RESUMO
INTRODUCTION: Trypsin inhibitors (TIs) have the ability to competitively or non-competitively bind to trypsin and inhibit its action. These inhibitors are commonly found in plants and are used in protease inhibition studies involved in biochemical pathways of pharmacological interest. OBJECTIVES: This work aimed to purify a trypsin inhibitor from Bauhinia pulchella seeds (BpuTI), describing its kinetic mechanism and anticoagulant effect. METHODS: Affinity chromatography, protein assay, and SDS-PAGE were used to purify the inhibitor. Mass spectrometry, inhibition assays, and enzyme kinetics were used to characterize the inhibitor. In vitro assays were performed to verify its ability to prolong blood clotting time. RESULTS: Affinity chromatography on a Trypsin-Sepharose 4B column gave a yield of 43.1. BpuTI has an apparent molecular mass of 20 kDa with glycosylation (1.15%). Protein identification was determined by MS/MS, and BpuTI showed similarity to several Kunitz-type trypsin inhibitors. BpuTI inhibited bovine trypsin as an uncompetitive inhibitor with IC50 (3 x 10-6 M) and Ki (1.05 x 10-6 M). Additionally, BpuTI showed high stability to temperature and pH variations, maintaining its activity up to 100ºC and in extreme pH ranges. However, the inhibitor was susceptible to reducing agents, such as DTT, which completely abolished its activity. BpuTI showed an anticoagulant effect in vitro at a concentration of 33 µM, prolonging clotting time by 2.6 times. CONCLUSION: Our results suggest that BpuTI can be a biological tool to be used in blood clotting studies.
Assuntos
Bauhinia , Inibidores da Tripsina , Animais , Bovinos , Inibidores da Tripsina/farmacologia , Inibidores da Tripsina/química , Bauhinia/metabolismo , Tripsina/análise , Tripsina/química , Tripsina/metabolismo , Espectrometria de Massas em Tandem , Sementes/química , Anticoagulantes/farmacologia , Anticoagulantes/análise , Anticoagulantes/químicaRESUMO
Introducción: la fibrosis quística (FQ) es una enfermedad genética, multisistémica, caracterizada por la disfunción de las glándulas de secreción exocrina secundaria a la mutación de una proteína transmembrana que actúa como canal de cloro. La inclusión de su búsqueda en el Sistema Nacional de Pesquisa Neonatal (SNPN) determinó un gran impacto sobre la morbimortalidad de esta enfermedad, permitiendo un diagnóstico y tratamiento precoz. El íleo meconial (IM) es la manifestación más precoz de la enfermedad, ocurriendo en un 10% a 18% de los pacientes. Objetivo: describir una forma de presentación precoz de FQ con resultado de pesquisa neonatal normal. Caso clínico: 18 meses. Sexo femenino; 33 semanas de edad gestacional. Retraso en la expulsión del meconio. Al tercer día de vida íleo meconial con oclusión intestinal, vólvulo de intestino medio y necrosis intestinal. Se realiza resección quirúrgica. Tripsina inmunorreactiva (TIR) normal a los 7 y 23 días de vida. No se solicita proteína asociada a la pancreatitis (PAP). Desnutrición crónica, bronquiolitis grave a los 3 meses, neumonías virales a los 7 y 11 meses. Ingreso con diagnóstico de neumonía aguda comunitaria con insuficiencia respiratoria. Test del sudor alterado en dos oportunidades. Elastasa pancreática insuficiente. Se aísla Staphylococcus aureus y Pseudomonas aeruginosa en exudado nasal y de secreciones respiratorias. Estudio genético: mutación Delta F508. Con diagnóstico de FQ se inició abordaje multidisciplinario, tratamiento y derivación al centro especializado. Conclusiones: la existencia de IM puede dar falsos negativos en el valor de la TIR, siendo necesaria la dosificación de la PAP. El pronóstico de estos pacientes va a depender de un diagnóstico precoz y el manejo terapéutico oportuno en centros especializados.
Introduction: cystic fibrosis (CF) is a genetic, multisystemic disease, characterized by dysfunction of the exocrine secretion glands secondary to the mutation of a transmembrane protein that acts as a chloride channel. Including its research by the National Neonatal Screening System (SNPN) caused a great impact on the morbidity and mortality of this disease, enabling early diagnosis and treatment. Meconium ileus (MI) is the earliest manifestation of the disease, occurring in 10% to 18% of patients. Objective: to describe a form of early presentation of CF with normal neonatal screening results. Clinical case: 18 months. Female, 33 weeks gestational age. Delay in the expulsion of meconium. On the third day of life, meconium ileus with intestinal obstruction, midgut volvulus and intestinal necrosis. Surgical resection is performed. Trypsin immunoreactive (TIR) normal at 7 and 23 days of age. Pancreatitis-associated protein (PAP) is not requested. Chronic malnutrition, severe bronchiolitis at 3 months, viral pneumonia at 7 and 11 months. Admission with a diagnosis of acute community pneumonia with respiratory failure. Sweat test altered on two occasions. Insufficient pancreatic elastase. Staphylococcus aureus and Pseudomonas aeruginosa were isolated in nasal exudate and respiratory secretions. Genetic study: Delta F508 mutation. With the CF diagnosis, a multidisciplinary approach, treatment and referral to a specialized center began. Conclusions: the existence of MI can provide false negatives in the IRR value, making PAP dosage necessary. The prognosis of these patients will depend on early diagnosis and timely therapeutic management at specialized centers.
Introdução: a fibrose cística (FC) é uma doença genética, multissistêmica, caracterizada pela disfunção das glândulas secretoras exócrinas secundária à mutação de uma proteína transmembrana que age como canal de cloreto. A inclusão da sua pesquisa no Sistema Nacional de Triagem Neonatal (SNPN) determinou grande impacto na morbimortalidade desta doença, permitindo diagnóstico e tratamento precoces. O íleo meconial (IM) é a manifestação mais precoce da doença, ocorrendo em 10 a 18% dos pacientes. Objetivo: descrever uma forma de apresentação precoce da FC com resultados normais de triagem neonatal. Caso clínico: 18 meses. Sexo feminino. 33 semanas de idade gestacional. Atraso na expulsão do mecônio. No terceiro dia de vida, íleo meconial com obstrução intestinal, vólvulo de intestino médio e necrose intestinal. A ressecção cirúrgica é realizada. Imunorreativo à tripsina (TIR) normal aos 7 e 23 dias de vida. A proteína associada à pancreatite (PAP) não é solicitada. Desnutrição crônica, bronquiolite grave aos 3 meses, pneumonia viral aos 7 e11 meses. Internação com diagnóstico de pneumonia comunitária aguda com insuficiência respiratória. Teste do suor alterado em duas ocasiões. Elastase pancreática insuficiente. Staphylococcus aureus e Pseudomonas aeruginosa foram isolados em exsudato nasal e secreções respiratórias. Estudo genético: mutação Delta F508. Com o diagnóstico de FC iniciou-se abordagem multidisciplinar, tratamento e encaminhamento para centro especializado. Conclusões: a existência de IM pode dar falsos negativos no valor da TIR, tornando necessária a dosagem de PAP. O prognóstico destes pacientes dependerá do diagnóstico precoce e do manejo terapêutico oportuno em centros especializados.
Assuntos
Humanos , Feminino , Lactente , Tripsina/análise , Fibrose Cística/complicações , Fibrose Cística/diagnóstico , Íleo Meconial/etiologia , Recém-Nascido Prematuro , Diagnóstico Precoce , Reações Falso-Negativas , Íleo Meconial/cirurgia , Obstrução Intestinal/cirurgiaRESUMO
Exocrine pancreatic insufficiency (EPI), a common disease indogs, is mainly caused by pancreatic acinar atrophy, followedby chronic pancreatitis and pancreatic duct obstruction, withconsequent inadequate secretion of pancreatic enzymes.The present paper is a case report observed in a male eightyear-old dog that had the diagnosis of EPI suggested by thefecal proteolytic activity test (X-ray film proof). To date, thetreatment given with pancreatic enzyme supplementation andadequate diet has been able to promote the resolution of theclinical condition.(AU)
A insuficiência pancreática exócrina (IPE), doença comum principalmenteem cães, é causada, usualmente, pela atrofia acinar dopâncreas, seguida de pancreatite crônica e obstrução do ductopancreático, com consequente secreção inadequada de enzimaspancreáticas. O caso clínico descrito foi observado em um cão,macho, de oito anos de idade, em que o diagnóstico de IPE foilevantado pelo resultado do teste da atividade proteolítica fecal(prova do filme de raio-x). Até o presente momento, o tratamentoinstituído com suplementação de enzimas pancreáticas eadequação da dieta propiciou a resolução do quadro clínico.(AU)
Assuntos
Animais , Cães , /classificação , Insuficiência Pancreática Exócrina/diagnóstico , Insuficiência Pancreática Exócrina/veterinária , Estudos Clínicos como Assunto , Tripsina/análiseRESUMO
Exocrine pancreatic insufficiency (EPI), a common disease indogs, is mainly caused by pancreatic acinar atrophy, followedby chronic pancreatitis and pancreatic duct obstruction, withconsequent inadequate secretion of pancreatic enzymes.The present paper is a case report observed in a male eightyear-old dog that had the diagnosis of EPI suggested by thefecal proteolytic activity test (X-ray film proof). To date, thetreatment given with pancreatic enzyme supplementation andadequate diet has been able to promote the resolution of theclinical condition.
A insuficiência pancreática exócrina (IPE), doença comum principalmenteem cães, é causada, usualmente, pela atrofia acinar dopâncreas, seguida de pancreatite crônica e obstrução do ductopancreático, com consequente secreção inadequada de enzimaspancreáticas. O caso clínico descrito foi observado em um cão,macho, de oito anos de idade, em que o diagnóstico de IPE foilevantado pelo resultado do teste da atividade proteolítica fecal(prova do filme de raio-x). Até o presente momento, o tratamentoinstituído com suplementação de enzimas pancreáticas eadequação da dieta propiciou a resolução do quadro clínico.
Assuntos
Animais , Cães , Insuficiência Pancreática Exócrina/diagnóstico , Insuficiência Pancreática Exócrina/veterinária , Estudos Clínicos como Assunto , Tripsina/análiseRESUMO
Herein described is the biochemical characterisation, including in vitro and in vivo assays, for a proteinase inhibitor purified from Clitoria fairchildiana seeds (CFPI). Purification was performed by hydrophobic interaction and gel filtration chromatography. Kinetic studies of the purified inhibitor showed a competitive-type inhibitory activity against bovine trypsin and chymotrypsin, with an inhibition stoichiometry of 1:1 for both enzymes. The inhibition constants against trypsin and chymotrypsin were 3.3 × 10(-10) and 1.5 × 10(-10)M, respectively, displaying a tight binding property. SDS-PAGE showed that CFPI has a single polypeptide chain with an apparent molecular mass of 15 kDa under non-reducing conditions. However, MALDI-TOF analysis demonstrated a molecular mass of 7.973 kDa, suggesting that CFPI is dimeric in solution. The N-terminal sequence of CFPI showed homology with members of the Bowman-Birk inhibitor family. CFPI remained stable to progressive heating for 30 min to each temperature range of 37 up to 100 °C and CD analysis exhibited no changes in spectra at 207 nm after heating at 90 °C and subsequent cooling. Moreover, CFPI was active over a wide pH range (2-10). In contrast, reduction with DTT resulted in a loss of inhibitory activity against trypsin and chymotrypsin. CFPI also exhibited significant inhibitory activity against larval midgut trypsin enzymes from Anagasta kuehniella (76%), Diatraea saccharalis (59%) and Heliothis virescens (49%). Its insecticidal properties were further analysed by bioassays and confirmed by negative impact on A. kuehniella development.
Assuntos
Clitoria/química , Inseticidas , Inibidores de Proteases , Sementes/química , Animais , Bovinos , Quimotripsina/análise , Inseticidas/química , Inseticidas/isolamento & purificação , Inseticidas/farmacologia , Cinética , Larva/efeitos dos fármacos , Larva/metabolismo , Peso Molecular , Mariposas/efeitos dos fármacos , Mariposas/metabolismo , Inibidores de Proteases/química , Inibidores de Proteases/isolamento & purificação , Inibidores de Proteases/farmacologia , Tripsina/análiseRESUMO
The purpose of this study was to develop a silica nanoparticle-based immunosensor with laser-induced fluorescence (LIF) as a detection system. The proposed device was applied to quantify the immunoreactive trypsin (IRT) in cystic fibrosis (CF) newborn screening. A new ultrasonic procedure was used to extract the IRT from blood spot samples collected on filter papers. After extraction, the IRT reacted immunologically with anti-IRT monoclonal antibodies immobilized on a microfluidic glass chip modified with 3-aminopropyl functionalized silica nanoparticles (APSN-APTES-modified glass chips). The bounded IRT was quantified by horseradish peroxidase (HRP)-conjugated anti-IRT antibody (anti-IRT-Ab) using 10-acetyl-3,7-dihydroxyphenoxazine (ADHP) as enzymatic mediator. The HRP catalyzed the oxidation of nonfluorescent ADHP to highly fluorescent resorufin, which was measured by LIF detector, using excitation lambda at 561nm and emission at 585nm. The detection limits (LODs) calculated for LIF detection and for a commercial enzyme-linked immunosorbent assay (ELISA) test kit were 0.87 and 4.2ngml(-1), respectively. The within- and between-assay variation coefficients for the LIF detection procedure were below 6.5%. The blood spot samples collected on filter papers were analyzed with the proposed method, and the results were compared with those of the reference ELISA method, demonstrating a potential usefulness for the clinical assessment of IRT during the early neonatal period.
Assuntos
Imunoensaio , Nanopartículas/química , Dióxido de Silício/química , Tripsina/análise , Anticorpos Imobilizados/química , Anticorpos Imobilizados/imunologia , Anticorpos Monoclonais/imunologia , Fibrose Cística/diagnóstico , Fibrose Cística/patologia , Teste em Amostras de Sangue Seco , Vidro/química , Peroxidase do Rábano Silvestre/metabolismo , Humanos , Recém-Nascido , Lasers , Técnicas Analíticas Microfluídicas , Tripsina/imunologiaRESUMO
Este estudo teve como objetivo determinar as atividades dos inibidores de tripsina, hemaglutinante e teores de taninos no farelo de soja e na soja crua e processada, e avaliar o coeficiente de digestibilidade aparente da fração proteica para juvenis de pacu. Oscoeficientes de digestibilidade aparente da proteína da soja crua, extrusada, tostada e macerada foram determinadas usando óxido de cromo (0,5%) como marcador. Foi elaborada dieta de referência com 26% de proteína bruta e 4.352 kcal kg-1, e a cada alimentoavaliado foram substituídas 30% da dieta-teste. As fezes foram coletadas por pressão abdominal. Todos os produtos analisados apresentaram fatores antinutricionais, mas foiobservada menor atividade de inibidor de tripsina no farelo de soja. Sojas que receberam tratamento térmico apresentaram os melhores coeficientes de digestibilidade e menoresvalores de atividade hemaglutinante do que a soja crua. Não foram observados efeitos dos inibidores de tripsina e taninos sobre o coeficiente de digestibilidade da proteína, mas foiobservada relação negativa entre os teores de hemaglutinina com a digestibilidade da proteína bruta. Para a alimentação do pacu recomenda-se a utilização do farelo de soja e dasoja processada por extrusão ou tostada.
This study aimed to determine theactivities of trypsin inhibitors, hemagglutinant and tannin levels in soybean meal and in raw andprocessed soy, as well evaluate the protein apparent digestibility coefficient for pacu juveniles. The apparent coefficients of raw, extruded, toasted and milled soy were determined usingchromium oxide (0.5%) as marker. A reference diet was created with 26% crude protein and 4,352 kcal kg-1, with each feed containing 30% of the test diet. Feces were collected by abdominalpressure. All analyzed products presented anti-nutritional factors, but the lowest trypsin inhibitoractivity was observed in soybean meal. Soy that received thermal treatment presented better digestibility coefficients and lower hemagglutinating activity values than raw soy. No effects of trypsin and tannin inhibitor were observed on the protein digestibility coefficient, but a negative relationship was observed between hemagglutinin levels and protein digestibility coefficient. The use of soybean meal and extruded or toasted soy is recommended for pacu feeding.
Assuntos
Animais , Alimentos de Soja/análise , Digestão/fisiologia , Peixes/metabolismo , Hemaglutininas/administração & dosagem , Taninos/administração & dosagem , Tripsina/análiseRESUMO
Este estudo teve como objetivo determinar as atividades dos inibidores de tripsina, hemaglutinante e teores de taninos no farelo de soja e na soja crua e processada, e avaliar o coeficiente de digestibilidade aparente da fração proteica para juvenis de pacu. Oscoeficientes de digestibilidade aparente da proteína da soja crua, extrusada, tostada e macerada foram determinadas usando óxido de cromo (0,5%) como marcador. Foi elaborada dieta de referência com 26% de proteína bruta e 4.352 kcal kg-1, e a cada alimentoavaliado foram substituídas 30% da dieta-teste. As fezes foram coletadas por pressão abdominal. Todos os produtos analisados apresentaram fatores antinutricionais, mas foiobservada menor atividade de inibidor de tripsina no farelo de soja. Sojas que receberam tratamento térmico apresentaram os melhores coeficientes de digestibilidade e menoresvalores de atividade hemaglutinante do que a soja crua. Não foram observados efeitos dos inibidores de tripsina e taninos sobre o coeficiente de digestibilidade da proteína, mas foiobservada relação negativa entre os teores de hemaglutinina com a digestibilidade da proteína bruta. Para a alimentação do pacu recomenda-se a utilização do farelo de soja e dasoja processada por extrusão ou tostada.(AU)
This study aimed to determine theactivities of trypsin inhibitors, hemagglutinant and tannin levels in soybean meal and in raw andprocessed soy, as well evaluate the protein apparent digestibility coefficient for pacu juveniles. The apparent coefficients of raw, extruded, toasted and milled soy were determined usingchromium oxide (0.5%) as marker. A reference diet was created with 26% crude protein and 4,352 kcal kg-1, with each feed containing 30% of the test diet. Feces were collected by abdominalpressure. All analyzed products presented anti-nutritional factors, but the lowest trypsin inhibitoractivity was observed in soybean meal. Soy that received thermal treatment presented better digestibility coefficients and lower hemagglutinating activity values than raw soy. No effects of trypsin and tannin inhibitor were observed on the protein digestibility coefficient, but a negative relationship was observed between hemagglutinin levels and protein digestibility coefficient. The use of soybean meal and extruded or toasted soy is recommended for pacu feeding.(AU)
Assuntos
Animais , Alimentos de Soja/análise , Peixes/metabolismo , Digestão/fisiologia , Hemaglutininas/administração & dosagem , Tripsina/análise , Taninos/administração & dosagemRESUMO
The existence of a diverse serine proteinase gene family in lepidopteran insects suggests they play a significant role in the insect adaptation to plant proteinase inhibitors. These proteinases have been shown to be involved in the process of proteolytic digestion in insect larvae. We carried out a selective transcriptome study of midguts from Spodoptera frugiperda larvae fed on a diet supplemented with soybean proteinase inhibitor (SPI). Using subtracted cDNA libraries made of gut-expressed transcripts, a total of 2100 partial sequences were obtained, of those 38% were related to digestive process. Two large and diverse groups of chymotrypsins and trypsins were obtained, and some of these proteinase-encoding genes were further characterized by quantitative RT-PCR. The transcription analyses revealed two groups: one group of genes constitutively expressed in the control larvae that is up regulated by introducing SPI to the diet, and a second group that is absent in the control but is induced by the SPI-rich diet. This observation suggests that adaptation of S. frugiperda to SPI involves de novo synthesis and also up regulation of existing enzymes. Proteases from intestines of larvae reared on a diet with SPI showed insensitivity to the inhibitor. The proteases were also insensitive to a broad-spectrum potato proteinase inhibitor preparation. We propose that adaptation of S. frugiperda to SPI follows a "shotgun" approach, based on a general up regulation of a large set of endoproteinases.
Assuntos
Endopeptidases/metabolismo , Inibidores de Proteases/metabolismo , Spodoptera/enzimologia , Sequência de Aminoácidos , Animais , Quimotripsina/análise , Quimotripsina/genética , Endopeptidases/genética , Larva/enzimologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Inibidores de Proteases/isolamento & purificação , Glycine max/química , Spodoptera/genética , Tripsina/análise , Tripsina/genética , Regulação para CimaRESUMO
A review dedicated mainly to the results obtained by the authors on the use of cyclodextrin (CD) derivatives on protein (enzyme) stabilization through covalent and non-covalent interactions (host-guest supramolecular interactions) is presented here. This latter procedure served to introduce a new method for enzyme immobilization on metallic surfaces that can be used to prepare biosensors and therapeutic nanodevices. The surfaces of gold (and silver) electrodes and nanoparticles were modified with sulphur-containing cyclodextrin derivatives. The protein (enzyme) was then supramolecularly immobilized on the modified surface when one or more of its bulky hydrophobic moieties was included into the CD cavity. The protein can also be modified with a typical CD guest, such as adamantane, to achieve a more stable immobilization. Different examples are presented, such as a biosensor based on monolayers of adamantane-modified cytochrome c and a bienzymatic nanodevice comprising gold nanoparticles stabilized with CD associated to catalase and superoxide dismutase modified with complementary host-guest residues. The possibilities of this new approach for the development of biosensors and therapeutic nanodevices are analyzed.
Assuntos
Técnicas Biossensoriais/instrumentação , Ciclodextrinas/análise , Ciclodextrinas/química , Sistemas de Liberação de Medicamentos/métodos , Nanomedicina/instrumentação , Tripsina/análise , Tripsina/química , Técnicas Biossensoriais/métodos , Técnicas Biossensoriais/tendências , Materiais Revestidos Biocompatíveis/química , Sistemas de Liberação de Medicamentos/instrumentação , Sistemas de Liberação de Medicamentos/tendências , Desenho de Equipamento , Substâncias Macromoleculares/química , Nanomedicina/métodos , Nanomedicina/tendênciasRESUMO
A monoclonal antibody (MAb) directed against human immunoaffinity purified trypsinogen has been produced by hybridization of myeloma cells with spleen cells of BALB/c immunized mice. Antibodies were screened by ultramicro-enzyme-linked immunosorbent assay (UMELISA). The MAb was purified by affinity chromatography on protein A-sepharose, and MAb had a high affinity for trypsinogen with the affinity constant equal 2.06 x 10(9) L/mol. Specificity was studied by UMELISA using cross-reactant proteins; MAb gave a positive reaction with native trypsinogen-1 but did not react with the same protein after reduction. The antibody seem to be directed against conformational epitope. The MAb obtained was characterized immunologically and used to develop UMELISA for detection Trypsin. This monoclonal assay enabled the detection of 2.8 ng/mL.
Assuntos
Anticorpos Monoclonais/imunologia , Tripsina/imunologia , Animais , Fibrose Cística/diagnóstico , Ensaio de Imunoadsorção Enzimática , Humanos , Recém-Nascido , Camundongos , Camundongos Endogâmicos BALB C , Triagem Neonatal , Tripsina/análise , Tripsina/sangue , Tripsinogênio/análise , Tripsinogênio/sangue , Tripsinogênio/imunologiaRESUMO
We describe a patient in whom newborn immunoreactive trypsin screening and mutation analysis suggested a diagnosis of cystic fibrosis; however, the clinical course and sweat test results were not consistent with the diagnosis. Direct sequencing of the patient's genomic DNA showed compound heterozygosity for delta F508 and F508C, a polymorphism not associated with clinical disease.
Assuntos
Fibrose Cística/diagnóstico , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Análise Mutacional de DNA , Reações Falso-Positivas , Técnicas Genéticas , Humanos , Recém-Nascido , Masculino , Mutação Puntual , Reação em Cadeia da Polimerase , Tripsina/análiseRESUMO
The albumin and globulin fractions from lentil seeds were isolated and characterised by gel filtration. The latter was shown to be homogeneous and the formet heterogeneous on PAGE, the aminoacid analysis revealed high values of amidic amino acids for both fractions with great differences in the sulphur-containing amino acids. Native albumin, globulin and salt-soluble proteins were markedly resistant to trypsin hydrolysis compared to casein. The SDS-PAGE of native salt-soluble proteins indicated that the globulin fragments (20 to 30kD) were slowly digested in the presence of albumin. The heating increased the hydrolysis of the proteins in the order: salt-soluble, albumin and globulin. The facilitated hydrolysis of the heated salt-soluble fraction seemed to be protein-protein interactions induced by heat
Assuntos
Fabaceae/anatomia & histologia , Hidrólise , Técnicas In Vitro , Proteínas/análise , Tripsina/análiseRESUMO
O objetivo deste trabalho foi analisar a lesäo hepática desenvolvida durante a PA através do estudo da funçäo mitocondrial hepática. Para tanto, 54 ratos machos Wistar foram distribuídos em seis grupos: GI (controle n=6), GII (PA duas horas n=12), GIII (PA quatro horas n=6), GIV (PA 12 horas n=8), GV (PA 24 horas n=7) e GVI (PA 48 horas n=7). A PA foi induzida por injeçäo retrógrada de taurocolato de sódio a 5 por cento. A avaliaçäo da funçäo mitocondrial hepática foi determinada polarograficamente com eletrodo de Clark, medindo-se o consumo de O2 na ausência de ADP (S4-basal) e na presença de ADP (S3 ativado), utilizando-se succinato de potássio como sunstrato. A razäo do controle respiratório (RCR) e a relaçäo ADP/O foram calculadas. Na fase precoce da PA (duas e quatro horas), existe desacoplamento das mitocôndrias evidenciado por aumento do S4 e diminuiçäo do RCR e da relaçäo ADP/O, No período de 12 e 24 horas após a PA, o RCR apresenta valores iguais aos do controle. Conclui-se que as alteraçöes mitocondriais na PA säo bifásicas: as alteraçöes precoces säo caracterizadas por desacoplamento da oxigenaçäo fosforilativa e talvez resulte da açäo à distância de enzimas de produtos da açäo enzimática, enquanto as alteraçöes tardias säo decorrentes de lesöes estruturais e graves, resultantes de isquemia tecidual. Estudos hemodinâmicos devem ser efetuados para esclarecer estas alteraçöes
Assuntos
Animais , Masculino , Ratos , Amilases/análise , Líquido Ascítico/química , Mitocôndrias Hepáticas , Pancreatite/induzido quimicamente , Proteínas/análise , Tripsina/análise , Doença Aguda , Modelos Animais de Doenças , Polarografia , Ratos WistarRESUMO
Gut absorption is one of the first requirements for the study of the mechanism of a possible anti-inflammatory action of proteases, such as orally administered trypsin. Porcine trypsin absorption was studied in isolated jejunal loops of rats (female Holtzman and male Wistar) and guinea pigs (males) by open-loop perfusion. Trypsin was dissolved in Tyrode solution and the solution perfused at a rate of 0.5 ml/min, at 37 degrees C. Trypsin activity, total protein, and sodium and potassium concentrations were assayed in the jejunal effluent; the values were unchanged throughout the experiments, which lasted 45 to 120 min. Using a high sensitivity ELISA (i.e. pg/ml), trypsin absorption could be demonstrated by determination of the enzyme in the mesenteric venous blood (samples of 0.5 ml); the enzyme concentration increased with time of perfusion. The linear range-specificity for intact trypsin varied from 1 to 500 ng/well. In this assay polyclonal antibodies prepared against trypsin-TLCK were utilized. Whereas trypsin concentration in the perfused lumen was practically constant at 0.12 mg/ml, the concentration of absorbed trypsin in mesenteric vein blood increased from about 100 ng/ml at time zero to 1.8 micrograms/ml, after 45 min of perfusion. Histological and ultrastructural examination of the jejunal mucosa before and after perfusion revealed that the brush-border, basal membrane, and junctional complexes were fully preserved, thus eliminating the possibility that trypsin might have destroyed the structures, thereby reaching the blood circulation. The present data indicate that micrograms quantities of trypsin were absorbed by the isolated jejunal loop of the rat.
Assuntos
Absorção Intestinal , Jejuno/metabolismo , Tripsina/metabolismo , Animais , Ensaio de Imunoadsorção Enzimática , Feminino , Cobaias , Jejuno/ultraestrutura , Masculino , Perfusão/métodos , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Tripsina/análiseRESUMO
Gut absorption is one of the first requirements for the study of the mechanism of a possible anti-inflammatory action of proteases, such as orally administered trypsin. Porcine trypsin absorption was studied in isolated jejunal loops of rats (female Holtzman and male Wistar) and guinea pig (males) by open-loop perfusion. Trypsin was dissolved in Tyrode solution and the solution perfused at a rate of 0.5 ml/min, at 37§C. Trypsin activity, total protein, and sodium and potassium concentrations were assayed in the jejunal effluent; the values were unchanged throughout the experiments, which lasted 45 to 120 min. Using a high sensitivity ELISA (i.e. pg/ml), trypsin absorption could be demonstrated by determination of the enzyme in the mesenteric venous blood (samples of 0.5 ml); the enzyme concentration increased with time of perfusion. The linear range-specificity for intact trypsin varied from 1 to 500 ng/well. In this assay polyclonal antibodies prepared against trypsin-TLCK were utilized. Whereas trypsin concentration in the perfused lumen was practically constant at 0.12 mg/ml, the concentration of absorbed trypsin in mesenteric vein blood increased from about 100 ng/ml at time zero to 1.8 µg/ml, after 45 min of perfusion. Histological and ultrastructural examination of the jejunal mucosa before and after perfusion revealed that the brush-border, basal membrane, and junctional complexes were fully preserved, thus eliminating the possibility that trypsin might have destroyed the structures, thereby reaching the blood circulation. The present data indicate that µg quantities of trypsin were absorbed by the isolated jejunal loop of the rat
Assuntos
Animais , Masculino , Feminino , Ratos , Cobaias , Absorção Intestinal , Jejuno/metabolismo , Tripsina/metabolismo , Ensaio de Imunoadsorção Enzimática , Jejuno/ultraestrutura , Perfusão/métodos , Ratos Sprague-Dawley , Ratos Wistar , Tripsina/análiseRESUMO
We compared pancreatic acinar and ductal secretion in two patients with Johanson-Blizzard syndrome, age-matched control subjects, and patients with other primary pancreatic diseases. Patients with Johanson-Blizzard syndrome had preservation of ductular output of fluid and electrolytes, as in patients with Shwachman syndrome but differing from those with cystic fibrosis, who have a primary ductular defect. They also had decreased acinar secretion of trypsin, colipase and total lipase, and low serum immunoreactive trypsinogen levels, consistent with a primary acinar cell defect.
Assuntos
Insuficiência Pancreática Exócrina/fisiopatologia , Pâncreas/fisiopatologia , Estudos de Casos e Controles , Colipases/análise , Consanguinidade , Fibrose Cística/enzimologia , Fibrose Cística/fisiopatologia , Insuficiência Pancreática Exócrina/enzimologia , Feminino , Humanos , Lactente , Recém-Nascido , Lipase/análise , Síndromes de Malabsorção/enzimologia , Síndromes de Malabsorção/fisiopatologia , Masculino , Pâncreas/enzimologia , Pâncreas/metabolismo , Pancreatopatias/enzimologia , Pancreatopatias/fisiopatologia , Ductos Pancreáticos/enzimologia , Ductos Pancreáticos/metabolismo , Síndrome , Tripsina/análise , Tripsinogênio/sangueRESUMO
Este estudio es un análisis retrospectivo de 23 pacientes con carcinoma de mama en estadio avanzado, seguidos entre seis y 51 meses con promedio de 31 meses. El receptor estrogénico (RE) se cuantificó con el método de carbón cubierto con dextrán y la actividad semejante a tripsina por medio de espectrofluorometría. El porcentaje de muestras con RE+ en pacientes ò 50 años fue 83 por ciento y la mediana de 148 fmoles/mg de proteína (intervalo de 10 a 1,847), mientras que en pacientes ó 49 años las muestras RE+ fueron 64 por ciento y la mediana de 33 fmoles/mg de proteína (intervalo de 12 a 147) (p<0.05). Por otro lado, el análisis estadístico de los valores de la actividad semejante a tripsina no mostró diferencias significativas entre estos grupos. La respuesta al tratamiento con tamoxifeno fue favorable en 92 por ciento de las pacientes ò 50 años y en 55 por ciento de ó 49 años (p<0.05). La presencia de un porcentaje más alto de positividad al RE, así como de valores intrínsecos más altos de RE en pacientes ò 50 años en comparación con los de las enfermas ó 49 años, parece tener relación con una mejor respuesta al tamoxifeno en las primeras. Por otro lado, no encontramos correlación entre los valores de actividad semejante a tripsina y la respuesta al tamoxifeno.