RESUMO
An antigenic surface protein of Eimeria tenella sporozoites has been identified that is the target of two neutralizing monoclonal antibodies Ptn 7.2A4/4 and Ptn 9.9D12. The antigen as isolated from the parasite is composed of a 17 kDa polypeptide and a 8 kDa polypeptide linked by a disulfide bridge. De novo synthesis of the antigen does not begin until approximately 16-20 h after the initiation of oocyst sporulation. A cDNA library was constructed using mRNA from sporulated oocysts and a clone encoding the antigen was isolated. The Ta4 gene encodes a single polypeptide of 25 kDa which contains the 17 and 8 kDa polypeptides. The protein has been synthesized in Escherichia coli either directly or as part of a beta-galactosidase fusion protein. The products synthesized in E. coli are single polypeptides and are not cleaved to two polypeptides as is seen in the parasite. The products accumulate in bacteria in an insoluble form which can be solubilized and renatured to an immunoreactive form.
Assuntos
Antígenos de Protozoários/genética , Eimeria/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/análise , Antígenos de Protozoários/biossíntese , Antígenos de Superfície/análise , Antígenos de Superfície/biossíntese , Antígenos de Superfície/genética , Sequência de Bases , Clonagem Molecular , DNA/genética , Eimeria/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Imunoensaio , Dados de Sequência Molecular , Peptídeos/genética , Peptídeos/imunologia , Plasmídeos , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologiaRESUMO
Trypanosoma cruzi (Peru strain) trypomastigotes and epimastigotes were biosynthetically labeled with [35S]methionine, and the proteins were analyzed by two dimensional polyacrylamide gel electrophoresis (2D-PAGE). 2D-PAGE analysis of the trypomastigotes showed a complex array of polypeptides with distinct clusters at Mr 88 000-92 000, isoelectric point (pI) 5.6-6.0, and Mr 72 000-76 000, pI 5.6-5.8. 2D-PAGE analysis of the epimastigotes did not show the cluster of polypeptides at Mr 90 000. When the trypomastigote lysate was reacted with sera from either mice or humans chronically infected with T. cruzi, 10-50 polypeptides were immunoprecipitated. Five of these polypeptides were recognized by all sera tested. However, of these polypeptides, only three, two of Mr 90 000 and one of Mr 150 000, can be identified by immunoreaction of [35S]methionine-labeled live parasites as surface proteins of T. cruzi trypomastigotes. 125I-iminobiotinylated surface proteins isolated from T. cruzi trypomastigotes were immunoprecipitated with the same series of sera as described above. Chagasic sera immunoprecipitated an antigen of Mr 90 000. The [35S]methionine and 125I-labeled Mr 90 000 polypeptides were not immunoprecipitated with sera from individuals infected with Leishmania donovani, Leishmania braziliensis, Leishmania tropica or Leishmania mexicana. These data indicate that a surface polypeptide of Mr 90000, pI 5.8-5.9 is a viable candidate for a Chagas' disease diagnostic antigen.
Assuntos
Antígenos de Superfície/análise , Doença de Chagas/diagnóstico , Trypanosoma cruzi/imunologia , Animais , Argentina , Brasil , Doença de Chagas/imunologia , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Humanos , Lectinas , México , Camundongos , Peso Molecular , VenezuelaAssuntos
3',5'-AMP Cíclico Fosfodiesterases/genética , 3',5'-GMP Cíclico Fosfodiesterases/genética , Linfoma/enzimologia , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , 3',5'-GMP Cíclico Fosfodiesterases/metabolismo , Animais , Linhagem Celular , Indução Enzimática , Variação Genética , Células Híbridas/enzimologia , Cinética , Peso Molecular , Mutação , Fenótipo , Especificidade por SubstratoAssuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , 3',5'-GMP Cíclico Fosfodiesterases/metabolismo , Linfoma/enzimologia , Mutação , 3',5'-AMP Cíclico Fosfodiesterases/genética , 3',5'-GMP Cíclico Fosfodiesterases/genética , Animais , Ligação Competitiva , Linhagem Celular , Centrifugação com Gradiente de Concentração , Cromatografia em Gel , Cromatografia por Troca Iônica , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Cinética , Camundongos , Neoplasias Experimentais/enzimologiaRESUMO
Pseudohypoparathyroidism, type I (PHP-I) is an inherited disorder of primary resistance to multiple hormones that work by stimulating adenylate cyclase. In an attempt to clarify the mode of inheritance of PHP-I, we measured the activity of the N protein, a receptor-cyclase coupling component, in erythrocyte membranes. Erythrocyte N-protein activity was reduced by approximately 50% in erythrocytes of 15 PHP-I patients and was normal in 19 of their clinically normal first degree relatives. Reduced N-protein activity and the PHP-I phenotype in these families exhibited both dominant and recessive patterns of inheritance. This suggests that at least two distinct genetic loci are involved in inheritance of N-protein deficiency. In two additional families, dominant inheritance of the PHP-I phenotype was associated with normal activities of erythrocyte N protein. Thus, it appears that mutation of at least one additional genetic locus, not involving the N protein, can produce PHP-I.
Assuntos
Adenilil Ciclases/deficiência , Pseudo-Hipoparatireoidismo/genética , Adenilil Ciclases/metabolismo , Adolescente , Adulto , Cálcio/sangue , Pré-Escolar , AMP Cíclico/urina , Eritrócitos/análise , Feminino , Genes Dominantes , Genes Recessivos , Humanos , Lactente , Masculino , Proteínas de Membrana/deficiência , Pessoa de Meia-Idade , Hormônio Paratireóideo/sangue , Linhagem , Valores de ReferênciaRESUMO
Hormone-sensitive adenylate cyclase contains a recently discovered protein component that is required for stimulation of cyclic AMP synthesis by hormones and guanine nucleotides. We measured this protein in erythrocyte membranes of ten patients with pseudohypoparathyroidism (PHP), using assays of its biochemical activity and of its susceptibility to radiolabeling in the presence of 32P-NAD and cholera toxin. By both assays, the protein was reduced by 50% in erythrocytes of 4 PHP patients, as compared with normal and hypoparathyroid subjects. These 4 subjects, in contrast to the 6 PHP patients (5 in one family) whose erythrocytes contained apparently normal amounts of the cyclase component, exhibited the full spectrum of skeletal abnormalities found in PHP. We conclude that partial deficiency of the guanine nucleotide regulatory protein is a biochemical marker for a subset of PHP patients. If present in other tissues, this deficiency could explain the resistance of target organs in PHP to parathormone and other hormones that work via cyclic AMP.
Assuntos
Adenilil Ciclases/metabolismo , Membrana Eritrocítica/enzimologia , Eritrócitos/enzimologia , Mutação , Pseudo-Hipoparatireoidismo/enzimologia , Toxina da Cólera/farmacologia , AMP Cíclico/urina , Guanosina 5'-O-(3-Tiotrifosfato) , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/farmacologia , Humanos , Isoproterenol/farmacologia , Tionucleotídeos/farmacologiaRESUMO
Hormone-sensitive adenylate cyclase contains a recently discovered protein component that is required for stimulation of cyclic AMP synthesis by hormones and guanine nucleotides; the component presumably couples the membrane receptor to the cyclase. We studied this protein (termed "N") in erythrocyte membranes of patients with pseudohypoparathyroidism, using assays of the protein's biochemical activity and of its susceptibility to radiolabeling in the presence of [32P]NAD and cholera toxin. By both assays, the protein's activity was reduced by 40 to 50 per cent in erythrocytes of five of 10 patients with Type I pseudohypoparathyroidism as compared with those of normal and hypoparathyroid subjects and one patient with Type II pseudohypoparathyroidism. If activity of the N protein is reduced in other tissues, this deficiency could cause the resistance of target organs in pseudohypoparathyroidism to parathyroid hormone and other hormones that work via cyclic AMP. Erythrocytes of five patients with Type I pseudohypoparathyroidism, all in one family, showed no defect in activity of the N protein; the biochemical defect of this family remains undefined.
Assuntos
Adenilil Ciclases/metabolismo , Proteínas de Membrana/deficiência , Hormônio Paratireóideo/metabolismo , Pseudo-Hipoparatireoidismo/sangue , Receptores de Superfície Celular/metabolismo , Adolescente , Adulto , Toxina da Cólera/metabolismo , AMP Cíclico/biossíntese , AMP Cíclico/urina , Membrana Eritrocítica/análise , Feminino , Humanos , Hipoparatireoidismo/sangue , Hipoparatireoidismo/metabolismo , Masculino , Proteínas de Membrana/sangue , Pessoa de Meia-Idade , Hormônio Paratireóideo/urina , Fosfatos/urina , Ligação Proteica , Pseudo-Hipoparatireoidismo/metabolismoAssuntos
Adenilil Ciclases/sangue , Membrana Eritrocítica/enzimologia , Eritrócitos/enzimologia , Adenilil Ciclases/isolamento & purificação , Toxina da Cólera/farmacologia , Humanos , Cinética , Substâncias Macromoleculares , Proteínas de Membrana/sangue , Proteínas de Membrana/isolamento & purificação , Peso Molecular , NAD , Conformação ProteicaRESUMO
Glutamine-dependent CPSase, ATCase, and DHOase from Drosophila, the first three enzymes in pyrimidine biosynthesis, show coordinate variation in activity throughout development. The three activities were highest in first instar larvae and decreased as development proceeded. The three activities cosediment in sucrose gradients as a single peak with a relative sedimentation coefficient of approximately 30S. CPSase, ATCase, and DHOase copurify during (NH4)2SO4 fractionation and during DEAE-cellulose and hydroxylapatite chromatography.