalloprotease presenting prothrombin activating activity was purified from Bothrops insularis venom and named insularinase A. It is a single-chain protease with a molecular mass of 22 639 Da. cDNA sequence analysis revealed that the disintegrin domain of the precursor protein is post-translationally processed, producing the mature insularinase A. Analysis of its deduced amino acid sequence showed a high similarity with several fibrin(ogen)olytic metalloproteases and only a moderate similarity with prothrombin activators. However, SDS-PAGE of prothrombin after activation by insularinase A showed fragment patterns similar to those generated by group A prothrombin activators, which convert prothrombin into meizothrombin independently of the prothrombinase complex. In addition, insularinase A activates factor X and hydrolyses fibrinogen and fibrin. Chelating agents fully inhibit all insularinase A activities. Insularinase A induced neither detachment nor apoptosis of human endothelial cells and was also not able to trigger an endothelial proinflammatory cell response. Nitric oxide and prostacyclin levels released by endothelial cells were significantly increased after treatment with insularinase A. Our results show that, although its primary structure is related to class P-I fibrin(ogen)olytic metalloproteases, insularinase A is functionally similar to group A prothrombin activators.
Asunto(s)
Masculino , Humanos , Animales , Ratones , Bothrops/clasificación , Bothrops/metabolismo , Protrombina/metabolismo , Venenos de Crotálidos/farmacología , Venenos de Crotálidos/aislamiento & purificación , Venenos de Crotálidos/química , Afibrinogenemia/metabolismo , Factor X/metabolismo , Secuencia de AminoácidosRESUMEN
The specific Kunitz Bauhinia ungulata factor Xa inhibitor (BuXI) and the Bauhinia variegata trypsin inhibitor (BvTI) blocked the activity of trypsin, chymotrypsin, plasmin, plasma kallikrein and factor XIIa, and factor Xa inhibition was achieved only by BuXI (K(i) 14 nM). BuXI and BvTI are highly homologous (70%). The major differences are the methionine residues at BuXI reactive site, which are involved in the inhibition, since the oxidized protein no longer inhibits factor Xa but maintains the trypsin inhibition. Quenched fluorescent substrates based on the reactive site sequence of the inhibitors were synthesized and the kinetic parameters of the hydrolysis were determined using factor Xa and trypsin. The catalytic efficiency k(cat)/K(m) 4.3 x 10(7) M(-1)sec(>-1) for Abz-VMIAALPRTMFIQ-EDDnp (lead peptide) hydrolysis by factor Xa was 10(4)-fold higher than that of Boc-Ile-Glu-Gly-Arg-AMC, widely used as factor Xa substrate. Lengthening of the substrate changed its susceptibility to factor Xa hydrolysis. Both methionine residues in the substrate influence the binding to factor Xa. Serine replacement of threonine (P(1)') decreases the catalytic efficiency by four orders of magnitude. Factor Xa did not hydrolyze the substrate containing the reactive site sequence of BvTI, that inhibits trypsin inhibitor but not factor Xa. Abz-VMIAALPRTMFIQ-EDDnp prolonged both the prothrombin time and the activated partial thromboplastin time, and the other modified substrates used in this experiment altered blood-clotting assays.
Asunto(s)
Bauhinia/química , Inhibidores del Factor Xa , Proteínas de Plantas/metabolismo , Inhibidores de Serina Proteinasa/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Bovinos , Factor Xa/química , Colorantes Fluorescentes , Humanos , Cinética , Datos de Secuencia Molecular , Proteínas de Plantas/aislamiento & purificación , Semillas/química , Homología de Secuencia , Inhibidores de Serina Proteinasa/aislamiento & purificación , Especificidad por SustratoRESUMEN
A highly purified trypsin inhibitor was obtained from Echinodorus paniculatus when an extract prepared from E. paniculatus seed flour (25 gl(-1), with 0.1 M ammonium acetate buffer, pH 8.3, under agitation for 6 min at 28 degrees C) was chromatographed on Sephadex G-25 (12 mlh(-1)), followed by affinity chromatography on immobilized Cratylia mollis isolectins (Cra Iso 1,2,3-Sepharose). The column chromatography was performed at 24 degrees C; the matrix was washed (30 mlh(-1)) with 0.1 M sodium phosphate buffer, pH 7.4 or with the same buffer containing 0.2 M glucose, followed by application of inhibitor sample and elution with 0.015 M sodium borate buffer, pH 7.4, or 1.0 M NaCl. A purified fraction of inhibitor was obtained by gel filtration chromatography (GF-450/HPLC column). Trypsin inhibitory activity was eliminated when the inhibitor was treated with metaperiodate showing that the carbohydrate moiety was important for trypsin inhibition. Binding of inhibitor was also evaluated on immobilized concanavalin A (Con A-Sepharose) using previously described chromatographic conditions with results similar to Cra Iso 1,2,3-Sepharose chromatography.
Asunto(s)
Alismataceae/química , Inhibidores Enzimáticos/aislamiento & purificación , Fabaceae/química , Proteínas de Plantas/aislamiento & purificación , Cromatografía de Afinidad , Concentración de Iones de Hidrógeno , Lectinas/química , Semillas/química , Inhibidores de Tripsina , alfa-Amilasas/antagonistas & inhibidoresRESUMEN
The saline extract of Bauhinia bauhinioides dry seeds was shown to inhibit cruzipain, a cysteine proteinase from Trypanosoma cruzi. The inhibitory activity was assigned to a protein with 164 amino acid residues and molecular mass of 18 034 Da that was purified by chromatography on DEAE-Sephadex, trypsin-Sepharose (removal of trypsin inhibitors), Mono Q and a reversed-phase C4 column. The primary structure is homologous to other plant Kunitz-type inhibitors, but it lacks cysteine residues and therefore the disulfide bridges. No methionine residue was identified by amino acid sequencing. The inhibition of cruzipain fits into a slow-tight binding mechanism with a low dissociation constant (Ki 1.2 nM). The studied Bauhinia protein also inhibits cruzain (Ki 0.3 nM), a C-terminally truncated recombinant species of cruzipain. Cathepsin L, a cysteine proteinase with high homology to cruzipain, is also inhibited (Ki 0.22 nM), but not cathepsin B, papain, bromelain or ficin.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/química , Proteínas de Plantas/química , Proteínas Protozoarias/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Cisteína Endopeptidasas/efectos de los fármacos , Inhibidores de Cisteína Proteinasa/aislamiento & purificación , Cinética , Datos de Secuencia Molecular , Estructura Molecular , Proteínas de Plantas/aislamiento & purificación , Semillas/química , Alineación de SecuenciaRESUMEN
Increasing occurrence of hemorrhagic syndrome in man, caused by contact with Lonomia obliqua caterpillars, has been reported in Southern Brazil in the past 10 years. The L. obliqua venom causes a severe consumptive coagulopathy, which can lead to hemorrhagic syndrome. L. obliqua prothrombin activator protease (Lopap) is a 69-kDa prothrombin activator serine protease isolated from L. obliqua caterpillar bristle extract, which is able to evoke thrombus formation, unclottable blood, and fibrinogen depletion when injected into the blood stream of rats. The purified protein generated thrombin from prothrombin, able to clot purified human fibrinogen and plasma. A decrease in platelet count and inhibition of collagen-induced platelet aggregation were observed, as well as leukocyte infiltration in the lungs. In addition, we observed congestion and hemorrhage in renal glomeruli and necrosis in renal distal tubules. These data support the hypothesis that Lopap contributes to the clinical syndrome caused by human contact with L. obliqua, most probably through prothrombin activation, resulting in a consumption coagulopathy.
Asunto(s)
Serina Endopeptidasas/farmacología , Animales , Venenos de Artrópodos/aislamiento & purificación , Venenos de Artrópodos/farmacología , Coagulación Sanguínea/efectos de los fármacos , Humanos , Riñón/irrigación sanguínea , Riñón/efectos de los fármacos , Riñón/patología , Larva , Lepidópteros , Pulmón/irrigación sanguínea , Pulmón/efectos de los fármacos , Pulmón/patología , Masculino , Microcirculación/efectos de los fármacos , Microcirculación/patología , Microscopía por Video , Especificidad de Órganos , Recuento de Plaquetas , Ratas , Ratas Wistar , Serina Endopeptidasas/aislamiento & purificación , Trombosis/inducido químicamenteRESUMEN
A serine proteinase inhibitor was purified from Delonix regia seeds a Leguminosae tree of the Caesalpinioideae subfamily. The inhibitor named DrTI, inactivated trypsin and human plasma kallikrein with K(i )values 2.19x10(-8) M and 5.25 nM, respectively. Its analysis by SDS-PAGE 10-20% showed that the inhibitor is a protein with a single polypeptide chain of M(r) 22 h Da. The primary sequence of the inhibitor was determined by Edman degradation, thus indicating that it contained 185 amino acids and showed that it belongs to the Kunitz type family; however, its reactive site did not contain Arg or Lys at the putative reactive site (position 63, SbTI numbering) or it was displaced when compared to other Kunitz-type inhibitors.
Asunto(s)
Fabaceae/embriología , Péptidos , Proteínas de Plantas , Plantas Medicinales , Semillas/química , Inhibidores de Tripsina/química , Secuencia de Aminoácidos , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Inhibidores de Tripsina/aislamiento & purificaciónRESUMEN
A serine proteinase inhibitor was purified from Bauhinia bauhinioides seeds after extraction with 0.15M NaCl by ion-exchange column chromatography on DEAE-Sephadex, gel filtration on Superose 12 column, Mono Q chromatography or alternatively by affinity chromatography on trypsin- Sepharose. The inhibitor is a single polypeptide chain with molecular mass 20 kDa by gel filtration on Superose 12, but was resolved into two peaks by ion - exchange chromatography on Mono Q (FPLC system). The main eluted peak inhibits trypsin (Ki = 0.6 nM), plasma kallikrein (Ki = 0.35 nM), plasmin (Ki = 33.1 nM), and weakly chymotrypsin (Ki = 2,700 nM), being the most effective plasma kallikrein inhibitor isolated from Bauhinia seeds. Therefore, it was denominated Bauhinia bauhinioides kallikrein inhibitor (BbKI). Activity is thermolabile and on trypsin inhibition optimum pH is 8.0. BbKI displays high homology to other plant Kunitz inhibitors, except for the absence of disulfide bridges, and the only cysteine residue is at the C-terminal position (residue 154) characterizes a distinct member of the Kunitz family. The affinity of the inhibitor to trypsin was confirmed by adsorption to trypsin-Sepharose resin and by isolation of the trypsin-inhibitor complex by gel filtration. Peptides with variations around the reactive site of BbKI (GLPVRFESPLRINIIKESY) were synthesized containing a quenched fluorogenic group. Trypsin but not plasma kallikrein substrates, these peptides strongly inhibited plasma kallikrein.
Asunto(s)
Proteínas de Plantas/farmacología , Calicreína Plasmática/antagonistas & inhibidores , Rosales/química , Inhibidores de Serina Proteinasa/aislamiento & purificación , Inhibidores de Tripsina/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Sitios de Unión , Bovinos , Cromatografía de Afinidad , Colorantes Fluorescentes/metabolismo , Humanos , Hidrólisis , Cinética , Datos de Secuencia Molecular , Peso Molecular , Péptidos/síntesis química , Péptidos/química , Péptidos/farmacología , Proteínas de Plantas/aislamiento & purificación , Calicreína Plasmática/metabolismo , Análisis de Secuencia de Proteína , Homología de Secuencia de Aminoácido , Inhibidores de Serina Proteinasa/genética , Inhibidores de Serina Proteinasa/farmacología , Tripsina/química , Inhibidores de Tripsina/genética , Inhibidores de Tripsina/farmacologíaRESUMEN
We have previously described Kunitz-type serine proteinase inhibitors purified from Bauhinia seeds. Human plasma kallikrein shows different susceptibility to those inhibitors. In this communication, we describe the interaction of human plasma kallikrein with fluorogenic and non-fluorogenic peptides based on the Bauhinia inhibitors' reactive site. The hydrolysis of the substrate based on the B. variegata inhibitor reactive site sequence, Abz-VVISALPRSVFIQ-EDDnp (Km 1.42 microM, kcat 0.06 s(-1), and kcat/Km 4.23 x 10(4) M(-1) s(-1)), is more favorable than that of Abz-VMIAALPRTMFIQ-EDDnp, related to the B. ungulata sequence (Km 0.43 microM, kcat 0.00017 s(-1), and kcat/Km 3.9 x 10(2) M(-1) s(-1)). Human plasma kallikrein does not hydrolyze the substrates Abz-RPGLPVRFESPL-EDDnp and Abz-FESPLRINIIKE-EDDnp based on the B. bauhinioides inhibitor reactive site sequence, the most effective inhibitor of the enzyme. These peptides are competitive inhibitors with Ki values in the nM range. The synthetic peptide containing 19 amino acids based on the B. bauhinioides inhibitor reactive site (RPGLPVRFESPL) is poorly cleaved by kallikrein. The given substrates are highly specific for trypsin and chymotrypsin hydrolysis. Other serine proteinases such as factor Xa, factor XII, thrombin and plasmin do not hydrolyze B. bauhinioides inhibitor related substrates.
Asunto(s)
Colorantes Fluorescentes/farmacología , Calicreínas/metabolismo , Péptidos/farmacología , Plantas/química , Inhibidor de la Tripsina de Soja de Kunitz/farmacología , Secuencia de Aminoácidos , Animales , Sitios de Unión/efectos de los fármacos , Colorantes Fluorescentes/síntesis química , Humanos , Hidrólisis , Calicreínas/efectos de los fármacos , Datos de Secuencia Molecular , Péptidos/síntesis química , Porcinos , Inhibidor de la Tripsina de Soja de Kunitz/aislamiento & purificaciónRESUMEN
Enterolobium contortisiliquum trypsin inhibitor (EcTI) belongs to the Kunitz family of plant inhibitors, which are widely distributed in nature, especially in plant seeds. EcTI is composed of two polypeptide chains with a total of 174 residues, homologous to other inhibitors from the same family. EcTI crystals, which were obtained with the acupuncture-gel technique, diffract to 2.0 A resolution and belong to space group P2(1), with unit-cell parameters a = 37.12, b = 38.42, c = 54.08 A, beta = 98.08 degrees. Molecular-replacement techniques using Erythrina caffra trypsin inhibitor (PDB code 1tie) as the search model indicate one monomer in the asymmetric unit. The secondary-structure content of EcTI was determined by circular dichroism spectroscopy, yielding values compatible with the expected topology.
Asunto(s)
Magnoliopsida/química , Semillas/química , Inhibidores de Serina Proteinasa/química , Dicroismo Circular , Cristalización , Estructura Secundaria de Proteína , Difracción de Rayos XRESUMEN
A second trypsin inhibitor (DMTI-II) was purified from the seed of Dimorphandra mollis (Leguminosae-Mimosoideae) by ammonium sulfate precipitation (30-60%), gel filtration, and ion-exchange and affinity chromatography. A molecular weight of 23 kDa was estimated by gel filtration on a Superdex 75 column SDS-PAGE under reduced conditions showed that DMTI-II consisted of a single polypeptide chain, although isoelectric focusing revealed the presence of three isoforms. The dissociation constant of 1.7 x 10(-9) M with bovine trypsin indicated a high affinity between the inhibitor and this enzyme. The inhibitory activity was stable over a wide pH range and in the presence of DTT. The N-terminal sequence of DMTI-II showed a high degree of homology with other Kunitz-type inhibitors.