RESUMO
Endopeptidase 24.15 (EP24.15) and 24.16 (EP24.16) are closely related metalloendopeptidases implicated in the metabolism of several neuropeptides and widely expressed in mammalian brain. To gain insight into the functional role of these two enzymes in the central nervous system, we examined their cellular and subcellular distribution in rat brain by using electron microscopic immunogold labeling. In all areas examined, EP24.15 and EP24.16 immunoreactivity were observed in selective subpopulations of neuronal and glial cells. Subcellular localization of EP24.15 in neurons revealed that this enzyme was predominantly concentrated in the nucleus, whereas EP24.16 was almost exclusively cytoplasmic. The amount of EP24.15 found in the nucleus was inversely correlated with that found in the cytoplasm, suggesting that the enzyme could be mobilized from one compartment to the other. Within the cytoplasm, EP24.15 and EP24.16 immunoreactivity showed comparable distributional patterns. Both enzymes were detected throughout perikarya and dendrites, as well as within axons and axon terminals. In all neuronal compartments, EP24.15 and EP24.16 showed a major association with membranes of neurosecretory elements, including Golgi cisternae, tubulovesicular organelles, synaptic vesicles, and endosomes. However, whereas EP24.15 always faced the cytoplasmic face of the membranes, EP24.16 was observed on both cytoplasmic and luminal sides, suggesting that the latter was more likely to contribute to the processing of peptides or to the degradation of internalized ligands. Taken together, the present results suggest that EP24.15 could play a major role in the hydrolysis of intranuclear substrates, whereas EP24.16 would be predominantly involved in the processing and inactivation of signaling peptides.
Assuntos
Encéfalo/enzimologia , Metaloendopeptidases/metabolismo , Neuroglia/enzimologia , Neurônios/enzimologia , Neuropeptídeos/metabolismo , Animais , Encéfalo/ultraestrutura , Compartimento Celular/fisiologia , Estruturas do Núcleo Celular/enzimologia , Estruturas do Núcleo Celular/ultraestrutura , Córtex Cerebelar/enzimologia , Córtex Cerebelar/ultraestrutura , Córtex Cerebral/enzimologia , Córtex Cerebral/ultraestrutura , Citoesqueleto/enzimologia , Citoesqueleto/ultraestrutura , Dendritos/enzimologia , Dendritos/ultraestrutura , Imuno-Histoquímica , Membranas Intracelulares/enzimologia , Membranas Intracelulares/ultraestrutura , Masculino , Microscopia Eletrônica , Neuroglia/ultraestrutura , Neurônios/ultraestrutura , Organelas/enzimologia , Organelas/ultraestrutura , Terminações Pré-Sinápticas/enzimologia , Terminações Pré-Sinápticas/ultraestrutura , Ratos , Ratos Wistar , Núcleo Solitário/enzimologia , Núcleo Solitário/ultraestruturaRESUMO
Thimet oligopeptidase (EC 3.4.24.15; EP24.15) and neurolysin (EC 3.4.24.16; EP24.16) are closely related enzymes involved in the metabolic inactivation of bioactive peptides. Both of these enzymes were previously shown to be secreted from a variety of cell types, although their primary sequence lacks a signal peptide. To investigate the mechanisms responsible for this secretion, we examined by confocal microscopy the subcellular localization of these two enzymes in the neuroendocrine cell line AtT20. Both EP24.15 and EP24.16 were found by immunohistochemistry to be abundantly expressed in AtT20 cells. Western blotting experiments confirmed that the immunoreactivity detected in the soma of these cells corresponded to previously cloned isoforms of the enzymes. At the subcellular level, both enzymes colocalized extensively with the integral trans-Golgi network protein, syntaxin-6, in the juxtanuclear region. In addition, both EP24.15 and EP24.16 were found within small vesicular organelles distributed throughout the cell body. Some, but not all, of these organelles also stained positively for ACTH. These results demonstrate that both EP24.15 and EP24.16 are present within the classical secretory pathway. Their colocalization with ACTH further suggests that they may be targeted to the regulated secretory pathway, even in the absence of a signal peptide.
Assuntos
Metaloendopeptidases/metabolismo , Microscopia Confocal/métodos , Animais , Western Blotting , CoelhosRESUMO
To assess relationships between neuropeptide-binding sites and receptor proteins in rat brain, the distribution of radioautographically labeled somatostatin and neurotensin-binding sites was compared to that of immunolabeled sst2A and NTRH receptor subtypes, respectively. By light microscopy, immunoreactive sst2A receptors were either confined to neuronal perikarya and dendrites or diffusely distributed in tissue. By electron microscopy, areas expressing somatodendritic sst2A receptors displayed only low proportions of membrane-associated, as compared to intracellular, receptors. Conversely, regions displaying diffuse sst2A labeling exhibited higher proportions of membrane-associated than intracellular receptors. Furthermore, the former showed only low levels of radioautographically labeled somatostatin-binding sites whereas the latter contained high densities of somatostatin-binding suggesting that membrane-associated receptors are preferentially recognized by the radioligand. In the case of NTRH receptors, there was a close correspondence between the light microscopic distribution of NTRH immunoreactivity and that of labeled neurotensin-binding sites. Within the substantia nigra, the bulk of immuno- and autoradiographically labeled receptors were associated with the cell bodies and dendrites of presumptive DA neurons. By electron microscopy, both markers were detected inside as well as on the surface of labeled neurons. At the level of the plasma membrane, their distribution was highly correlated and characterized by a lack of enrichment at the level of synaptic junctions and by a homogeneous distribution along the remaining neuronal surface, in conformity with the hypothesis of an extra-synaptic action of this neuropeptide. Inside labeled dendrites, there was a proportionally higher content of immunoreactive than radiolabeled receptors. Some of the immunolabeled receptors not recognized by the radioligand were found in endosome-like organelles suggesting that, as in the case of sst2A receptors, they may have undergone endocytosis subsequent to binding to the endogenous peptide.
Assuntos
Autorradiografia , Células Receptoras Sensoriais , Animais , Imuno-Histoquímica , Microscopia Eletrônica , Ratos , Receptores de Neuropeptídeos , Receptores de Neurotensina , Receptores de Somatostatina , Formação de Roseta , Estatísticas não ParamétricasRESUMO
This paper reviews the use of confocal microscopy as it pertains to the identification of G-protein coupled receptors and the study of their dynamic properties in cell cultures and in mammalian brain following their tagging with specific fluorescent ligands. Principles that should guide the choice of suitable ligands and fluorophores are discussed. Examples are provided from the work carried out in the authors' laboratory using custom synthetized fluoresceinylated or BODIPY-tagged bioactive peptides. The results show that confocal microscopic detection of specifically bound fluorescent ligands permits high resolution appraisal of neuropeptide receptor distribution both in cell culture and in brain sections. Within the framework of time course experiments, it also allows for a dynamic assessment of the internalization and subsequent intracellular trafficking of bound fluorescent molecules. Thus, it was found that neurotensin, somatostatin and mu- and delta-selective opioid peptides are internalized in a receptor-dependent fashion and according to receptor-specific patterns into their target cells. In the case of neurotensin, this internalization process was found to be clathrin-mediated, to proceed through classical endosomal pathways and, in neurons, to result in a mobilization of newly formed endosomes from neural processes to nerve cell bodies and from the periphery of cell bodies towards the perinuclear zone. These mechanisms are likely to play an important role for ligand inactivation, receptor regulation and perhaps also transmembrane signaling.
Assuntos
Receptores de Neuropeptídeos/metabolismo , Endocitose , Fluorescência , Proteínas de Ligação ao GTP , Ligantes , Microscopia Confocal , Entorpecentes/metabolismo , Neurotensina/metabolismo , Peptídeos , Ligação Proteica , Somatostatina/metabolismoAssuntos
Edema Encefálico/etiologia , Doença da Urina de Xarope de Bordo/complicações , Adolescente , Edema Encefálico/líquido cefalorraquidiano , Feminino , Humanos , Leucina/sangue , Leucina/líquido cefalorraquidiano , Doença da Urina de Xarope de Bordo/dietoterapia , Pseudotumor Cerebral/etiologiaRESUMO
This report summarizes our experience with DNA analysis using a complementary DNA probe for ornithine transcarbamylase in 24 individuals or families with deficiency of this enzyme. In four cases, including three reported elsewhere, a Taql restriction site alteration directly detected the mutation. In 10 additional cases, only an affected male was available, and results of DNA analysis using the Taql enzyme were normal. In 10 cases, family studies were performed with the use of restriction fragment length polymorphisms. Prenatal diagnostic studies were performed for three informative pregnancies, and two affected male fetuses were identified. Analysis of two restriction fragment length polymorphisms, Mspla and BamHl, was informative in 14 of 19 (74%) known carrier females and in 21 of 35 (60%) females (the total number studied). One female previously predicted to be a noncarrier by protein-loading test was determined to be a carrier by analysis of restriction fragment length polymorphisms. The frequency of Taql site alterations was 4 of 24 families (17%). These data illustrate the importance of DNA analysis, pedigree analysis, and biochemical testing in families with ornithine transcarbamylase deficiency to detect carriers and establish the diagnosis prenatally.
Assuntos
Triagem de Portadores Genéticos , Doença da Deficiência de Ornitina Carbomoiltransferase , Diagnóstico Pré-Natal , Southern Blotting , DNA/análise , Feminino , Humanos , Masculino , Linhagem , Polimorfismo de Fragmento de Restrição , GravidezRESUMO
The expression and activity of phenylalanine hydroxylase was studied in the liver of a fetus aborted after prenatal diagnosis of phenylketonuria. No phenylalanine hydroxylase enzymatic activity or immunoreactive protein was detectable in the PKU liver specimen, though both enzymatic activity and immunoreactive protein were detectable in control specimens of similar gestational age. Phenylalanine hydroxylase messenger RNA of normal size was present in the PKU fetal liver at normal abundance. These results confirm the genetic diagnosis of PKU in this fetus and indicate that the mutations in this fetus affect translation or stability of the phenylalanine hydroxylase protein.
Assuntos
Fígado/enzimologia , Fenilalanina Hidroxilase/metabolismo , Fenilcetonúrias/enzimologia , Aborto Induzido , Feminino , Feto/enzimologia , Humanos , Recém-Nascido , Fígado/embriologia , Fenilalanina Hidroxilase/genética , Fenilcetonúrias/diagnóstico , Fenilcetonúrias/genética , Polimorfismo de Fragmento de Restrição , Gravidez , Diagnóstico Pré-NatalAssuntos
Fibrose Cística/diagnóstico , Doenças Fetais/diagnóstico , Diagnóstico Pré-Natal , Alelos , Líquido Amniótico/enzimologia , Cromossomos Humanos Par 7 , Ensaios Enzimáticos Clínicos , Fibrose Cística/genética , DNA/genética , Erros de Diagnóstico , Feminino , Marcadores Genéticos , Humanos , Intestinos/enzimologia , Polimorfismo de Fragmento de Restrição , GravidezRESUMO
Ornithine transcarbamylase (OTC) deficiency is an X-linked disorder associated with hyperammonemia. Heterozygous females have variable clinical expression, ranging from asymptomatic illness to recurrent episodes of hyperammonemic coma. We studied 17 OTC-deficient kindreds containing 114 women at risk for heterozygosity. Sixty-one of these women were designated heterozygotes by pedigree analysis, history of protein intolerance, protein tolerance tests, or DNA probe studies. Eleven (18%) of the 61 heterozygotes had experienced encephalopathic episodes; nine (82%) girls died during these episodes. Our findings indicate that there is a significant risk of symptomatic hyperammonemia in females heterozygous for OTC deficiency. We suggest that, within OTC-deficient kindreds, females at risk should be identified early, by means of protein tolerance tests and DNA probe studies. Those who develop significant hyperammonemia after a protein load should be considered for long-term alternate pathway therapy and should receive aggressive therapy during hyperammonemic episodes.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/genética , Amônia/sangue , Heterozigoto , Doença da Deficiência de Ornitina Carbomoiltransferase , Erros Inatos do Metabolismo dos Aminoácidos/complicações , Pré-Escolar , Coma/etiologia , Feminino , Triagem de Portadores Genéticos , Ligação Genética , Humanos , Lactente , Recém-Nascido , Masculino , Linhagem , Risco , Cromossomo XRESUMO
The association of neutropenia with type IB glycogen storage disease was observed in siblings. Biochemical studies of liver demonstrated a defect in glucose-6-phosphate transport. Neutrophil mobilization in vivo was impaired but the bone marrow was normal histologically. In vitro studies of neutrophils indicated a defect in random and directed cell migration. The studies suggest that a role for glucose-6-phosphate transport in neutrophils should be considered.
Assuntos
Agranulocitose/etiologia , Quimiotaxia de Leucócito , Doença de Depósito de Glicogênio Tipo I/sangue , Neutropenia/etiologia , Glucose-6-Fosfatase/análise , Doença de Depósito de Glicogênio Tipo I/complicações , Humanos , Lactente , Fígado/análise , Masculino , NeutrófilosRESUMO
The occurrence in a family of three spontaneous abortions and two siblings with Saldino-Noonan dwarfism supports an autosomal recessive etiology and raises the possibility of lethality early in utero. No long bones were visualized in radiographs at 19 weeks' gestation in the second affected pregnancy. highlighting the difficulties of distinguishing technical limitations from fetal anatomic abnormalities. Ultrasound studies demonstrated oligohydramnios. Radiologic prenatal diagnosis can rule out this condition prior to 20 to 24 weeks' gestation and affected fetuses can be suspected if not proved by radiographs and ultrasound examination.
Assuntos
Nanismo/diagnóstico , Doenças Fetais/diagnóstico , Diagnóstico Pré-Natal , Líquido Amniótico , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/patologia , Nanismo/diagnóstico por imagem , Feminino , Humanos , Gravidez , Radiografia , Recidiva , Crânio/diagnóstico por imagemRESUMO
Of three siblings affected with cholesterol ester storage disease, two died at ages 7 and 9 years, respectively, with hepatic scarring and portal hypertension. Lipid storage was documented in both patients, as were esophageal varices and aortic plaques in the older child. The third affected sibling, followed to 13 years of age, has hepatomegaly, hyperlipidemia, short stature, adrenal calcification, and acid lipase deficiency. Leukocyte extracts demonstrated deficiency of acid lipase in this patient. This autosomal recessive condition may be allelic with Wolman disease with a more malignant course in this family than in most reported cases.
Assuntos
Ésteres do Colesterol/metabolismo , Colesterol/análogos & derivados , Erros Inatos do Metabolismo Lipídico/metabolismo , Criança , Colesterol/metabolismo , Feminino , Fibroblastos/enzimologia , Hepatomegalia/etiologia , Histocitoquímica , Humanos , Lipase/deficiência , Erros Inatos do Metabolismo Lipídico/enzimologia , Erros Inatos do Metabolismo Lipídico/patologia , Fígado/metabolismo , Masculino , Esplenomegalia/etiologia , Triglicerídeos/metabolismoRESUMO
Defiency of beta-glucuronidase was demonstrated in serum, leukocytes, and cultured skin fibroblasts of two unrelated patients. One patient died at 2 9/12 years with a phenotype consistent with severe mucopolysaccharidosis; the other is 14 years of age and well, except for hypertension and obstructive lesions of large blood vessels. Analysis of urinary mucopolysaccharides revealed impaired degradation of dermatan sulfate and, to a lesser extent, of heparan sulfate. Cultured skin fibroblasts accumulated excess glycosaminoglycans (the term glycosaminoglycans is synonymous with acid mucopolysaccharides) as indicated by 35-SO-4 uptake.