Obtainment and characterization of digestive aspartic proteases from the fish Caranx hippos (Linnaeus, 1766) / Obtenção e caracterização de proteases aspárticas digestórias do peixe Caranx hippos (Linnaeus, 1766)

This work aimed to obtain aspartic proteases of industrial and biotechnological interest from the stomach of the crevalle jack fish (Caranx hippos). In order to do so, a crude extract (CE) of the stomach was obtained and subjected to a partial purification by salting-out, which resulted in the enzyme extract (EE) obtainment. EE proteases were characterized physicochemically and by means of zymogram. In addition, the effect of chemical agents on their activity was also assessed. By means of salting-out it was possible to obtain a purification of 1.6 times with a yield of 49.4%. Two acid proteases present in the EE were observed in zymogram. The optimum temperature and thermal stability for EE acidic proteases were 55 ºC and 45 °C, respectively. The optimum pH and pH stability found for these enzymes were pH 1.5 and 7.0, respectively. Total inhibition of EE acid proteolytic activity was observed in the presence of pepstatin A. dithiothreitol (DTT) and Ca2+ did not promote a significant effect on enzyme activity. In the presence of heavy metals, such as Al3+, Cd2+ and Hg2+, EE acidic proteases showed more than 70% of their enzymatic activity. The results show that it is possible to obtain, from the stomach of C. hippos, aspartic proteases with high proteolytic activity and characteristics that demonstrate potential for industrial and biotechnological applications.

Este trabalho objetivou obter proteases aspárticas de interesse industrial e biotecnológico a partir do estômago do peixe xaréu (Caranx hippos). Para isso, foi obtido um extrato bruto do estômago, o qual foi submetido a uma purificação parcial por salting-out onde se obteve o extrato enzimático (EE). As proteases do EE foram caracterizadas físico-quimicamente e através de zimograma. Além disso, o efeito de agentes químicos sobre sua atividade também foi avaliado. Através de salting-out foi possível obter uma purificação de 1,6 vezes com rendimento de 49,4%. Foram observadas duas proteases ácidas presentes no EE através de zimograma. A temperatura ótima e a estabilidade térmica para as proteases ácidas do EE foram de 55 ºC e 45 °C, respectivamente. O pH ótimo e a estabilidade ao pH encontrados para estas enzimas foram o pH 1,5 e 7,0, respectivamente. Observou-se a inibição total da atividade proteolítica ácida do EE na presença de pepstatina A. O ditiotreitol (DTT) e o Ca2+ não promoveram efeito significativo na atividade enzimática. Na presença de metais pesados, como Al3+, Cd2+ e Hg2+, o EE manteve mais de 70% de atividade enzimática do EE. Os resultados mostram que é possível obter, a partir do estômago de C. hippos, proteases aspárticas com alta atividade proteolítica e características que demonstram potencial para aplicações industriais e biotecnológicas.

Histological, biochemical and pharmacologycal characterization of the gastric muscular layer in Chagas disease / Caracterização histológica, bioquímica e farmacológica da musculatura gástrica na doença de Chagas

PURPOSE: To assess in vitro the correlation between the number of neurons and the sensitivity to cholinergic drugs and acetylcholinesterase activity in chagasic patients. METHODS: A 3x1 cm strip of the muscle layer of the anterior part of the stomach, always close to the angular incisure, was removed from 10 chronic chagasic patients (6 men) submitted to megaesophagus or megacolon surgery and from 10 non-chagasic patients (4 men) submitted to other types of surgery (control group), aged on average 52.3 and 50.1 years, respectively, for histological and pharmacological studies. The action of cholinergic drugs was investigated in isolated preparations according to the superfusion method of Ferreira and Costa, and acetylcholinesterase activity was determined by the method of Ellman. For neuron count, the strips were cut into 8 µm sections according to the method standardized by Alcântara. RESULTS: There was a difference in number of neurons between the chagasic (5,6) and control (7,3) groups. Acetylcholinesterase activity, in moles of hydrolyzed substrate per minute per gram tissue, was reduced in chagasic patients (4,32) compared to the controls (7,30). No hypersensitivity of the gastric musculature to cholinergic drugs was detected, with a reduced maximum response to carbachol and betanechol in the chagasic group. CONCLUSIONS: The reduction of neurons in the myenteric plexus of the stomach of chronic chagasic patients can be demonstrated even in the absence of clinical chagasic gastropathy. The hypersensitivity of the gastric musculature to cholinergic drugs probably depends on intense denervation. The reduced acetylcholinesterase activity demonstrates the involvement of the cholinergic innervation in the stomach of chronic chagasic patients. There was no correlation between number of neurons, sensitivity to cholinergic drugs and acetylcholinesterase activity in the gastric musculature of chagasic and non-chagasic patients.

OBJETIVO: Avaliar in vitro a correlação entre o número de neurônios e a sensibilidade a drogas colinérgicas e a atividade da acetilcolinesterase em pacientes chagásicos. MÉTODOS: Em 10 pacientes chagásicos crônicos (6 homens) submetidos à cirurgia de megaesôfago ou de megacólon e em 10 pacientes não chagásicos (4 homens) submetidos a outros tipos de cirurgia (grupo controle), respectivamente com idade média de 52,3 e 50,1 anos, retirou-se uma tira de 3x1 cm da camada muscular da parede anterior do estômago, sempre junto á cisura angular, que serviu para os estudos histológicos e farmacológicos. A ação de drogas colinérgicas foi feita em preparação isolada de acordo com o método de superfusão de Ferreira e Costa, e a determinação da atividade da acetilcolinesterase pelo método de Ellman. Para a contagem de neurônios a tira muscular foi submetida a cortes de 8 micra segundo método padronizado por Alcântara. RESULTADOS: Houve diferença do número de neurônios entre os grupos chagásico (5,6) e controle (7,3). A atividade da acetilcolinesterase mostrou-se diminuída nos chagásicos (4,32) expressa como número de moles do substrato hidrolisado por minuto por grama de tecido, em relação aos controles (7,30). Não se encontrou hipersensibilidade da musculatura gástrica a drogas colinérgicas, encontrando-se inclusive efeito máximo reduzido ao carbacol e betanecol no grupo chagásico. CONCLUSÕES: A redução de neurônios no plexo mioentérico do estômago de pacientes chagásicos crônicos pode ser demonstrada mesmo na ausência de gastropatia chagásica clínica. A hipersensibilidade da musculatura gástrica a drogas colinérgicas provavelmente depende de desnervação intensa. A redução da atividade da acetilcolinesterase demonstra o comprometimento da inervação colinérgica no estômago de pacientes chagásicos crônicos. Não houve correlação entre número de neurônios, sensibilidade a drogas colinérgicas e atividade da acetilcolinesterase na musculatura gástrica de pacientes chagásicos ou não chagásicos.

Azadirachta indica leaf extract modulates initiation phase of murine forestomach tumorigenesis.

The effects of aqueous Azadirachta indica leaf extract (AAILE) on benzo(a)pyrene [B(a)P]-induced forestomach tumorigenesis, B(a)P-DNA adduct formation and certain parameters of carcinogen biotransformation system in mice have been reported earlier from our laboratory. In this study, the effects of AAILE on the enzymes of B(a)P biotransformation, which play crucial role in initiation of chemical carcinogenesis - aryl hydrocarbon hydroxylase (AHH) and uridinediphosphoglucuronosyltransferase (UDP-glucuronosyltransferase) have been evaluated in murine forestomach and liver. In addition, lipid peroxidation (LPO) levels in forestomach as well as liver and the activities of tissue injury marker enzymes - lactate dehydrogenase, aspartate aminotransferase and alkaline phosphatase in the serum have also been evaluated. Oral administration of AAILE (100 mg/kg body wt for 2 weeks) reduces the AHH activity and enhances the UDP-glucuronosyltransferase activity in both the tissues, suggesting its potential in decreasing the activation and increasing the detoxification of carcinogens. The LPO levels decrease upon AAILE treatment in the hepatic tissue, suggesting its antioxidative and hence anti-carcinogenic effects. Non-significant alterations have been observed in tissue injury marker enzymes upon AAILE treatment, suggesting its safety at the given dose. In conclusion, AAILE appears to modulate initiation phase of carcinogenesis and may be suggested as safe and an effective agent for chemoprevention.

Common Whelk (Buccinum undatum)Allergy:Identification of IgE-binding Components and Effects of Heating and Digestive Enzymes

In Korea, common whelk (Buccinum undatum) is a popular edible shellfish. The aim of this study was to observe the sensitization rate to common whelk and to characterize its allergens. We carried out skin prick test (SPT) in 1,700 patients with various allergic diseases. Specific IgE were detected by ELISA in the patient sera and ELISA inhibition tests were conducted. IgE-binding components were identified by means of SDS-PAGE and IgE-immunoblotting. The effects of diges-tive enzymes were evaluated in both raw and thermally treated extracts. SPT to common whelk was positive (> or =2+) in 83 (4.9%) patients studied. Twenty-four (38.7%) out of 62 SPT positive patients had high serum specific IgE to common whelk. ELISA inhibition test showed significant inhibitions by abalone as well as by common whelk. IgE-immunoblotting demonstrated three IgE-binding components (40, 71, 82 kDa), which were digested by simulated intestinal fluid and moderately digested by simulated gastric fluid, and the digestibility of allergens remained unchanged after thermal treatment. In conclusion, IgE-sensitization rate to com-mon whelk was 4.9% in allergy patients. IgE-immunoblotting demonstrated three IgE-binding components, which were degraded by digestive enzymes. Further studies are needed to evaluate the clinical significance of the sensitized patients to common whelk.

Measurement of Telomerase Activity and Telomerase Reverse Transcriptase Expression in Gastric Fluid and Tissue for Early Diagnosis of Stomach Cancer / 대한소화기학회지

BACKGROUND/AIMS: Telomerase activity and telomerase reverse transcriptase (TERT) expression have been proposed as a marker for malignancy. However, little is known about those markers in intestinal metaplasia (IM). This study was performed to evaluate the usefulness of telomerase activity in gastric washing fluid and TERT expression in tissue as a marker for early diagnosis of stomach cancer. METHODS: Gastric washing fluid and biopsies were taken endoscopically. We examined the telomerase activity by telomeric repeat amplification protocol (TRAP) and the TERT expression by semiquantitative reverse transcription-polymerase chain reaction in 26, 21 and 15 cases of cancer, IM, and normal mucosa respectively. RESULTS: The telomerase activity was positive in 65% of cancer, 44% of incomplete IM, and 33% of complete IM. The TERT was expressed in 89% of cancer, 81% of IM, but not in normal mucosa. The TERT expression level was higher in cancer and incomplete IM than in complete IM and normal mucosa (p<0.05). CONCLUSIONS: Telomerase activity in gastric washing fluid and TERT expression in tissue may have limited usefulness as a marker for the early diagnosis of stomach cancer. However, the increased levels of TERT expression in IM and cancer suggest that TERT expression may be associated with carcinogenesis in stomach cancer.

NADH diaphorase positive myenteric neurons of the aglandular region of the stomach of rats subjected to desnutrition

El estómago realiza movimientos caracterizados por potentes ondas peristálticas lentas, útiles en la mezcla del bolo alimenticio, la digestión mecánica y su lenta liberación por el píloro. El control de este movimiento se realiza por acción hormonal y por actividad nerviosa, destacando la inervación intrísica que representan las neuronas del plexo miotérico. Considerando que un estado de desnutrición puede provocar alteraciones morfológicas, incluso neuronas, se ha propuesto este trabajo, con el objetivo de verificar los efectos provocados por la desnutrición proteínica durante el período de 120 días, en el aspecto cuantiativo de las neuronas NADH-diaforasa positivas. Para este estudio se utilizaron 10 ratones adultos, los cuales, a los 90 días de vida fueron divididos en dos grupos; control y desnutridos. A los ratones del grupo desnutridos (n=5) se les alimentó con una dieta proteínica de, aproximadamente, 8 por ciento y a los ratones del grupo control (n=5) con dieta de valor proteínico normal, 22 por ciento. Transcurridos 120 días, al estómago aglandular se le hizo un ensayo de histoenzimología del NADH-diaforasa, para verificar características neuronales. La región glandular fue dividida en dos áreas: una proxima al lóbulo gástrico mayor y la otra, anterior al repliegue límite. En el recuento por muestreo, las neuronas observadas en 40 campos microscópicos (6,64mm²) de cada área, de las muestas de los ratones de los grupos control y desnutridos, fueron contadas con un microscopio de 40x. Constatamos entonces que las neuronas pueden estar aisladas o agrupadas en sus ganglios. Cerca del repliegue límite encontramos un promedio de 570,8 neuronas en el grupo control y 718,4 en el grupo desnutridos. Por otra parte, cerca del lóbulo gástrico mayor, encontramos 25,8 neuronas en el grupo control y 52,6 en el grupo desnutridos. La dieta hipoproteínica implicó un menor crecimiento físico, con una descompensación de alrededor de 30,33 por ciento, al compararlo con ratones del grupo control, como también hubo una reducción de la superficie del perfil estomacal, de aproximadamente 20.13 por ciento. Concluimos que, las neuronas no están distribuidas uniformemente en la pared estomacal y que en los ratones desnutridos presentaron una menor dispersión, presentando por lo tanto una mayor densidad neuronal por mm²

Mecanismos de açäo e controle da digestäo de proteínas e peptídios em humanos / Human protein digestive activity and control

Este trabalho objetiva rever os principais mecanismos controladores da digestão de proteínas e peptídeos de interesse especial para humanos. O processo de digestão e aproveitamento protéico tem início no estômago com ações não tão indispensáveis como as do lúmen duodeno/jejunal. Entretanto, mesmo esses processos intestinais estão parcialmente sob controle da secreção gástrica. As atividades proteolíticas das enzimas são relacionadas à estrutura protéica e seus aminoácidos constituintes. Estruturas terciárias e quaternárias necessitam da desnaturação com ácido clorídrico antes de sofrerem hidrólise enzimática. A seguir, há ação das exopeptidases guiadas pelos terminais NH2 (amino peptidases) e COOH (carboxipeptidases) da molécula, enquanto as endopeptidases são orientadas pelos aminoácidos constituintes da molécula peptídica. Deste modo, tanto os polipeptídios e proteínas dietéticas como as secretadas no lúmen sofrem proteólise limitada ou completa resultando di-octapeptídios (60 por cento) ou aminoácidos livres (40 por cento). As peptidases da borda em escova continuam a degradação até di-tripeptídios ou aminoácidos livres (neutros). Alguns peptídios escapam a essa ação e são captados pelo enterócito, cujas peptidases citosólicas completam a hidrólise. Desta forma, os produtos da digestão, circulantes na veia porta, são constituídos de aminoácidos livres e alguns di-tripeptídios. Os processos mecânicos e químicos da digestão sofrem controle neural (vagal), neuroendócrino (acetilcolina), endócrino (gastrina, secretina, colecistocinina) ou parácrino (histamina). A fase gástrica (secreções de ácido clorídrico e pepsinogênio) é ativada pela gastrina, histamina e acetilcolina em resposta tanto aos aminoácidos dietéticos (triptofano e fenilalanina) como a distensão mecânica do estômago. A secreção pancreática é estimulada pelas fases cefálica e gástrica e influencia a fase intestinal da digestão. As células intestinais tipos S e I liberam secretina e colecistocinina, respectivamente, em resposta ao quimo ácido (célula S) ou aminoácidos e peptídios no lúmen (célula I). A secretina estimula a lilberação de água, bicarbonato e enteropeptidases enquanto a colecistocinina libera as enzimas pancreáticas.

Purificacion y actividad de la pepsina de pollo / Purification and assay of chicken pepsin

The proteolitic enzyme pepsin (EC 3.4.23.1) was purified from chicken stomach by a modification of the method of Bohak (1970): after homogenazing the raw material, the zymogen was extracted with NaCl and NaHCO3, activated with 3N HCl and precipitated with NaCl (28 per cent final concentration). The precipitate was lyophilised; fractions of it were suspended in 0.02N HCl. The solution was filtered through a column (2.6 x 80 cm) of Sephadex G-100 at an elution rate of 8-10 ml x cm-2 x h-1. Two protein peaks were obtained, the first one corresponding to the pepsin (2.0 mg/ml in pooled fractions). The milk clotting activity of the enzyme was determined on skimmed milk as a substrate (Berridge, 1955). Its proteolitic activity on the artificial substrate N-acetyl-L-phenylalanyl-L-3, 5-diiodo tyrosin (APD) also was determined (Rick-Fritsch, 1974). Mean clotting activity value was 5.52 UC, higher (P < 0.01) than that of the reference chymosin (0.64 UC). The activity with APD was unsatisfactory, due to very high absorbance values of the blanks. It is concluded, that the purification steps followed in this trial are simple and rapid, conferring a strong stimulus to using chicken pepsin as a clotting agent for the industrial production of pasteurized white cheese.

Porcine gastric mucin: effect of carbohydrate moieties on susceptility to pancreatic proteases

Digestion of porcine gastric, containing an average of one hexasaccharide per three amino acids, with a mixture of glycosidases gave a partial loss of carbohydrate and an increase in turbidity. The original mucin was completely resistant to digestion, but the carbohydrate-depleted mucin was degraded by pancreatic proteases. Amino acid analyses of the glycosidase-treated mucin indicate that it different from the original mucin. These results indicate that the closely spaced oligosaccharide chains on gastrointestinal mucins may be responsible both for maintaining their solubility and for their resistance to digestion by glycosidases and proteases. Since the carbohydrate-depleted mucin is much more susceptible to proteolysis than the mucin-glycoprotein subunit, bacterial glycosidases rather than proteases are apparently limiting in mucin digestion

Aldehído Deshidrogenasa humana en una muestra de la población costarricense / Human aldehyde dehydrogenase in a sample of the Costa Rica population

This is an electrophoretic study of ALDH isozymes in post-mortem tissue extracts. There differente electrophoretic variants of the isosyme ALDH3 were found in the 100 individuals examined. One liver sample showed lack of ALDH1 activity, but it remains unknown whether this is due to genetic mechanism. The other two isosymes - ALDH2 and ALDH4 - did not show any variations.