RESUMEN
The paper examines progress towards integrated catchment management and sustainable agriculture at Whaingaroa (Raglan), New Zealand. Application of the Canadian "Atlantic Coastal Action Program" model (ACAP) has been only partially successful within New Zealand's bicultural setting. Even before the introduction of the ACAP process there existed strong motivation and leadership by various sectors of the community. A merging of resource management planning and implementation processes of the larger community and that of the Maori community has not occurred. Research carried out by Crown Research Institutes has clearly shown the actions required to make pastoral farming more sustainable. There are difficulties in the transference to, and uptake of, these techniques by farmers. An examination of the socio-economic context is required. There has been a requirement on local government bodies to tighten their focus as part of recent reform. This has occurred concurrently with a widening of vision towards integrated and sustainable forms of management. This (as well as a clear belief in empowerment of local communities) has lead to Council reliance on voluntary labour. There is a need to account for the dynamic interaction between social and political history and the geological and biophysical history of the area. As part of a re-examination of sustainable development, New Zealand needs to reconcile the earning of the bulk of its foreign income from primary production, with the accelerating ecological deficit that it creates. A sustainability strategy is required linking consumer demand, property rights and responsibilities.
Asunto(s)
Agricultura , Conservación de los Recursos Naturales , Contaminación Ambiental/prevención & control , Participación de la Comunidad , Contaminación Ambiental/economía , Humanos , Renta , Liderazgo , Política , Condiciones Sociales , Movimientos del AguaRESUMEN
OBJECTIVES: The KCNQ1 gene encodes the KvLQT1 potassium channel, which generates in the human heart the slow component of the cardiac delayed rectifier current, I(Ks). Mutations in KCNQ1 are the most frequent cause of the congenital long QT syndrome. We have previously cloned a cardiac KCNQ1 human isoform, which exerts a strong dominant-negative effect on KvLQT1 channels. We took advantage of this dominant-negative isoform to engineer an in vivo model of KvLQT1 disruption, obtained by overexpressing the dominant-negative subunit under the control of the alpha-myosin heavy chain promoter. RESULTS: Three different transgenic lines demonstrated a phenotype with increasing severity. Functional suppression of KvLQT1 in transgenic mice led to a markedly prolonged QT interval associated with sinus node dysfunction. Transgenic mice also demonstrated atrio-ventricular block leading to occasional Wenckebach phenomenon. The atrio-ventricular block was associated with prolonged AH but normal HV interval in His recordings. Prolonged QT interval correlated with prolonged action potential duration and with reduced K(+) current density in patch-clamp experiments. RNase protection assay revealed remodeling of K(+) channel expression in transgenic mice. CONCLUSIONS: Our transgenic mouse model suggests a role for KvLQT1 channels not only in the mouse cardiac repolarisation but also in the sinus node automaticity and in the propagation of the impulse through the AV node.
Asunto(s)
Síndrome de QT Prolongado/metabolismo , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/metabolismo , Potenciales de Acción/fisiología , Animales , Electrocardiografía , Humanos , Canales de Potasio KCNQ , Canal de Potasio KCNQ1 , Síndrome de QT Prolongado/genética , Síndrome de QT Prolongado/fisiopatología , Ratones , Ratones Transgénicos , Técnicas de Placa-Clamp , FenotipoRESUMEN
A 469-base pair (bp) upstream regulatory fragment (URF) and the proximal promoter of the carbamoylphosphate synthetase I (CPS) gene were analyzed for their role in the regulation of spatial, developmental, and hormone-induced expression in vivo. The URF is essential and sufficient for hepatocyte-specific expression, periportal localization, perinatal activation and induction by glucocorticoids, and cAMP in transgenic mice. Before birth, the transgene is silent but can be induced by cAMP and glucocorticoids, indicating that these compounds are responsible for the activation of expression at birth. A 102-bp glucocorticoid response unit within the URF, containing binding sites for HNF3, C/EBP, and the glucocorticoid receptor, is the main determinant of the hepatocyte-specific and hormone-controlled activity. Additional sequences are required for a productive interaction between this minimal response unit and the core CPS promoter. These results show that the 469-bp URF, and probably only the 102-bp glucocorticoid response unit, functions as a regulatory module, in that it autonomously executes a correct spatial, developmental and hormonal program of CPS expression in the liver.
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Carbamoil-Fosfato Sintasa (Amoniaco)/genética , Regulación Enzimológica de la Expresión Génica , Hígado/enzimología , Secuencias Reguladoras de Ácidos Nucleicos , Células 3T3 , Animales , Secuencia de Bases , Células CHO , Cricetinae , Cartilla de ADN , Regulación del Desarrollo de la Expresión Génica , Ratones , Regiones Promotoras Genéticas , Células Tumorales CultivadasRESUMEN
The carbamoyl-phosphate synthetase I gene is expressed in the periportal region of the liver, where it is activated by glucocorticosteroids and glucagon (via cyclic AMP), and in the crypts of the intestinal mucosa. The enhancer of the gene is located 6.3 kilobase pairs upstream of the transcription start site and has been shown to direct the hormone-dependent hepatocyte-specific expression in vitro. To analyze the function of the upstream region in vivo, three groups of transgenic mice were generated. In the first group the promoter drives expression of the reporter gene, whereas the promoter and upstream region including the far upstream enhancer drive expression of the reporter gene in the second group. In the third group the far upstream enhancer was directly coupled to a minimized promoter fragment. Reporter-gene expression was virtually undetectable in the first group. In the second group spatial, temporal, and hormonal regulation of expression of the reporter gene and the endogenous carbamoyl-phosphate synthetase gene were identical. The third group showed liver-specific periportal reporter gene expression, but failed to activate expression in the intestine. These results show that the upstream region of the carbamoyl-phosphate synthetase gene controls four characteristics of its expression: tissue specificity, spatial pattern of expression within the liver and intestine, hormone sensitivity, and developmental regulation. Within the upstream region, the far upstream enhancer at -6.3 kilobase pairs is the determinant of the characteristic hepatocyte-specific periportal expression pattern of carbamoyl-phosphate synthetase.
Asunto(s)
Carbamoil-Fosfato Sintasa (Amoniaco)/genética , Regulación Enzimológica de la Expresión Génica , Animales , Northern Blotting , Dexametasona/farmacología , Elementos de Facilitación Genéticos , Genes Reporteros , Hibridación in Situ , Intestino Delgado/enzimología , Hígado/enzimología , Ratones , Ratones Transgénicos , ARN Mensajero/metabolismoRESUMEN
Major histocompatibility complex (MHC) class I and II molecules are loaded with peptides in distinct subcellular compartments. The transporter associated with antigen processing (TAP) is responsible for delivering peptides derived from cytosolic proteins to the endoplasmic reticulum, where they bind to class I molecules, while the invariant chain (Ii) directs class II molecules to endosomal compartments, where they bind peptides originating mostly from exogenous sources. Mice carrying null mutations of the TAP1 or Ii genes (TAP10) or Ii0, respectively) have been useful tools for elucidating the two MHC/peptide loading pathways. To evaluate to what extent these pathways functionally intersect, we have studied the biosynthesis of MHC molecules and the generation of T cells in Ii0TAP10 double-mutant mice. We find that the assembly and expression of class II molecules in Ii0 and Ii0TAP10 animals are indistinguishable and that formation and display of class I molecules is the same in TAP10 and Ii0TAP10 animals. Thymic selection in the double mutants is as expected, with reduced numbers of both CD4+ CD8- and CD4- CD8+ thymocyte compartments. Surprisingly, lymph node T-cell populations look almost normal; we propose that population expansion of peripheral T cells normalizes the numbers of CD4+ and CD8+ cells in Ii0TAP10 mice.
Asunto(s)
Presentación de Antígeno , Antígenos de Diferenciación de Linfocitos B/fisiología , Antígenos H-2/biosíntesis , Antígenos de Histocompatibilidad Clase II/biosíntesis , Antígenos de Histocompatibilidad Clase II/fisiología , Síndromes de Inmunodeficiencia/inmunología , Fragmentos de Péptidos/metabolismo , Subgrupos de Linfocitos T/citología , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/fisiología , Animales , Antígenos de Diferenciación de Linfocitos B/genética , Transporte Biológico , División Celular , Supresión Clonal , Retículo Endoplásmico/metabolismo , Endosomas/metabolismo , Antígenos H-2/inmunología , Antígenos H-2/metabolismo , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Síndromes de Inmunodeficiencia/genética , Ganglios Linfáticos/inmunología , Recuento de Linfocitos , Ratones , Ratones Mutantes , Fragmentos de Péptidos/inmunología , Subgrupos de Linfocitos T/inmunología , Timo/inmunologíaRESUMEN
In previous studies of the glutamine synthetase gene, the promoter and two enhancer elements, one in the upstream region and one within the first intron, were identified. To analyze the role of the far-upstream enhancer element in the regulation of the expression of the glutamine synthetase gene, two classes of transgenic mice were generated. In GSK mice, the basal promoter directs the expression of the chloramphenicol acetyltransferase reporter gene. In GSL mice reporter gene expression is driven, in addition, by the upstream regulatory region, including the far-upstream enhancer. Whereas chloramphenicol acetyltransferase expression was barely detectable in GSK mice, high levels were detected in GSL mice. By comparing chloramphenicol acetyltransferase expression with that of endogenous glutamine synthetase in GSL mice, three groups of organs were distinguished in which the effects of the upstream regulatory region on the expression of glutamine synthetase were quantitatively different. The chloramphenicol acetyltransferase mRNA in the GSL mice was shown to be localized in the pericentral hepatocytes of the liver. The developmental changes in chloramphenicol acetyltransferase enzyme activity in the liver were similar to those in endogenous glutamine synthetase. These results show that the upstream region is a major determinant for three characteristics of glutamine synthetase expression: its organ specificity, its pericentral expression pattern in the liver, and its developmental appearance in the liver.
Asunto(s)
Envejecimiento/metabolismo , Elementos de Facilitación Genéticos , Glutamato-Amoníaco Ligasa/biosíntesis , Glutamato-Amoníaco Ligasa/genética , Hígado/enzimología , Animales , Secuencia de Bases , Cloranfenicol O-Acetiltransferasa/biosíntesis , Cartilla de ADN , Regulación Enzimológica de la Expresión Génica , Tamización de Portadores Genéticos , Homocigoto , Intrones , Hígado/citología , Hígado/crecimiento & desarrollo , Masculino , Ratones , Ratones Endogámicos , Ratones Transgénicos , Datos de Secuencia Molecular , Especificidad de Órganos , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Ratas , Virus 40 de los Simios/genética , Transcripción GenéticaRESUMEN
We report the generation and characterization of mice lacking adenosine deaminase (ADA). In humans, absence of ADA causes severe combined immunodeficiency. In contrast, ADA-deficient mice die perinatally with marked liver-cell degeneration, but lack abnormalities in the thymus. The ADA substrates, adenosine and deoxyadenosine, are increased in ADA-deficient mice. Adenine deoxyribonucleotides are only modestly elevated, whereas S-adenosylhomocysteine hydrolase activity is reduced more than 85%. Consequently, the ratio of S-adenosylhomocysteine (AdoMet) to S-adenosyl homocysteine (AdoHcy) is reduced threefold in liver. We conclude that ADA plays a more critical role in murine than human fetal development. The murine liver pathology may be due to AdoHcy-mediated inhibition of AdoMet-dependent transmethylation reactions.
Asunto(s)
Adenosina Desaminasa/deficiencia , Adenosina Desaminasa/genética , Intestino Delgado/patología , Hígado/patología , Atelectasia Pulmonar/genética , Animales , Animales Recién Nacidos , Secuencia de Bases , Muerte Celular , Cartilla de ADN/genética , Modelos Animales de Enfermedad , Desarrollo Embrionario y Fetal/genética , Desarrollo Embrionario y Fetal/fisiología , Femenino , Marcación de Gen , Homocigoto , Humanos , Masculino , Metilación , Ratones , Datos de Secuencia Molecular , Mutación , Embarazo , Purinas/metabolismo , Inmunodeficiencia Combinada Grave/etiología , Subgrupos de Linfocitos T/inmunologíaRESUMEN
E12 and E47 are two helix-loop-helix transcription factors that arise by alternative splicing of the E2A gene. Both have been implicated in the regulation of immunoglobulin gene expression. We have now generated E2A (-/-) mice by gene targeting. E2A-null mutant mice fail to generate mature B cells. The arrest of B cell development occurs at an early stage, since no immunoglobulin DJ rearrangements can be detected in homozygous mutant mice. While immunoglobulin germline I mu RAG-1, mb-1, CD19, and lambda 5 transcripts are dramatically reduced in fetal livers of E2A (-/-) mice, B29 and mu degrees transcripts are present, but at lower levels. In addition, we show that Pax-5 transcripts are significantly reduced in fetal livers of E2A (-/-) mice. These data suggest a crucial role for E2A products as central regulators in early B cell differentiation.
Asunto(s)
Linfocitos B/fisiología , Proteínas de Unión al ADN/metabolismo , Genes de Inmunoglobulinas/genética , Proteínas de Homeodominio , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Diferenciación Celular , Proteínas de Unión al ADN/genética , Reordenamiento Génico , Homocigoto , Cadenas lambda de Inmunoglobulina/genética , Tejido Linfoide/inmunología , Ratones , Ratones Mutantes , Datos de Secuencia Molecular , Proteínas/genética , ARN Mensajero/análisis , ARN Mensajero/genética , Factores de Transcripción TCF , Proteína 1 Similar al Factor de Transcripción 7 , Factores de Transcripción/genéticaRESUMEN
The requirement for a functional retinoblastoma gene, Rb-1, in murine development around days 12-15 of gestation precludes monitoring the effect of loss of Rb-1 function on later stages of development and on tumorigenesis in adult mice. Here we describe the developmental rescue of embryonic stem cells carrying two inactive Rb-1 alleles in chimeric mice. Rb-1- cells contributed substantially to most tissues in adult chimeras, including blood, liver and central nervous system, which were severely affected in pure Rb-1- embryos. The adult chimeric erythroid compartment appeared completely normal, but an increased number of nucleated red cells was observed during fetal liver erythropoiesis in highly chimeric embryos. No ostensive abnormalities were seen in the developing and adult CNS. However, the developing retina of chimeric Rb-1- embryos showed ectopic mitoses and substantial cell degeneration, while the contribution of Rb-1- cells to the adult retina was much reduced. Moreover, the formation of lens fibre cells was severely disturbed. No retinoblastomas developed in any of these mice. Instead, nearly all animals died of pituitary gland tumours which were exclusively derived from Rb-1- cells.
Asunto(s)
Embrión de Mamíferos/patología , Genes Letales/genética , Genes de Retinoblastoma/genética , Mutación , Animales , Sistema Nervioso Central/patología , Eritropoyesis , Glucosa-6-Fosfato Isomerasa/análisis , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica , Neoplasias Hipofisarias/genética , Retina/patología , Distribución Tisular , Quimera por TrasplanteRESUMEN
Transgenic mice overexpressing the interleukin 9 gene were generated to study the biological activity of this cytokine in vivo. Although no major histological or morphological modifications of the lymphoid system were observed in most animals, approximately 7% of transgenic mice developed thymic lymphomas at the age of 3-9 months. The tumor cells, which were clonal, with unique T cell rearrangements, were double positive for the expression of CD4 and CD8. The need for additional transforming events, suggested by the low incidence of spontaneous tumors, was further indicated by the high susceptibility of the transgenic animals to injections of low doses of N-methyl-N-nitrosourea, a chemical carcinogen with a thymic tropism. Expression of interleukin 9 was required for optimal tumor growth in vivo, as one of the tumors studied, which had lost the transgene, was much more efficiently transplanted into transgenic than in normal mice. Moreover, the in vitro proliferative activity of interleukin 9 on cell lines derived from such transgene-negative tumors suggests that an autocrine loop mediates the proliferation of these cells in vivo. Taken together, these results indicate that dysregulated IL-9 expression could be involved in the development of some T cell malignancies.
Asunto(s)
Interleucina-9/fisiología , Linfoma de Células T/etiología , Neoplasias del Timo/etiología , Animales , Interleucina-9/genética , Interleucina-9/metabolismo , Linfoma de Células T/inducido químicamente , Linfoma de Células T/genética , Linfoma de Células T/metabolismo , Metilnitrosourea , Ratones , Ratones Transgénicos , Trasplante de Neoplasias , Neoplasias del Timo/inducido químicamente , Neoplasias del Timo/genética , Neoplasias del Timo/metabolismoRESUMEN
The bmi-1 proto-oncogene has been implicated in B-cell lymphomagenesis in E mu-myc transgenic mice. Distinct domains of the Bmi-1 protein are highly conserved within the Drosophila protein Posterior Sex Combs, a member of the Polycomb group involved in maintaining stable repression of homeotic genes during development. We have inactivated the bmi-1 gene in the germ line of mice by homologous recombination in ES cells. Null mutant mice display three phenotypic alterations: (1) a progressive decrease in the number of hematopoietic cells and an impaired proliferative response of these cells to mitogens; (2) neurological abnormalities manifested by an ataxic gait and sporadic seizures; and (3) posterior transformation, in most cases along the complete anteroposterior axis of the skeleton. The observations indicate that Mbi-1 plays an important role in morphogenesis during embryonic development and in hematopoiesis throughout pre- and postnatal life. Furthermore, these data provide the first evidence of functional conservation of a mammalian Polycomb group homolog.
Asunto(s)
Encéfalo/anomalías , Sistema Hematopoyético/anomalías , Proteínas Nucleares/fisiología , Proteínas Proto-Oncogénicas/fisiología , Proteínas Represoras , Columna Vertebral/anomalías , Animales , Ataxia/genética , Células de la Médula Ósea , Desarrollo Embrionario y Fetal , Femenino , Regulación del Desarrollo de la Expresión Génica , Marcación de Gen , Mutación de Línea Germinal/fisiología , Sistema Hematopoyético/citología , Activación de Linfocitos , Masculino , Ratones , Ratones Transgénicos , Mitógenos/farmacología , Proteínas Nucleares/genética , Complejo Represivo Polycomb 1 , Proteínas Proto-Oncogénicas/genética , Proto-Oncogenes , Recombinación Genética , Costillas/anomalías , Convulsiones/genética , Bazo/citología , Timo/citologíaRESUMEN
Two types of P-glycoprotein have been found in mammals: the drug-transporting P-glycoproteins and a second type, unable to transport hydrophobic anticancer drugs. The latter is encoded by the human MDR3 (also called MDR2) and the mouse mdr2 genes, and its tissue distribution (bile canalicular membrane of hepatocytes, B cells, heart, and muscle) suggests a specialized metabolic function. We have generated mice homozygous for a disruption of the mdr2 gene. These mice develop a liver disease that appears to be caused by the complete inability of the liver to secrete phospholipid into the bile. Mice heterozygous for the disrupted allele had no detectable liver pathology, but half the level of phospholipid in bile. We conclude that the mdr2 P-glycoprotein has an essential role in the secretion of phosphatidylcholine into bile and hypothesize that it may be a phospholipid transport protein or phospholipid flippase.
Asunto(s)
Bilis/metabolismo , Proteínas Portadoras/genética , Hepatopatías/genética , Glicoproteínas de Membrana/genética , Fosfolípidos/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Alelos , Animales , Secuencia de Bases , Bilirrubina/sangre , Proteínas Portadoras/análisis , Enzimas/sangre , Homocigoto , Hígado/fisiopatología , Glicoproteínas de Membrana/análisis , Ratones , Ratones Mutantes , Datos de Secuencia Molecular , Mutagénesis , ARN Mensajero/análisis , Recombinación GenéticaRESUMEN
Human retinoblastomas can occur both as hereditary and as sporadic cases. Knudson's proposal that they result from two mutational events, of which one is present in the germ line in hereditary cases, has been confirmed by more recent molecular analysis, which has shown both events to involve loss or mutational inactivation of the same gene, RB-1 (ref. 2). RB-1 heterozygosity also predisposes to osteosarcoma, and RB-1 allele losses are seen in sporadic lung, breast, prostate and bladder carcinomas. RB-1 is expressed in most, if not all, tissues and codes for a nuclear phosphoprotein which becomes hypophosphorylated in the G0 growth arrest state and in the G1 phase of the cell cycle. To gain a further insight into the role of RB-1 we and other groups have generated mice carrying an inactivated allele of the homologous gene, Rb-1 (ref. 10), by gene targeting. We report here that young heterozygous mice do not appear abnormal and do not develop retinoblastoma at a detectable frequency. However, homozygous mutant embryos fail to reach term and show a number of abnormalities in neural and haematopoietic development. Broadly similar results are reported by the other groups.
Asunto(s)
Anomalías Múltiples/genética , Desarrollo Embrionario y Fetal/genética , Genes de Retinoblastoma , Anomalías Múltiples/embriología , Animales , Células Cultivadas , Quimera , Mapeo Cromosómico , Cruzamientos Genéticos , Exones , Hematopoyesis/genética , Humanos , Ratones , Ratones Endogámicos , Células Madre/citologíaRESUMEN
The effects of pretreatment with symmetrically dihalogenated biphenyls (DXBs, X-F, Cl(C), Br(B) and I) on rat liver drug metabolism enzymes were investigated. 4,4'-DFB, -DCB, and -DBB as well as 2,2'-DFB appeared to be inducers of microsomal cytochrome P-450-linked monoxygenases (N-demethylases of aminopyrine and ethylmorphine). However, no structure-induction relationship was found. 4,4'-DXBs also induced a cytochrome P-448-linked mono-oxygenase (ethoxyresorufin O-deethylase), and their order of induction potential seemed to parallel the increase of the size of the halogen substituent. Therefore, 4,4'-DXB's may be categorized as mixed-type inducers, the cytochrome P-450 component being the more pronounced. Data on the cytochrome P-448 induction by dihalogenated biphenyls with only para substituents may be considered as a refinement of the previously described structure-activity relationship in this respect. All of the DXBs except 3,3'-DCB and 4,4'-DIB, enhanced, like phenobarbital, the activity of UDP-glucuronyltransferase toward 4-hydroxybiphenyl. Only 4,4'-DFB was able to induce the activity of glutathione S-transferase toward 1,2-epoxy-3-(p-nitrophenoxy)propane. Studies after 4,4'-DBB-treatment revealed, like phenobarbital, a preferential induction of ethylmorphine N-demethylase on rough endoplasmic reticulum-derived microsomes, whereas UDP-glucuronyltransferase activity toward 4-hydroxybiphenyl was induced to a larger extent on smooth endoplasmic reticulum microsomes, suggesting a dissimilar enzyme induction in microsomal subfractions.
Asunto(s)
Hidrocarburos Halogenados/farmacología , Microsomas Hepáticos/enzimología , Animales , Compuestos de Bifenilo/farmacología , Sistema Enzimático del Citocromo P-450/biosíntesis , Sistema Enzimático del Citocromo P-450/metabolismo , Citosol/enzimología , Inducción Enzimática , Glucuronosiltransferasa/biosíntesis , Glucuronosiltransferasa/metabolismo , Glutatión Transferasa/biosíntesis , Glutatión Transferasa/metabolismo , Hígado/ultraestructura , Masculino , Oxigenasas de Función Mixta/biosíntesis , Oxigenasas de Función Mixta/metabolismo , Oxigenasas/biosíntesis , Oxigenasas/metabolismo , Bifenilos Policlorados/farmacología , Ratas , Ratas Endogámicas , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The aim of this study was to see whether the rat embryo can serve as a model system for hepatocyte-specific gene expression in the human embryo. Carbamoylphosphate synthetase was used as a hepatocyte-specific marker molecule. Despite the earlier developmental appearance of this enzyme in human than in murine liver, the hormonal regulation of gene expression in cultures of embryonic hepatocytes was found to be the same. Therefore, a relatively early developmental appearance of regulatory hormones rather than differences in regulatory mechanisms of gene expression appears to be responsible for the early accumulation of the enzyme in human liver, when compared to murine liver.
Asunto(s)
Embrión de Mamíferos/fisiología , Regulación de la Expresión Génica , Hígado/fisiología , Animales , Carbamoil-Fosfato Sintasa (Amoniaco)/genética , Carbamoil-Fosfato Sintasa (Amoniaco)/metabolismo , Células Cultivadas , Humanos , Inmunohistoquímica , Hígado/citología , Hígado/embriología , Hibridación de Ácido Nucleico , ARN Mensajero/metabolismo , RatasRESUMEN
To gain insight into the mechanisms that govern the first steps of liver-specific enzyme accumulation upon hormone exposure, the initial accumulation of carbamoylphosphate synthetase, phosphoenolpyruvate carboxykinase, and arginase in monolayer cultures of Embryonic Day 14 rat hepatocytes was studied. By using different fluorescent labels the initial accumulation of two enzymes could be studied simultaneously in individual cells. Both microscopic and flow cytometric analyses showed that the initial expression of genes that are under the same hormonal control appears to lack the coordinated regulation of expression that is seen later in development. The coordination is gradually established during exposure to hormones. Once gene expression becomes coordinated, the enzyme content appears to increase continuously with time. Therefore, we postulate that within individual embryonic hepatocytes the initial intercellular heterogeneity in rate of accumulation of a particular protein may be the result of competition of different genes for an initially limiting supply of common regulatory factors, leading to random differences in the rate of accumulation of the respective gene products. This makes the initiation of liver-specific gene expression within the hepatocytes a stochastic event.
Asunto(s)
Regulación de la Expresión Génica , Hígado/embriología , 1-Metil-3-Isobutilxantina/farmacología , Animales , Arginasa/metabolismo , Bucladesina/farmacología , Carbamoil-Fosfato Sintasa (Glutamina-Hidrolizante)/metabolismo , Células Cultivadas , ADN/metabolismo , Dexametasona/farmacología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica/efectos de los fármacos , Hígado/enzimología , Hígado/fisiología , Fosfoenolpiruvato Carboxiquinasa (GTP)/metabolismo , Ratas , Procesos Estocásticos , Triyodotironina/farmacologíaRESUMEN
The hormone-induced expression of the hepatocyte-specific enzyme carbamoylphosphate synthetase can take place in each phase of the cell cycle and is not restricted to the G1 or the G0 phase. To arrive at this conclusion, the cell cycle parameters of embryonic day 14 rat hepatocytes in vitro were determined by autoradiography after labeling with (3H)-TdR or with (3H)- and (14C)-TdR. An S-phase of approximately 14 h, a G2 + M-phase of 8 h, a G1-phase of 8-13 h and a total cell cycle of 30-35 h were measured. Freshly isolated embryonic hepatocytes have exponential growth parameter values, but shift to a steady state growth under culture conditions in the presence of hormones (glucocorticosteroids, thyroid hormones and cyclic AMP). The length of the S-phase and of the total cell cycle remain constant during the culture time. The time course of accumulation of carbamoylphosphate synthetase protein in embryonic hepatocytes is identical in all phases of the cell cycle. It is suggested that hormones, in particular glucocorticosteroids, simultaneously and independently regulate growth mode and gene expression in developing hepatocytes. The nucleotide-analogue 5-bromodeoxyuridine inhibits the hormone-induced expression of carbamoylphosphate synthetase only in cells that are exposed to the drug during early S-phase, indicating replication of the carbamoylphosphate synthetase gene in that part of the cell cycle.
Asunto(s)
Carbamoil-Fosfato Sintasa (Glutamina-Hidrolizante)/biosíntesis , Hígado/enzimología , Animales , Autorradiografía , Carbamoil-Fosfato Sintasa (Glutamina-Hidrolizante)/análisis , Ciclo Celular , División Celular , Células Cultivadas , Interfase , Hígado/embriología , Ratas , Ratas Endogámicas , Factores de TiempoRESUMEN
The aim of this study was to establish whether the initial accumulation of hepatocyte-specific proteins after hormone induction is regulated at the pretranslational and/or the translational level. To this end, mRNA molar concentrations were determined and compared with rates of protein synthesis from previous studies [van Roon, M.A., Charles, R. & Lamers, W.H. (1987) Eur. J. Biochem. 165, 229-234]. In vivo, carbamoylphosphate-synthetase mRNA starts to accumulate at day 17 of pregnancy. Phosphoenolpyruvate-carboxykinase mRNA starts to accumulate only just prior to birth. Embryonic day 14 (i.e. 8 days before the expected day of birth), livers were chosen to study the regulation of the initiation of hepatocyte-specific mRNA accumulation in vitro. Accumulation of carbamoylphosphate-synthetase and phosphoenolpyruvate-carboxykinase mRNA is regulated by the same hormones as accumulation of the respective proteins. The rate at which carbamoylphosphate-synthetase and phosphoenolpyruvate-carboxykinase mRNA molecules accumulate in cultured embryonic hepatocytes is relatively low, compared to that of postnatal hepatocytes. However, the increase of the rate of synthesis of carbamoylphosphate-synthetase and phosphoenolpyruvate-carboxykinase protein is even 3-6-fold slower than that of mRNA. This shows that initially mRNAs accumulate intracellularly to a relatively high concentration without being efficiently translated or translatable. Only after the mRNA concentration reaches a plateau of 72 h and 48 h respectively, the cellular capacity to synthesize the respective proteins increases. Therefore, the translational efficiency is certainly one of the major rate-limiting factors of the initial phases of expression of the hepatocyte-specific genes for carbamoylphosphate synthetase and phosphoenolpyruvate carboxykinase.
Asunto(s)
Carbamoil-Fosfato Sintasa (Glutamina-Hidrolizante)/genética , Regulación de la Expresión Génica , Hígado/enzimología , Hibridación de Ácido Nucleico , Fosfoenolpiruvato Carboxiquinasa (GTP)/genética , ARN Mensajero/metabolismo , Transcripción Genética , Animales , Células Cultivadas , Femenino , Feto/metabolismo , Hormonas/farmacología , Hígado/embriología , Masculino , Ratas , Ratas EndogámicasRESUMEN
Glucocorticosteroid, thyroid hormones and cyclic AMP can induce the synthesis of carbamoylphosphate synthetase and phosphoenolpyruvate carboxykinase in cultures of hepatocytes as soon as these cells differentiate from the embryonic foregut. The low levels of both enzymes that can accumulate in such still protodifferentiated hepatocytes are due to low levels of enzyme synthesis. In cultures, the rate of synthesis of both enzymes increases continually in the presence of hormones, showing that maturation of the capacity for synthesis towards the postnatal, fully differentiated situation is occurring in these cells. The turnover rate of both enzymes in embryonic hepatocytes is lower in the presence of hormones than in the absence, but does not change during the culture period. In the presence of hormones the turnover rate is comparable to that found in adult rat liver in vivo. The development of the capacity to accumulate organ-specific enzymes in vitro (and hence the rate of enzyme synthesis) is found to be comparable to that in utero.
Asunto(s)
Carbamoil-Fosfato Sintasa (Amoniaco)/metabolismo , Ligasas/metabolismo , Hígado/embriología , Fosfoenolpiruvato Carboxiquinasa (GTP)/metabolismo , 1-Metil-3-Isobutilxantina/farmacología , Animales , Bucladesina/farmacología , Células Cultivadas , Dexametasona/farmacología , Feto , Edad Gestacional , Cinética , Hígado/efectos de los fármacos , Hígado/enzimología , Ratas , Ratas Endogámicas , Triyodotironina/farmacologíaRESUMEN
A completely defined medium (EHM-1), which reflects the amino acid composition of fetal rat serum and contains albumin as the sole proteinaceous compound, allows the accumulation of carbamoylphosphate synthetase and phosphoenolpyruvate carboxykinase in the presence of dexamethasone, dibutyryl cyclic AMP, and triiodothyronine to approximately twice the level attained in a standard culture medium (RPMI 1640) supplemented with 10% fetal bovine serum (and hormones). Using the EHM-1 medium we could show that the capacity of hepatocytes to synthesize phosphoenolpyruvate carboxykinase in the presence of hormones is manifest as soon as the cells differentiate from the embryonic foregut (embryonic Day 11). Furthermore we could show that embryonic hepatocytes can become binuclear or polyploid when cultured in the presence of thyroid hormone.