Efectos de la desnutrición proteica y de su recuperación nutricional en el timo de rata / Effects of protein malnutrition and its nutritional recovery in rat thymus

El objetivo de este trabajo fue estudiar el contenido de ADN y la actividad de Adenosina Deaminasa (ADA), Purina Nucleósido Fosforilasa (PNP) y su 5ïNucleotidasa (5ïNT)-enzimas relacionadas con el desarrollo y funcionamiento de los linfocito T- en el timo de ratas con desnutrición proteica leve, moderada y several al destete y la posterior recuperación nutricional. El estrés nutricional causado por la administración desde el destete de dietas carentes de proteínas provocó una disminución del peso del timo, del contenido de DNA y un aumento en la actividad de ADA y PNP sin modificar la actividad de 5ïNT. Este aumento de actividad podría explicarse como un mecanismo para evitar la formación de productos potencialmente tóxicos para los linfocitos T. La administración de la dieta de recuperación fue capaz de revertir los efectos provocados por la desnutrición proteica al destete; no se observaron diferencias significativas en los parámetros estudiados, entre el grupo realimentado y su contro bien nutrido de igual edad. Estos hallazgos avalan la hipótesis relacionada con el equilibrio de nutrientes en la dieta y la evolución del desarrollo celular tímico

Ectonucleotidase activities in Sertoli cells from immature rats

Sertoli cells have been shown to be targets for extracellular purines such as ATP and adenosine. These purines evoke responses in Sertoli cells through two subtypes of purinoreceptors, P2Y2 and P A1. The signals to purinoreceptors are usually terminated by the action of ectonucleotidases. To demonstrate these enzymatic activities, we cultured rat Sertoli cells for four days and then used them for different assays. ATP, ADP and AMP hydrolysis was estimated by measuring the Pi released using a colorimetric method. Adenosine deaminase activity (EC 3.5.4.4) was determined by HPLC. The cells were not disrupted after 40 min of incubation and the enzymatic activities were considered to be ectocellularly localized. ATP and ADP hydrolysis was markedly increased by the addition of divalent cations to the reaction medium. A competition plot demonstrated that only one enzymatic site is responsible for the hydrolysis of ATP and ADP. This result indicates that the enzyme that acts on the degradation of tri- and diphosphate nucleosides on the surface of Sertoli cells is a true ATP diphosphohydrolase (EC 3.6.1.5) (specific activities of 113 + or - 6 and 21 + or - 2 nmol Pi mg-1 min-1 for ATP and ADP, respectively). The ecto-5'-nucleotidase (EC 3.1.3.5) and ectoadenosine deaminase activities (specific activities of 32 + or - 2 nmol Pi mg-1 min-1 for AMP and 1.52 + or - 0.13 nmol adenosine mg-1 min-1, respectively) were shown to be able to terminate the effects of purines and may be relevant for the physiological control of extracellular levels of nucleotides and nucleosides inside the seminiferous tubules

Brain ischemia alters platelet ATP diphosphohydrolase and 5'-nucleotidase activities in naive and preconditioned rats

The effects of transient forebrain ischemia, reperfusion and ischemic preconditioning on rat blood platelet ATP diphosphohydrolase and 5'-nucleotidase activities were evaluated. Adult Wistar rats were submitted to 2 or 10 min of single ischemic episodes, or to 10 min of ischemia 1 day after a 2-min ischemic episode (ischemic preconditioning) by the four-vessel occlusion method. Rats submitted to single ischemic insults were reperfused for 60 min and for 1, 2, 5, 10 and 30 days after ischemia; preconditioned rats were reperfused for 60 min 1 and 2 days after the long ischemic episode. Brain ischemia (2 or 10 min) inhibited ATP and ADP hydrolysis by platelet ATP diphosphohydrolase. On the other hand, AMP hydrolysis by 5'-nucleotidase was increased after 2, but not 10, min of ischemia. Ischemic preconditioning followed by 10 min of ischemia caused activation of both enzymes. Variable periods of reperfusion distinctly affected each experimental group. Enzyme activities returned to control levels in the 2-min group. However, the decrease in ATP diphosphohydrolase activity was maintained up to 30 days of reperfusion after 10-min ischemia. 5'-Nucleotidase activity was decreased 60 min and 1 day following 10-min ischemia; interestingly, enzymatic activity was increased after 2 and 5 days of reperfusion, and returned to control levels after 10 days. Ischemic preconditioning cancelled the effects of 10-min ischemia on the enzymatic activities. These results indicate that brain ischemia and ischemic preconditioning induce peripheral effects on ecto-enzymes from rat platelets involved in nucleotide metabolism. Thus, ATP, ADP and AMP degradation and probably the generation of adenosine in the circulation may be altered, leading to regulation of microthrombus formation since ADP aggregates platelets and adenosine is an inhibitor of platelet aggregation

Effects of dietary polyunsaturated fatty acids on adenylate cyclase, 5'nucleotidasa and Na+K+-ATPase activities in rat brain-plasma membrane

The incidence of polyunsaturated fatty acids (PUFA) in human nutrition is now generally accepted. As essential membrane components, PUFA may act as enzyme activity modulators. In this study, four different diets, in which PUFA type was the only modifying factor, were evaluated on 5'nucleotidase, adenylate cyclase and Na+/K+ATPase activities in rat brain plasma membranes. Animals fed the total PUFA deficient diet exhibited significant lower body weight and lower brain weight than did the control group. The specific activities of 5'nucleotidase and Na+/K+ATPase in brain plasma membrane were slightly modified by dietary PUFA. The catalytic unit of adenylate cyclase in total PUFA deficient animals presented augmented enzyme activity and animals receiving diets deficient in n-6 PUFA showed reduced activity in relation to the control animals. Our results showed that the epinephrine receptors, in the case of adenylate cyclase are not modified by dietary PUFA, but rather the catalytic unit seems to be altered by dietary PUFA. These results can be partially explained by the fluidity that PUFA confers to membranes facilitating the proximity of enzyme-substrate. The physiological consequences of dietary PUFA incidence on enzyme activity needs further study.

Estandarización de un método citoquímico para el estudio de la enzima esto 5'-nucleotidasa en linfocitos / Standarization of a cytochemical method for the study of Ecto-5'nucleotidase enzyme on lymphocytes

Se describe un método para la detección de la enzima ecto 5'nucleotidasa (5'NT) en linfocitos de sangre periférica. El estudio se realizó en 25 adultos sanos (10 mujeres y 15 hombres) con un rango de edad entre 19 y 44 años y una edad promedio de 30 años. La cantidad de células y de reactivos se redujo con respecto a otras técnicas. El rango de células 5'NT+ obtenido es de 18 a 40 por ciento , lo que se corresponde con lo informado por otros autores

Immunogold labeling and cerium cytochemistry of the enzyme ecto-5'-nucleotidase in promastigote forms of Leishmania species

We have applied both enzyme cytochemistry and immunological labeling techniques to characterize the enzyme 5'-nucleotidase (5'-Nase), at the ultrastructural level, in promastigote forms of four Leishmania species: Leishmania amazonensis, Leishmania mexicana, Leishmania donovani and Leishmania chagasi. The cerium phosphate staining was localized at the surface of the cell body, the flagellum and the flagellar pocket membranes of all the parasites studied. The immunogold labelling technique confirmed these results. In this report we localized 5'-Nase in L. chagasi and L. amazonensis which have been implicated respectively in visceral and cutaneous forms of leishmaniasis. In addition, we confirmed the localization of this phosphomonoesterase in the other two species studied. The superior quality of the images, obtained with both methodologies, confirms that these parasites possess mechanisms capable of hydrolyzing nucleotide monophosphates, and that the expression of 5'-Nase is associated with the outer surface of the plasma membrane

Histoquímica do metabolismo dos nucleotídios no carcinoma mamário / Histoenzymological study of 5'nucleotidase activity in the human breast carcinoma

Utilizando técnica histoenzimológica, foi estudado, em 15 biópsias mamárias (seis provenientes de mamas com hipertrofia, uma com displasia - hiperplasia epitelial -, e oito com carcinoma ductal), o metabolismo dos nucleotídios, representado pela 5' nucleotidase. Observaram tendência para forte atividade dessa hidrolase no tecido estromal, com nítido predomínio sobre o componente epitelial. Nas amostras processadas histoquimicamente näo puderam evidenciar sinal que indicasse característica diferencial de comportamento entre os tumores cromatina-positivos e os cromatina-negativos. No único caso de displasia mamária a 5' nucleotidase näo mostrou atividade