RESUMO
Mesotocin is the oxytocin-like hormone found in most terrestrial vertebrates from lungfishes to marsupials, which includes all non-mammalian tetrapods (amphibians, reptiles, and birds). It has the largest distribution in vertebrates after vasotocin found in all non-mammalian vertebrates and isotocin identified in bony fishes. In this study, we report the cloning and functional characterization of the cDNA for the mesotocin receptor (MTR) from the urinary bladder of the toad Bufo marinus. The cloned cDNA encodes a polypeptide of 389 amino acids that shows the greatest similarity to the teleost fish isotocin receptor and to mammalian oxytocin receptors with mutations in extracellular loops which are involved in ligand binding. When expressed in COSM6 cells, MTR exhibits the following relative order of ligand affinity: mesotocin > vasotocin = oxytocin > vasopressin > hydrin 1, isotocin, hydrin 2. Injection of MTR cRNA into Xenopus laevis oocytes induces membrane chloride currents in response to mesotocin, which indicates the coupling of the mesotocin receptor to the inositol phosphate/calcium pathway. This response is inhibited by an oxytocin antagonist, but not by a vasopressin antagonist specific for V2 vasopressin receptors. MTR mRNA is not only found in toad urinary bladder, but also in kidney, muscle, and brain tissue of the toad as revealed by northern blot analysis and reverse-transcriptase PCR. The results suggest a variety of function for mesotocin and its receptor including, in particular, an involvement in the regulation of water and salt transport.
Assuntos
Bufo marinus/genética , Bufo marinus/metabolismo , Ocitocina/análogos & derivados , Receptores de Ocitocina/genética , Receptores de Ocitocina/metabolismo , Receptores de Vasopressinas/genética , Receptores de Vasopressinas/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , DNA Complementar/genética , Feminino , Humanos , Dados de Sequência Molecular , Oócitos/metabolismo , Ocitocina/metabolismo , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Distribuição Tecidual , Xenopus laevisRESUMO
The gene encoding the human homologue of the guinea pig uterine bombesin receptor [(1992) Eur. J. Biochem. 208, 405] was isolated from a genomic lambda library by the PCR/homology screening approach. The gene spans more than 4 kb and consists of 3 exons and 2 introns. The deduced amino acid sequence shows about 86% identity to that of guinea pig bombesin receptor. This subtype of bombesin receptor is expressed in the pregnant uterus and in two human tumour cell lines, T47D (ductal breast carcinoma) and A431 (epidermal carcinoma). PCR analysis of genomic DNA from human-mouse cell hybrids allows the cloned gene to be localized to the region q26-q28 on chromosome X.
Assuntos
Receptores da Bombesina/genética , Útero/metabolismo , Cromossomo X , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , DNA Complementar , Feminino , Cobaias , Humanos , Células Híbridas , Camundongos , Dados de Sequência Molecular , Gravidez , Homologia de Sequência de AminoácidosRESUMO
Primary infection of HEp-2 cells with rubella virus resulted in non-cytophatic long-term persistent infection. During four years of persistence the virus was produced in sufficient quantities (up to 6 logs PFU/ml) and did not differ from the parental variant in its pathogenicity for BHK-21 or RK-13 cells, or hemagglutinating activity, but formed smaller plaques. Persistent virus preserved the original antigenicity as judged from reciprocal hemagglutination-inhibition or plaque reduction-neutralization tests with polyclonal antisera. Both original and persistent rubella viruses were thermoresistant (T 56 degrees C) and slightly temperature-sensitive. Clonal analysis revealed presence of ts-mutants among both original and persistent virus clones with different degrees of plating efficiency at 40 degrees/34 degrees C. RNA fingerprinting showed only minor changes in persistent rubella virus.
Assuntos
Vírus da Rubéola/crescimento & desenvolvimento , Antígenos Virais/análise , Carcinoma de Células Escamosas , Linhagem Celular , Humanos , Vírus da Rubéola/isolamento & purificação , Temperatura , Ensaio de Placa ViralRESUMO
The application of different approaches for preparing DNAs coding for peptide hormones was demonstrated. The libraries of human, bovine and porcine pituitaries cDNA were obtained starting from their total mRNAs. Screening of these libraries revealed clones containing human, bovine and porcine growth hormone sequences, cDNAs for bovine ACTH-beta-lipotropin precursor and for bovine and porcine prolactin. The gene of human calcitonin was created by combination of chemical and enzymatic synthesis. This synthetic gene was further cloned in pBR322. The expression of cloned human growth hormone cDNA under control of different Escherichia coli promoters was studied and physico-chemical and biological properties of the growth hormone produced by E. coli were tested.