Cloning and expression of trypsin-encoding cDNA from Blattella germanica and its possibility as an allergen

In this study, the trypsin gene (bgtryp-1) from the German cockroach, Blattella germanica, was cloned via the immunoscreening of patients with allergies to cockroaches. Nucleotide sequence analysis predicted an 863 bp open reading frame which encodes for 257 amino acids. The deduced amino acid sequence exhibited 42-57% homology with the serine protease from dust mites, and consisted of a conserved catalytic domain (GDSGGPLV). bgtryp-1 was determined by both Northern and Southern analysis to be a 0.9 kb, single-copy gene. SDS-PAGE and Western blotting analyses of the recombinant protein (Bgtryp-1) over-expressed in Escherichia coli revealed that the molecular mass of the expressed protein was 35 kDa, and the expressed protein was capable of reacting with the sera of cockroach allergy patients. We also discussed the possibility that trypsin excreted by the digestive system of the German cockroach not only functions as an allergen, but also may perform a vital role in the activation of PAR-2.

Trypsin and aminopeptidase activities in blood-fed females Anopheles dirus (Diptera: Culicidae) of differing susceptibility to Plasmodium yoelii nigeriensis.

Midgut proteolytic enzymes contribute to the success or failure of Plasmodium infection of the mosquito. The present study investigated trypsin and aminopeptidase activities in the midgut of two strains of Anopheles dirus selected for susceptibility and refractoriness to Plasmodium yoelii nigeriensis. At intervals of 6 hours following a bloodmeal, the midguts of fully engorged female mosquitos were dissected, homogenized, and assayed for enzyme activity. No differences trypsin activity (nmole/min) were observed between the two strains throughout the course of blood digestion. By contrast, the aminopeptidase activity measured at 0 to 18 hours post-feeding was the same for the two strains, but at 24, 30 and 36 hours significantly less activity was observed in the refractory females. The results suggest neither trypsin nor aminopeptidase plays a role in the limitation of parasite development.

Isolation and in vitro hydrolysis of lentil protein fractions by trypsin / Aislamiento e hidrólisis \"in vitro\" de las fracciones proteínicas de la lenteja por la tripsina

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

Lesäo hepática na pancreatite aguda: estudo experimental em ratos / Hepatic lesion in acute pancreatitis: experimental study in rats

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

Jejunal absorption of trypsin in rat and guinea pig

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

Niveles de receptor estrogénico y actividad semejante a tripsina en cáncer mamario y respuesta a tamoxifeno / Estrogen receptor levels and trypsin-like activity in breast cancer and tamoxifen response

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.

Silanized silica bound trypsin as analytical probe.

Trypsin immobilized by covalent coupling to silanized silica shows significant activity (30-38%) and greater thermostability as compared to soluble trypsin. Proteolytic processing of albumin at varying periods suggest that the enzyme matrix can be used efficiently for limited proteolysis. Repeated use of the immobilized enzyme in protein digestion produces similar products as seen by electrophoretic analysis. Also, digestion of albumin by the immobilized enzyme follows similar pattern as that by soluble enzyme. The enzyme matrix can be easily removed from the incubation mixture. The results indicate the possibility of the immobilized enzyme for its effective application as analytical tool in peptide mapping and limited proteolytic processing.

Estudo duplo-cego, comparativo, sobre a disponibilidade sanguínea e em tecido amigdaliano da ampicilina trihidratada sob forma isolada e em combinaçäo com tripsína e quimotripsina / Controlled and double-blind study on blood and tonsil tissue bioavaibility of trihydrated ampicillin under isolated form and in combination with trypsin and chymotrypsin

Avaliaram-se em 50 pacientes os níveis sangüíneos e amigdalianos da ampicilina trihidratada administrada sob forma isolada e em combinaçäo com a tripsina e quimotripsina. Os resultados mostraram que: 1) em ambos os grupos os níveis sangüíneos foram sempre superiores aos teciduais; 2) com exceçäo da 1ª dose, todos os demais níveis séricos da ampicilina associada às enzimas foram superiores aos da ampicillina isolada; 3) todos os níveis teciduais da ampicilina com enzimas foram superiores aos da ampicilina isolada; 4) tanto os níveis séricos como teciduais da ampicilina com enzimas mostraram concentraçäo bem acentuada no tempo de 4 horas. Conclui-se que a associaçäo da ampicilina com as enzimas tripsina e quimotripsina deverá ser mais útil no tratamento de infecçöes amigdalianas do que a ampicilina isolada