RESUMEN
In the present study, we wished to demonstrate the ability of surface gametocyte antigens to induce protective immunity against Eimeria maxima infections in chickens. In order to accomplish this goal, we employed maternal immunization as a means of providing large amounts of specific antibodies to offspring chicks. Upon challenge with sporulated E. maxima oocysts, chicks from hens immunized with affinity-purified gametocyte antigens showed greatly reduced oocyst production compared with chicks from sham-immunized hens. These results suggest that maternal immunization with gametocyte antigens can be used as a means to provide transmission-blocking immunity against E. maxima infections.
Asunto(s)
Antígenos de Protozoos/inmunología , Animales , Anticuerpos Antiprotozoarios/análisis , Pollos , Coccidiosis/inmunología , Eimeria/crecimiento & desarrollo , Femenino , Inmunización , EmbarazoRESUMEN
We prepared a cDNA library from gametocytes of Eimeria maxima and screened it using antibodies raised against an 82-kDa gametocyte antigen. One cDNA clone designated pEM230 was isolated and characterized. It encodes a portion of a 230-kDa gametocyte protein and its DNA sequence shows the presence of several tandem repeats of 42 bp. In order to determine the stage and sex specificity of the mRNA for the 230-kDa protein, Northern blotting and in situ hybridization studies were performed. The 230-kDa protein is encoded for by a 7 kb mRNA, which is expressed exclusively during the macrogamete stage with no detectable expression seen in any other stage of parasite development.
Asunto(s)
Eimeria/genética , Regulación de la Expresión Génica , Proteínas Protozoarias/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Southern Blotting , Western Blotting , ADN Protozoario , Eimeria/crecimiento & desarrollo , Eimeria/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Biblioteca Genómica , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Oocitos/metabolismo , Proteínas Protozoarias/biosíntesis , Mapeo RestrictivoRESUMEN
Eimeria maxima gametocytes contain two major antigens with molecular masses of 56 and 82 kilodaltons (kDa) which are recognized by convalescent sera from immune chickens. Preparations enriched in these two antigens were used to immunize mice, and several monoclonal antibodies which specifically reacted with the 56-kDa antigen were produced. One of these monoclonal antibodies of the immunoglobulin M subclass, along with immune chicken sera raised against affinity-purified 56- and 82-kDa antigens, was used to passively immunize chicks. On the basis of the parameter of total oocyst output, it was found that these antibodies provided partial protection (40 to 50% inhibition) against E. maxima challenge infections.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos de Protozoos/inmunología , Eimeria/inmunología , Inmunización Pasiva , Animales , Antígenos de Protozoos/genética , Pollos , Coccidiosis/prevención & controlRESUMEN
Eimeria maxima gametocytes were isolated from infected chicken intestinal tissue by treatment with hyaluronidase and subsequent filtration through polymon filters. The isolated gametocytes were analyzed by microscopical and biochemical methods and shown to be highly enriched. The antigenicity of the gametocytes was analyzed in mice, rabbits, and chickens by ELISA and indirect immunofluorescence. Contrary to published results, we have found gametocytes to be highly immunogenic in all animals tested.
Asunto(s)
Anticuerpos Antiprotozoarios/biosíntesis , Antígenos de Protozoos/inmunología , Pollos/parasitología , Coccidiosis/veterinaria , Eimeria/inmunología , Animales , Coccidiosis/inmunología , Coccidiosis/parasitología , Eimeria/genética , Eimeria/aislamiento & purificación , Electroforesis en Gel de Agar , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Sueros Inmunes/inmunología , Inmunización , Ratones , Enfermedades de las Aves de Corral/inmunología , Enfermedades de las Aves de Corral/parasitología , ARN/análisis , ConejosRESUMEN
The antigenicity of Eimeria maxima gametocyte proteins during the course of an infection and when injected into mice and rabbits was demonstrated using the Western blotting technique. Serum taken from chickens at various times postinfection reacted to a few gametocyte proteins, with the strongest reactivity seen with serum taken 14-days postinfection. Two major antigens having molecular weights of 56,000 and 82,000 were consistently detected by these sera. Using immune rabbit or mouse sera to whole gametocyte detergent extracts, the 56,000 and 82,000 molecular weight proteins were again the immunodominant antigens, despite their representing only a small proportion of the extract which was used to immunize the animals. These results, together with those obtained by Rose (1971) using recovered chicken serum to passively immunize chickens, indicate that these two gametocyte antigens may play a role in protective immunity to E. maxima.
Asunto(s)
Antígenos de Protozoos/inmunología , Eimeria/inmunología , Animales , Western Blotting/veterinaria , Pollos , Coccidiosis/inmunología , Coccidiosis/veterinaria , Grupo Citocromo c/inmunología , Detergentes/farmacología , Eimeria/aislamiento & purificación , Inmunización , Parasitosis Intestinales/inmunología , Parasitosis Intestinales/veterinaria , Ratones , Peso Molecular , Enfermedades de las Aves de Corral/inmunología , Enfermedades de las Aves de Corral/parasitología , Conejos , Especificidad de la EspecieRESUMEN
RNA was extracted from isolated Eimeria maxima gametocytes and translated in a rabbit reticulocyte cell-free protein synthesis system. The major cell-free translation products from E. maxima gametocyte RNA ranged from 225 to 50 kDa, distinct and different from uninfected chicken intestine cell-free translation products. Rabbit antiserum to E. maxima gametocytes as well as recovered chicken sera specifically precipitated some of the major gametocyte cell-free products. A time course of infected intestine RNA indicated that these cell-free synthesized gametocyte antigens appear at 130 to 138 hr postinfection.
Asunto(s)
Antígenos de Protozoos/genética , Eimeria/inmunología , Biosíntesis de Proteínas , Animales , Antígenos de Protozoos/biosíntesis , Western Blotting/veterinaria , Sistema Libre de Células , Pollos , Coccidiosis/inmunología , Coccidiosis/veterinaria , Eimeria/genética , Eimeria/crecimiento & desarrollo , Parasitosis Intestinales/inmunología , Parasitosis Intestinales/veterinaria , Enfermedades de las Aves de Corral/inmunología , Enfermedades de las Aves de Corral/parasitología , Pruebas de Precipitina/veterinaria , ARN Mensajero/genéticaRESUMEN
Rat fetuses of 17-19-day gestation were injected in utero with 5-azacytidine (two to three daily injections of 40 micrograms/fetus). Neonates were injected with seven daily injections (1 mg/kg). DNA samples were isolated from the fetal and neonatal livers and neonatal spleen and subjected to analysis of their methylation status. Overall methylation was analyzed by the nearest-neighbor analysis (at CpG sites) and the pattern of methylation at CCGG sites by Southern blot analysis using phosphoenolpyruvate carboxykinase (PEPCK) sequences as probes. While DNAs from the liver and spleen undergo hypomethylation to the same extent in response to the 5-azacytidine treatment, the changes in the methylation patterns of the PEPCK gene in the two tissues are strikingly different. The changes observed indicate that a decrease in the methylase activity (inhibition by 5-azacytidine) results in site- and tissue-specific hypomethylation. The tissue-specific changes in the methylation pattern are associated with a tissue-specific expression of the PEPCK gene. Although the gene is hypomethylated by azacytidine in both liver and spleen, it is expressed only in the liver. The expression of already active genes (PEPCK in the kidney and albumin in the liver) is not further enhanced by the drug.
Asunto(s)
Azacitidina/farmacología , Genes/efectos de los fármacos , Fosfoenolpiruvato Carboxiquinasa (GTP)/genética , Transcripción Genética/efectos de los fármacos , Animales , Animales Recién Nacidos , ADN/aislamiento & purificación , Femenino , Feto , Hígado/efectos de los fármacos , Hígado/enzimología , Metilación , Hibridación de Ácido Nucleico , Embarazo , ARN/aislamiento & purificación , Ratas , Ratas Endogámicas , Bazo/efectos de los fármacos , Bazo/enzimologíaRESUMEN
Structural conservation of cytosolic phosphoenolpyruvate carboxykinase protein and mRNA sequence was found in all species examined from rodents to human. The mitochondrial isoenzyme, in all species tested, represents a distinct protein. Moreover, irrespective of the ratio of cytosolic to mitochondrial isoenzyme, cytosolic phosphoenolpyruvate carboxykinase activity in the human as in the rat is controlled at the level of gene expression and through the same multiple hormonal stimulation. This evolutionary conservation of the cytosolic phosphoenolpyruvate carboxykinase structure and mode of regulation supports the enzymes' physiological importance in mammals.
Asunto(s)
Regulación de la Expresión Génica , Fosfoenolpiruvato Carboxiquinasa (GTP)/genética , Animales , Bucladesina/farmacología , Gatos , Cricetinae , Cricetulus , Citosol/enzimología , ADN/análisis , Dexametasona/farmacología , Cobayas , Humanos , Riñón/enzimología , Hígado/enzimología , Mesocricetus , Ratones , Mitocondrias/enzimología , ARN Mensajero/análisis , Ratas , Especificidad de la EspecieRESUMEN
The cytosolic phosphenolpyruvate carboxykinase [PEPCK; GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32] gene was isolated from a rat genomic library, and a map of the methylatable sites C-C-G-G and G-C-G-C has been constructed. The extent of methylation of 18 sites in the PEPCK gene in adult liver, kidney, spleen, and heart muscle and in fetal liver has been analyzed using the 5-methylcytosine sensitive enzymes Hpa II and Hha I. This analysis revealed extensive undermethylation of the PEPCK gene in the adult liver and kidney (PEPCK-expressing tissue), whereas the gene in adult spleen and heart muscle as well as in fetal liver (PEPCK-nonexpressing tissues) was heavily methylated. However, unlike the gene in the adult nonexpressing tissues, a region in the middle of the gene was found to be partially hypomethylated in fetal liver. This hypomethylation correlates with the competence of the fetal liver gene to be expressed. Treatment of fetuses by in utero injection of 5-azacytidine causes a hypomethylation-associated activation of the PEPCK gene. Taken together, the present findings suggest a sequential loss of methyl groups during development. When related to PEPCK gene expression, the sequential loss of methyl groups demonstrates an early stage prior to transcription characterized by hypomethylation of discrete sites and a later developmental hypomethylation of all sites associated with the mature active PEPCK gene around the time of birth.
Asunto(s)
ADN/metabolismo , Fosfoenolpiruvato Carboxiquinasa (GTP)/genética , Factores de Edad , Animales , Azacitidina/farmacología , Secuencia de Bases , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Enzimas de Restricción del ADN/metabolismo , Inducción Enzimática , Femenino , Hígado/enzimología , Masculino , Metilación , Músculos/enzimología , Embarazo , Ratas , Ratas Endogámicas , Bazo/enzimologíaRESUMEN
The primary appearance of phosphoenolpyruvate (P-pyruvate) carboxykinase RNA transcripts in fetal liver was induced by a number of different stimulii . This may occur as rapidly as an hour after injection in utero of N6,O2'-dibutyryl-adenosine 3',5'-monophosphate (Bt2cAMP) to fetuses, suggesting that all stimulii predominantly affect activation of the P-pyruvate carboxykinase gene. Bt2cAMP treatment induces the appearance of the enzyme RNA transcripts, predominantly of the mature type in the cytoplasm. However, insulin deficiency by streptozotocin treatment causes the appearance of large-size as well as mature mRNA in the nucleus, in addition to the appearance of P-pyruvate carboxykinase mRNA in the cytoplasm. Insulin treatment of such diabetic fetuses, prior to causing the disappearance of P-pyruvate carboxykinase mRNA, reduces nuclear transcripts but increases the abundance of mature cytoplasmic enzyme mRNA. Bt2cAMP treatment of insulin-deficient fetuses causes an additive effect, increasing the abundance of not only the mature but the large P-pyruvate carboxykinase RNA transcripts as well. The results are best interpreted as insulin acting both to inhibit transcription of and accelerate post-transcriptional processes affecting P-pyruvate carboxykinase RNA.
Asunto(s)
Citosol/enzimología , Hígado/enzimología , Fosfoenolpiruvato Carboxiquinasa (GTP)/genética , ARN Mensajero/biosíntesis , Animales , Núcleo Celular/enzimología , Diabetes Mellitus Experimental/enzimología , Femenino , Feto/enzimología , Hígado/embriología , Fosfoenolpiruvato Carboxiquinasa (GTP)/biosíntesis , Embarazo , Ratas , Ratas Endogámicas , Transcripción GenéticaRESUMEN
Streptozotocin treatment produces a typical experimental diabetes in neonates exhibiting hyperglycemia, glucosuria, ketonemia and increased level of fatty acids in the blood. The liver is affected as well, with reduced activity of glycogen synthase and a corresponding decrease in the content of liver glycogen. In contrast, the activity of liver cytosolic phosphoenolpyruvate carboxykinase and the level of its mRNA are not affected. Using a cDNA containing P-pyruvate carboxykinase sequence, the relative abundance of the enzyme mRNA was estimated. The level of the mRNA was readily observed increasing by glucocorticoid treatment or decreasing in response to administered load of glucose. These parallel the changes observed in the activity of the enzyme under these treatments, indicating that the level of P-pyruvate carboxykinase mRNA actually determines that of the enzyme. The failure of diabetes to increase the level of enzyme mRNA and the limited response to glucose loading strongly suggest that the mechanisms controlling the level of P-pyruvate carboxykinase mRNA in neonates are relatively resistant to insulin. This is unique to neonates, since in both the adult and the fetal liver. P-pyruvate carboxykinase readily responds to insulin. The minimal levels of glucocorticoids characteristic of neonates may be associated with this phenomenon.
Asunto(s)
Animales Recién Nacidos/metabolismo , Diabetes Mellitus Experimental/enzimología , Glucocorticoides/farmacología , Glucosa/farmacología , Hígado/enzimología , Fosfoenolpiruvato Carboxiquinasa (GTP)/aislamiento & purificación , ARN Mensajero/aislamiento & purificación , Animales , Citosol/enzimología , Insulina/farmacología , Fosfoenolpiruvato Carboxiquinasa (GTP)/genética , Fosfoenolpiruvato Carboxiquinasa (GTP)/metabolismo , Ratas , Ratas EndogámicasRESUMEN
Injection of streptozotocin in utero to fetuses elicited a premature appearance of cytosolic hepatic activity of phosphoenol pyruvate carboxykinase. This was due to a precocious initiation of the synthesis of the enzyme. The streptozotocin-induced appearance of enzyme activity was not mediated by adenosine 3':5'-monophosphate since the concentration of the cyclic nucleotide in the liver was unaffected by the antibiotic, the administration of dibutyryladenosine 3':5'-monophosphate to streptozotocin-treated fetuses elicited an additive increase in enzyme activity, and insulin administration in utero repressed the streptozotocin effect while the effect due to dibutyryladenosine 3':5'-monophosphate was not inhibited by simultaneous insulin injection. Streptozotocin treatment also caused a small but consistent retardation of fetal growth and a marked reduction of liver wet weight. Histological analysis of the liver demonstrated a premature loss of some hematopoietic elements, while hepatocytes appeared normal. Hepatic protein synthesis was unaffected by the streptozotocin treatment. Streptozotocin treatment had no effect on fetal renal phosphoenol pyruvate carboxykinase activity or kidney wet weight.
Asunto(s)
AMP Cíclico/metabolismo , Hígado/enzimología , Fosfoenolpiruvato Carboxiquinasa (GTP)/biosíntesis , Estreptozocina/farmacología , Animales , Bucladesina/farmacología , Corticosterona/sangre , Inducción Enzimática/efectos de los fármacos , Femenino , Sangre Fetal/análisis , Feto , Insulina/sangre , Insulina/farmacología , Riñón/efectos de los fármacos , Riñón/enzimología , Hígado/efectos de los fármacos , Hígado/embriología , RatasRESUMEN
The effects of triamcinolone on renal and hepatic phosphoenolpyruvate carboxykinase activity in the developing rat were investigated. The hormone induced increases in pre-existing enzyme activity of both tissues in fetal and neonatal rats, yet did not cause the primary appearance of phosphoenolpyruvate carboxykinase activity in utero. Neonatal hepatic phosphoenolpyruvate carboxykinase activity was increased 2--3 fold by triamcinolone form the 3rd to the 15th postnatal day. This was shown to be additive to the effect of Bt2cAMP on enzyme activity. The increases in phosphoenolpyruvate carboxykinase activity were demonstrated to be due to increased synthesis of the enzyme, which was accompanied by a proportionate increase in the amount of functional phosphoenolpyruvate carboxykinase mRNA, as measured by the polyribosomal and poly(A)-containing RNA directed cell-free synthesis of the enzyme. The demonstration of a triamcinolone effect on kidney and liver phosphoenolpyruvate carboxykinase activity in fetal and neonatal rats provides support for a possible role of glucocorticoids in the regulation of phosphoenolpyruvate carboxykinase activity during development.