RESUMEN
The expression of PRB1, the gene that encodes the precursor to the soluble vacuolar proteinase B (PrB) in Saccharomyces cerevisiae, is regulated by carbon and nitrogen sources and by growth phase. Little or no PRB1 mRNA is detectable during exponential growth on glucose as the carbon source; it begins to accumulate as cells exhaust the glucose. Previous work has shown that glucose repression of PRB1 transcription is not mediated by HXK2 or by the SNF1, SNF4, and SNF6 genes (C. M. Moehle and E. W. Jones, Genetics 124:39-55, 1990). We analyzed the effects of mutations in the MIG1, TUP1, and GRR1 genes on glucose repression of PRB1 and found that mutations in each partially alleviate glucose repression. tup1 and mig1 mutants fail to translocate all of the Prb1p into the lumen of the endoplasmic reticulum. A screen for new mutants revealed mutations in MIG1 and REG1, genes already known to regulate glucose repression, as well as in three new genes that we have named PBD1 to PBD3; all cause derepressed expression. Mutations that result in failure to completely derepress PRB1 were also identified in two new genes, named PND1 and PND2. Good nitrogen sources, like ammonia, repress PRB1 transcription; mutations in URE2 do not affect this response. Derepression upon transfer to a poor nitrogen source is dependent upon GLN3.
Asunto(s)
Precursores Enzimáticos/genética , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Fosfoproteínas Fosfatasas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Serina Endopeptidasas/genética , Factores de Transcripción , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Retículo Endoplásmico/metabolismo , Precursores Enzimáticos/biosíntesis , Precursores Enzimáticos/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Reguladores , Glucosa/metabolismo , Mutación , Proteína Fosfatasa 1 , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/enzimología , Serina Endopeptidasas/biosíntesis , Serina Endopeptidasas/metabolismo , Transformación Genética , beta-Galactosidasa/biosíntesisRESUMEN
Serum antibodies reactive with eye muscle autoantigens, in particular a 64-kDa protein that is also expressed in the thyroid, and the TSH receptor, are associated with the ophthalmopathy that occurs in about 50% of patients with Graves' hyperthyroidism. We have had the opportunity to study a euthyroid, apparently normal, 35-year-old woman with a family history of thyroid autoimmunity and "colitis" but no clinical or biochemical evidence for thyroid disease or ophthalmopathy, who developed Graves' hyperthyroidism and ophthalmopathy together 18 months later. Serum taken when the patient was first seen was positive for antibodies reactive with (i) 9 different eye muscle proteins ranging in size from 15 to 130 kDa, notably those of 64, 55, and 50 kDa, by immunoblotting with eye muscle membranes, (ii) eye muscle and Müller's muscle cell membrane antigens in antibody-dependent cell-mediated cytotoxicity (ADCC), (iii) an eye muscle cytoplasmic antigen in indirect immunofluorescence, and (iv) the TSH receptor as measured in a radioreceptor binding inhibition assay. When she developed Graves' disease, serum concentration of antibodies to the 55-kDa protein had decreased from +2 to +/-, those reactive with other eye muscle antigens had not changed significantly, and TSH receptor antibodies had increased 3-fold. This case report suggests that antibodies reactive with eye muscle antigens and the TSH receptor are markers of the ophthalmopathy and able to predict its development in predisposed subjects. The significance of these findings needs to be confirmed in a prospective study of first-degree relatives of patients with thyroid-associated ophthalmopathy and patients with Graves' hyperthyroidism without eye signs.
Asunto(s)
Ojo/metabolismo , Enfermedad de Graves/metabolismo , Músculo Liso/metabolismo , Receptores de Tirotropina/metabolismo , Adulto , Citotoxicidad Celular Dependiente de Anticuerpos , Western Blotting , Electroforesis en Gel de Poliacrilamida , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Enfermedad de Graves/complicaciones , Humanos , Tirotropina/sangre , Tiroxina/sangreRESUMEN
We studied the human lysosomal alpha-mannosidase (MANB) by expressing the putative cDNA in mammalian cells, using the eucaryotic expression vector pCDE. The construct pCDE-MANB and pSV2-Neo were cotransfected into human alpha-mannosidase deficient fibroblasts and into a murine cell line and selected by culture in the presence of G418. Six G418 resistant 3T3 clones had increased alpha-mannosidase activity 2 to 3 times above the controls. Two clones from transfected human fibroblasts showed a 2 fold increase in enzyme activity. The human MANB cDNA gene was demonstrated in the target cells by Southern blot analysis and the expression of the gene was shown by RT-PCR analysis. This study is the first to successfully express the MANB gene in a human and a murine cell line. The results confirm that the putative MANB cDNA encodes the full length of lysosomal alpha-mannosidase. Molecular characterization of mannosidosis and approaches to gene therapy are now possible using this cDNA.
Asunto(s)
Lisosomas/enzimología , Manosidasas/genética , Células 3T3 , Animales , Secuencia de Bases , Southern Blotting , Células Clonales , ADN Complementario , Fibroblastos/enzimología , Humanos , Manosidasas/metabolismo , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Transfección , alfa-ManosidasaRESUMEN
We have carried out tests for antibody-dependent cell-mediated cytotoxicity (ADCC) against extra ocular muscle (EOM), Müller's muscle, orbital fibroblasts and skeletal muscle in patients with thyroid-associated ophthalmopathy (TAO) and related eye disorders. Cytotoxicity was measured as lactate dehydrogenase (LDH) release and results expressed as % cytotoxicity. Tests were positive, with EOM cells, in 65% of patients with TAO, 75% with ocular myopathy, a variant of TAO in which periorbital inflammation is minimal, 50% with euthyroid Graves' disease defined as ophthalmopathy associated with subclinical thyroiditis and in 50% of patients with stable lid lag and retraction but no other signs of progressive ophthalmopathy, but in only 13% of patients with Graves' hyperthyroidism without ophthalmopathy, 10% with Hashimoto's thyroiditis and 14% of patients with other thyroid disorders. Tests were positive, with Müller's muscle cells, in 40% of patients with TAO, 25% with ocular myopathy, 40% with euthyroid Graves' disease, 44% with lid lag, 19% with Graves'hyperthyroidism, 50% with Hashimoto's thyroiditis and in 37.5% of patients with other thyroid disorders. When skeletal muscle cells were used as target, tests were positive in 13% of patients with TAO, 31% with lid lag, 25% with Graves' hyperthyroidism and in 29% of patients with Hashimoto's thyroiditis, but in no patient with euthyroid Graves' disease or other thyroid disorders. Tests were negative in all patients and normals tested when EOM-derived fibroblasts were used as targets in ADCC. A significant positive correlation between % cytotoxicity against EOM cells and the severity of the eye muscle dysfunction expressed as an eye muscle index, was observed in patients with TAO. There was a significant negative correlation between the duration of eye disease and % cytotoxicity against EOM cells, suggesting higher titers of cytotoxic antibodies in the early stages of TAO. There was no correlation between % cytotoxicity and serum level of anti-TSH receptor antibodies, measured in a radioreceptor assay. These findings suggest that autoimmunity against Müller's muscle may play a role in the pathogenesis of persistent lid lag and retraction. The nature of the EOM and Müller's muscle autoantigens recognized by cytotoxic antibodies in the serum of patients with TAO and related eye disorders is unknown.
Asunto(s)
Citotoxicidad Celular Dependiente de Anticuerpos , Autoanticuerpos/sangre , Oftalmopatías/inmunología , Enfermedad de Graves/inmunología , Órbita/inmunología , Adulto , Anciano , Autoantígenos/inmunología , Células Cultivadas , Tejido Conectivo/inmunología , Femenino , Fibroblastos/inmunología , Humanos , L-Lactato Deshidrogenasa/metabolismo , Masculino , Persona de Mediana Edad , Músculo Esquelético/inmunología , Músculos/inmunología , Tiroiditis Autoinmune/inmunologíaRESUMEN
The relationship between the many immunologic abnormalities demonstrated in the peripheral blood of patients with Graves' disease (GD) and the broad spectrum of clinical features with which patients may present has not yet been addressed in detail. In this review we examine the evidence to support the notion that GD could be considered a multi-system autoimmune disorder in which tissue damage is restricted to the thyroid gland, connective tissue of the skin and orbit, extra-ocular and other skeletal muscles and, possibly, the lacrimal glands. Apart from the well recognized reactions of autoantibodies and sensitized T lymphocytes with epitopes on the thyroid specific TSH receptor, thyroid peroxidase and thyroglobulin, in patients with hyperthyroidism, there is also good evidence for autoantibody and, to a lesser extent, T lymphocyte reactivity with several eye muscle, other skeletal muscle and connective tissue, antigens in patients with ophthalmopathy, systemic myopathy, dermopathy and acropachy. There is also some evidence for immunoreactivity against lacrimal gland antigens in patients with ophthalmopathy associated with other features of GD. There are, in addition, a variety of organ non-specific reactions in GD; antinuclear antibodies are detected in serum from about one-third of patients with hyperthyroidism and ophthalmopathy, while from 5% to 10% have antibodies reactive with several other ubiquitous tissue proteins. Cloned proteins which are autoantigenic in some patients with hyperthyroidism or Hashimoto's thyroiditis and ophthalmopathy include collagen XIII, nebulin, the calcium binding protein calmitine, and the Mac-II antigen. All antibodies reactive with eye muscle antigens, except the 64 kDa protein which is also expressed in the thyroid, cross react with the same, or a related, protein in other skeletal muscle. Future research should focus on the underlying mechanisms for this broad loss of tolerance to self antigens and the effect of environmental factors such as stress, radioiodine and viral infection of the thyroid gland and other target tissues, in precipitating disease.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Enfermedad de Graves/inmunología , Animales , Especificidad de Anticuerpos , Enfermedades Autoinmunes/etiología , Proteínas de Unión al Calcio , Enfermedades de los Párpados/etiología , Enfermedades de los Párpados/inmunología , Enfermedades del Pie/complicaciones , Enfermedad de Graves/etiología , Hipertiroidismo/etiología , Hipertiroidismo/inmunología , Aparato Lagrimal/inmunología , Aparato Lagrimal/patología , Dermatosis de la Pierna/etiología , Dermatosis de la Pierna/inmunología , Enfermedades Musculares/etiología , Enfermedades Musculares/inmunología , Mixedema/etiología , Mixedema/inmunología , Músculos Oculomotores/inmunología , Enfermedades de la Piel/etiología , Enfermedades de la Piel/inmunologíaRESUMEN
We have studied a possible role of T cell sensitization to eye muscle antigens in patients with thyroid-associated ophthalmology (TAO). Peripheral blood mononuclear cell (PBMC) proliferation in response to crude porcine orbital tissue antigens, partially purified porcine eye muscle membrane proteins and predicted epitopic fragments of the recombinant 64 kDa protein 1D, was determined in patients with TAO and thyroid autoimmunity without eye disease. When membrane and cytosol fractions were used as antigen PBMC from 43% of patients with TAO but only 12.5% of normal subjects were responsive to a crude orbital connective tissue membrane fraction, although this difference was not significant. We were unable to demonstrate specific recognition of partially purified eye muscle membrane fractions; although most of the fractions tested were occasionally recognized by T cells from patients with ophthalmopathy, this was also the case for patients with autoimmune thyroid disease without ophthalmopathy and normal subjects. We did not clearly identify epitopic sequences within the 1D protein, most of the predicted peptides tested being recognized not only by T cells from a small proportion of patients with TAO, but also by those from some patients with autoimmune thyroid disease without ophthalmopathy and normal subjects. It is noteworthy however that approximately 22% of TAO patients, but no normal subjects, were positive to one or more of three peptides, suggesting that reactivity to the 1D protein may play a role in the pathogenesis of the eye disorder in some patients with TAO. The inconsistent and generally low T cell responses to crude and purified antigens noted in a few patients with TAO could be explained by low numbers of specifically sensitized lymphocytes in peripheral blood.
Asunto(s)
Antígenos/inmunología , Oftalmopatías/inmunología , Inmunidad Celular , Órbita/inmunología , Fragmentos de Péptidos/inmunología , Proteínas/inmunología , Enfermedades de la Tiroides/complicaciones , Adulto , Anciano , Secuencia de Aminoácidos , Animales , Autoantígenos , Enfermedades Autoinmunes/inmunología , Proteínas del Citoesqueleto , Epítopos/inmunología , Oftalmopatías/etiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Neutrófilos/inmunología , Proteínas/química , PorcinosRESUMEN
Graves' disease comprises hyperthyroidism, ophthalmopathy, pretibial myxedema and acropachy, which occur separately or in various combinations. We have used the indirect immunofluorescence test to investigate reactivity of sera from patients with autoimmune thyroid disorders with and without ophthalmopathy, with porcine extra ocular muscle (EOM) and control tissue substrates. Sera from 75% of patients with Graves' hyperthyroidism (GH) and ophthalmopathy, which we call thyroid-associated opthalmopathy (TAO), contained one or more antibodies reactive with EOM compared to 32% of those with GH without the eye disorder, 41% of patients with Hashimoto's thyroiditis (HT), and 16% of normals. Antibodies reactive with an EOM connective tissue antigen(s), seen as fluorescence of the interstitium and endomysium, were found in sera from 10% of patients with TAO and 16% of those with GH, but not from any patient with HT or normal subject. Similar patterns of connective tissue reactivity were also found in lacrimal gland, skeletal muscle, kidney and salivary gland. Antinuclear antibodies were detected in sera from 31% of patients with TAO, but from only 8% with HT, in no patient with GH and in only 3% of normal subjects. The most common pattern was a fine speckled fluorescence, found in 45% of sera, consistent with reactivity against the Sm antigen or nuclear RNP. The finding of a high prevalence of ANA and, less often, anti-connective tissue antibodies in patients with thyroid autoimmunity and ophthalmopathy, is consistent with Graves' disease being a "collagen-like disorder". The reason why inflammation and resulting tissue damage is limited to the thyroid, connective tissue of the skin and orbit, skeletal muscle and, possibly, the lacrimal gland, is unclear. One possibility is cross reaction of ANA with tissue specific membrane proteins in these sites. The extent of immunologic abnormalities, and the resulting clinical features, in patients with Graves' disease may reflect the severity of a putative defect in immune regulation.
Asunto(s)
Enfermedades del Colágeno/inmunología , Tejido Conectivo/inmunología , Enfermedad de Graves/inmunología , Músculo Esquelético/inmunología , Glándula Tiroides/inmunología , Anticuerpos Antinucleares/sangre , Autoantígenos/análisis , HumanosRESUMEN
Although sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting are widely used to detect serum antibodies in patients with autoimmune disorders, this procedure unfolds and denatures proteins and may alter antibody binding sites. We have used nondenaturing methods for the purification of a 64-kDa eye muscle (EM) membrane antigen associated with thyroid-associated ophthalmopathy (TAO). Pig EM membrane proteins were prepared from crude homogenates by high-speed centrifugation and solubilized by hand homogenization. The 64-kDa protein was further purified by isoelectric focusing performed in the absence of SDS, detergents, reducing agents, and urea. Sera from patients with active TAO of recent onset and thyroid autoimmunity without ophthalmopathy were tested for reactivity against purified native 64-kDa protein in immunoblotting. Tests were positive in 64% of patients with TAO, in 37.5% of those with Graves' hyperthyroidism without eye disease, in 11% of patients with Hashimoto's thyroiditis without eye disease, and in 13% of normal subjects. Many of the same sera were also tested for cytotoxic activity against human EM cells in an antibody-dependent cell-mediated cytotoxicity (ADCC) assay. ADCC tests were positive in 69% of patients with TAO but in no normal subject. The specificity and sensitivity of these two tests in TAO surpass those for all other published results for orbital tissue reactive autoantibodies. Although there was a tendency for a relationship between reactivity to the 64-kDa protein and cytotoxic activity against EM cells in ADCC there were many exceptions and overall the relationship between the two tests was not significant.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Anticuerpos/análisis , Antígenos/inmunología , Oftalmopatías/etiología , Oftalmopatías/inmunología , Músculos Oculomotores/inmunología , Enfermedades de la Tiroides/complicaciones , Adolescente , Adulto , Anciano , Anticuerpos/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos , Pruebas Inmunológicas de Citotoxicidad , Femenino , Enfermedad de Graves/inmunología , Humanos , Immunoblotting , Masculino , Persona de Mediana Edad , Tiroiditis Autoinmune/inmunologíaRESUMEN
Although sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting are widely used to detect serum antibodies in patients with autoimmune disorders, this procedure unfolds and denatures proteins and may alter antibody-binding sites. We have used a gentle protocol for the preparation and purification of a 64-kilodalton (kDa) eye muscle (EM) membrane antigen associated with thyroid-associated ophthalmopathy (TAO) for use as antigen in immunoblotting. Pig EM membrane proteins were prepared from crude homogenates by high speed centrifugation and solubilized by hand homogenization. These native membrane proteins (NMprot) were then electrophoresed on an 8.5% polyacrylamide gel in the absence of SDS, reducing agents, or urea, and proteins from individual bands were eluted, applied to standard SDS-PAGE, and immunoblotted with selected TAO patient sera. A prominent 64-kDa protein, present in most of the bands, was recognized by autoantibodies in sera from 35% of the patients with TAO and 47% of those with Graves' hyperthyroidism without evident ophthalmopathy, but in only 4% of normal subjects. To further purify the 64-kDa protein and increase the sensitivity of immunoblotting, NMprot were separated by isoelectric focusing (IEF) in the absence of SDS, reducing agent, and urea. The 64-kDa protein appeared mainly in IEF fraction 7 and had an isoelectric point of 6.1-6.2. Similar results were found for a human EM protein of 64 kDa. Sera from groups of patients and normal subjects were tested in immunoblotting against a pig EM 64-kDa protein prepared from NMprot and purified in IEF. Tests were positive in 67% of patients with TAO, in 37.5% of those with Graves' hyperthyroidism without eye disease, in 11% of patients with Hashimoto's thyroiditis without eye disease, and in 9% of normal subjects. The 64-kDa protein was not found in other skeletal muscle. The demonstration that a native 64-kDa protein that is specifically targeted by autoantibodies in the serum of patients with TAO is expressed in EM, but not other skeletal muscle, greatly enhances its possible significance in the pathogenesis of this eye disorder.
Asunto(s)
Autoanticuerpos/inmunología , Oftalmopatías/etiología , Proteínas Musculares/inmunología , Proteínas Musculares/metabolismo , Enfermedades de la Tiroides/complicaciones , Adulto , Animales , Electroforesis en Gel de Poliacrilamida , Oftalmopatías/inmunología , Oftalmopatías/metabolismo , Femenino , Humanos , Focalización Isoeléctrica , Masculino , Persona de Mediana Edad , Peso Molecular , Proteínas Musculares/química , Músculos Oculomotores , Porcinos , Tiroiditis Autoinmune/inmunologíaRESUMEN
The exact pathogenic mechanism of thyroid-associated ophthalmopathy (TAO) remains unclear, and extensive studies on this disorder have resulted in often conflicting data. Well-known technical difficulties including the limited access to orbital tissues from patients with active and early disease, lack of an animal model and poor reproducibility of some of the immunological techniques used are in part responsible for this confusing situation. Despite this there is considerable evidence for eye muscle (EM) tissue involvement in the autoimmune reactions of TAO. Although the primary EM antigen(s) recognized by immunocompetent cells and autoantibodies has not been definitely identified, some good candidates, among them a membrane antigen of 64 kD which is also expressed in the thyroid, have been partially characterized. While it is unclear which component of the autoimmune reaction against EM-humoral or cell mediated-plays the more important role, autoantibodies seem to be responsible at least in part for the clinical features of the eye disorder. On the other hand, the orbital connective tissue (OCT) cells, especially the fibroblasts surrounding the EM fibers, seem to be extremely sensitive to stimulation by cytokines and other soluble proteins and immunoglobulins released in the course of an immune reaction in the muscle cells. Fibroblasts secrete large amounts of glycosaminoglycans and also participate in maintaining the autoimmune reaction. It seems likely that the EM is the main and primary target of the orbital autoimmune process whereas inflammation of the OCT is probably secondary.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Oftalmopatías/inmunología , Músculos Oculomotores/inmunología , Enfermedades de la Tiroides/inmunología , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Tejido Conectivo/inmunología , Reacciones Cruzadas , Enfermedad de Graves/inmunología , Humanos , Músculos Oculomotores/patología , Músculos Oculomotores/ultraestructuraRESUMEN
The amino acid sequence of the human lysosomal alpha-mannosidase precursor has been deduced by PCR mediated cloning and sequencing of the cDNA. The protein has 961 amino acids and a molecular weight of 107,644 Daltons. The amino acid sequence shows 38% identity to the Dictyostelium discoideum lysosomal alpha-mannosidase. The cDNA maps proximal to the centromere on chromosome 19q, the same locus as the MANB gene. Our results provide valuable information for the study of the lysosomal storage disease alpha-mannosidosis, an inherited disorder caused by mutations in the MANB gene which encodes the human lysosomal alpha-mannosidase.
Asunto(s)
Cromosomas Humanos Par 19 , Lisosomas/enzimología , Manosidasas/química , Manosidasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Secuencia de Consenso , Cartilla de ADN , ADN Complementario/análisis , Dictyostelium/enzimología , Dictyostelium/genética , Biblioteca de Genes , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , Peso Molecular , Reacción en Cadena de la Polimerasa/métodos , Ratas , Homología de Secuencia de Aminoácido , alfa-ManosidasaRESUMEN
Proteinase B precursors are modified by an N-linked carbohydrate side chain at Asn 314. Glycosylation at this position is not required for proper localization, processing, or activation of the enzyme.
Asunto(s)
Precursores Enzimáticos/metabolismo , Saccharomyces cerevisiae/enzimología , Serina Endopeptidasas/metabolismo , Secuencia de Bases , ADN de Hongos/genética , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Glicosilación , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Plásmidos , Procesamiento Proteico-Postraduccional , Saccharomyces cerevisiae/genética , Serina Endopeptidasas/química , Serina Endopeptidasas/genética , Vacuolas/enzimologíaRESUMEN
Proteinase B (PrB) is a subtilisin-like serine protease found in the vacuole of the yeast Saccharomyces cerevisiae. It is first made as a large precursor that consists of a putative signal sequence, a 260-amino acid pro region, the serine protease domain, and two small COOH-terminal post regions (Moehle, C. M., Dixon, C. K., and Jones, E. W. (1989) J. Cell Biol. 108, 309-324). This precursor is glycosylated and proteolytically processed at least three times before mature enzyme is formed. To determine whether an intact PrB catalytic site is required for proteolytic processing of the precursor, point mutations were generated at the codons for the active site serine or aspartate residues by site-directed mutagenesis. The effect of these mutations on PrB processing suggests that the large pro region may be cleaved by an intramolecular, autocatalytic mechanism. The properties of a prb1 mutant that accumulates a 37-kDa precursor in addition to mature sized mutant PrB antigen suggests that the final proteolytic cleavage step is also autocatalytic. A prb1 deletion that lacks codons for the large pro region was made to test whether this part of the precursor is required for formation of mature PrB. Analysis of this mutant revealed two functions for this region: it prevents N-linked glycosylation of the serine protease domain and it allows the PrB precursor to be processed by proteinase A. The pro region can fulfill this latter function if added as a separate molecule, so long as glycosylation of the catalytic domain is prevented by other means.
Asunto(s)
Precursores de Proteínas/metabolismo , Saccharomyces cerevisiae/enzimología , Serina Endopeptidasas/metabolismo , Secuencia de Bases , Catálisis , ADN de Hongos , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Datos de Secuencia Molecular , Mutación , Precursores de Proteínas/genética , Procesamiento Proteico-Postraduccional , Saccharomyces cerevisiae/genética , Serina Endopeptidasas/genéticaRESUMEN
Dexamethasone rapidly stimulated transcription of the tyrosine aminotransferase and metallothionein-I genes--but not of the phosphoenolpyruvate carboxykinase gene--in rat hepatocytes cultured in serum-free medium. This differential response was not observed for cyclic AMP. The results suggest that the phosphoenolpyruvate carboxykinase gene--but not the tyrosine aminotransferase and metallothionein-I genes--requires a factor which is permissive for stimulation of transcription by the glucocorticoid receptor.
Asunto(s)
Dexametasona/farmacología , Genes/efectos de los fármacos , Hígado/metabolismo , Metalotioneína/genética , Fosfoenolpiruvato Carboxiquinasa (GTP)/genética , ARN Mensajero/biosíntesis , Transcripción Genética/efectos de los fármacos , Tirosina Transaminasa/genética , Animales , Northern Blotting , Células Cultivadas , AMP Cíclico/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reguladores , Cinética , Hígado/efectos de los fármacos , Masculino , ARN Mensajero/genética , Ratas , Ratas Endogámicas , Factores de TiempoRESUMEN
Primary cultures of rat hepatocytes produce tissue-type plasminogen activator (tPA) and plasminogen activator-inhibitor type 1 (PAI-1). Incubation of hepatocytes with 50 microM 8-(4-chlorophenylthio)cAMP (CPT-cAMP) results in a 4-fold increase in tPA activity, whereas the synthetic glucocorticoid dexamethasone (1 microM) causes a more than 90% decrease. In combination, dexamethasone completely overcomes the CPT-cAMP effect and markedly decreases PA activity. PAI-1 is induced by both CPT-cAMP and dexamethasone, and the effects of these agents are additive. Accumulation of tPA mRNA is increased more than 4-fold by CPT-cAMP and is greatly decreased by incubation with dexamethasone. Dexamethasone in combination with CPT-cAMP totally blocks this cAMP effect. The protein synthesis inhibitor cycloheximide does not prevent either the dexamethasone-induced decrease or the CPT-cAMP-induced increase in tPA message and, in fact, augments the cAMP-induced increase in tPA mRNA. Hepatocyte PAI-1 mRNA levels are increased 2-fold by incubation with either CPT-cAMP or dexamethasone; in combination, these effectors cause a 4-fold increase in PAI-1 mRNA. Cycloheximide alone causes a marked increase in PAI-1 mRNA, but does not block the induction by either CPT-cAMP or dexamethasone. We conclude that incubation of hepatocytes with CPT-cAMP induces tPA activity by increasing tPA mRNA accumulation and that dexamethasone causes a decrease in tPA activity by both decreasing tPA mRNA and increasing PAI-1 mRNA and activity. Concomitant protein synthesis is not required for the regulation of tPA or PAI-1 mRNA by either CPT-cAMP or dexamethasone, indicating a primary effect of these agents on gene transcription or mRNA stability.
Asunto(s)
AMP Cíclico/análogos & derivados , Dexametasona/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Glicoproteínas/genética , Hígado/metabolismo , Tionucleótidos/farmacología , Activador de Tejido Plasminógeno/genética , Animales , Células Cultivadas , AMP Cíclico/farmacología , Cicloheximida/farmacología , Interacciones Farmacológicas , Humanos , Masculino , Inactivadores Plasminogénicos , Biosíntesis de Proteínas , ARN Mensajero/biosíntesis , Ratas , Ratas EndogámicasRESUMEN
Chromosomal deletions at and around the albino locus on chromosome 7 of the mouse affect the enzyme activities and steady-state levels of mRNAs for five urea-cycle enzymes in liver. In newborn c3H homozygotes, activities of these enzymes were 43-62% of normal, while corresponding mRNA levels were 14-29% of normal. c14CoS deletion homozygotes expressed mRNA levels for these enzymes which were 32-48% of normal. However, transcription rates of these genes in hepatic nuclei of c3H/c3H mice were reduced only to 57-84% of normal. Since effects of the deletions had previously been noted in the kidney, mRNA levels for three enzymes expressed also in the kidney were examined. Mice homozygous for the c3H deletion, shown previously to have drastically reduced mRNA levels for phosphoenolpyruvate carboxykinase in the liver, expressed the same deficiency in the kidney, while mRNA levels for argininosuccinate synthetase and argininosuccinate lyase were reduced in the liver but remained unaffected in the kidney. However, mRNA levels for phosphoenolpyruvate carboxykinase, carbamyl phosphate synthetase I, and ornithine transcarbamylase were unaffected in the intestine of c3H homozygotes. The results suggest that a regulatory factor(s) encoded in the DNA encompassed by the deletion is involved in the normal developmental maturation of hepatocytes and certain cells in the kidney.
Asunto(s)
Deleción Cromosómica , Mapeo Cromosómico , Genes , Intestinos/enzimología , Riñón/enzimología , Hígado/enzimología , Fosfoenolpiruvato Carboxiquinasa (GTP)/genética , Urea/metabolismo , Animales , Cruzamientos Genéticos , Femenino , Homocigoto , Masculino , Ratones , Ratones EndogámicosRESUMEN
Cyclic AMP analogues induced expression of metallothionein-I (MT-I) mRNA in adult rat liver and in rat hepatocytes cultured in serum-free medium. This induction occurred via an increased rate of transcription of the MT-I gene. The effect of cyclic AMP analogues was tissue-specific since no induction of MT-I mRNA occurred in rat kidney. Induction of MT-I mRNA by a combination of cyclic AMP analogue and dexamethasone was additive in liver and cultured hepatocytes, indicating that induction occurred via independent regulatory pathways.
Asunto(s)
AMP Cíclico/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Riñón/metabolismo , Hígado/metabolismo , Metalotioneína/genética , Animales , Bucladesina/farmacología , Células Cultivadas , AMP Cíclico/análogos & derivados , Dexametasona/farmacología , Técnicas In Vitro , Riñón/efectos de los fármacos , Hígado/efectos de los fármacos , ARN Mensajero/genética , Ratas , Tionucleótidos/farmacologíaRESUMEN
In adult rat liver, amounts of the urea cycle enzymes are regulated by diet, glucocorticoids, and cAMP. Rat hepatocytes cultured in chemically defined medium were used to precisely define the roles of glucocorticoids and cAMP in regulation of these enzymes at the pretranslational level. With the exception of ornithine transcarbamylase mRNA, cultured rat hepatocytes retain the capacity to express mRNAs for the urea cycle enzymes at the same level observed for liver of intact rats. In the absence of added hormones, mRNAs for argininosuccinate synthetase and argininosuccinate lyase remained at or above normal in vivo levels, while mRNAs for the other three enzymes declined to very low levels. Messenger RNAs for carbamyl phosphate synthetase I, argininosuccinate synthetase, argininosuccinate lyase, and arginase increased in response to either dexamethasone or 8-(4-chlorophenylthio) cAMP (CPT-cAMP). Half-maximal responses occurred at 2-3 nM dexamethasone and at 2-7 microM CPT-cAMP. Cycloheximide abolished the response to dexamethasone but not to CPT-cAMP, suggesting that dexamethasone induced expression of an intermediate gene product required for induction of these mRNAs. The effects of a combination of both hormones were additive for argininosuccinate lyase mRNA and synergistic for carbamyl phosphate synthetase I, argininosuccinate synthetase, and arginase mRNAs. Messenger RNA for ornithine transcarbamylase showed little or no response to any condition tested. Depending on the particular mRNA and hormonal condition tested, increases in mRNA levels ranged from 1.4- to 70-fold above control values.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
AMP Cíclico/fisiología , Glucocorticoides/fisiología , Hígado/enzimología , ARN Mensajero/biosíntesis , Urea/metabolismo , Animales , Células Cultivadas , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacología , Cicloheximida/farmacología , Dexametasona/farmacología , Regulación de la Expresión Génica , Hígado/citología , Masculino , Biosíntesis de Proteínas , ARN Mensajero/genética , Ratas , Ratas Endogámicas , Tionucleótidos/farmacologíaRESUMEN
Dexamethasone is necessary and sufficient to induce mRNA for phosphoenolpyruvate carboxykinase (GTP) (PEPCK) by 19-fold in rat hepatocytes cultured in serum-free medium. However, the time required for maximum induction is 16 h. The slow induction suggested that glucocorticoids regulate the expression of an intermediate gene product(s) which is required for glucocorticoid stimulation of PEPCK-gene expression. Consistent with this notion was the finding that cycloheximide completely blocked the response to dexamethasone. In contrast, cycloheximide did not block the response to a cyclic AMP analogue.