RESUMEN
Exposure of rat pulmonary artery smooth muscle cells (rPASMC) to cytokines leads to nitric oxide (NO) production by NO synthase 2 (NOS2). NO stimulates cGMP synthesis by soluble guanylate cyclase (sGC), a heterodimer composed of alpha(1)- and beta(1)-subunits. Prolonged exposure of rPASMC to NO decreases sGC subunit mRNA and protein levels. The objective of this study was to determine whether levels of NO produced endogenously by NOS2 are sufficient to decrease sGC expression in rPASMC. Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) increased NOS2 mRNA levels and decreased sGC subunit mRNA levels. Exposure of rPASMC to IL-1beta and TNF-alpha for 24 h decreased sGC subunit protein levels and NO-stimulated sGC enzyme activity. L-N(6)-(1-iminoethyl)lysine (NOS2 inhibitor) or 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (sGC inhibitor) partially prevented the cytokine-mediated decrease in sGC subunit mRNA levels. However, cytokines also decreased sGC subunit mRNA levels in PASMC derived from NOS2-deficient mice. These results demonstrate that levels of NO and cGMP produced in cytokine-exposed PASMC are sufficient to decrease sGC subunit mRNA levels. In addition, cytokines can decrease sGC subunit mRNA levels via NO-independent mechanisms.
Asunto(s)
Citocinas/metabolismo , Guanilato Ciclasa/metabolismo , Músculo Liso Vascular/metabolismo , Óxido Nítrico/metabolismo , Arteria Pulmonar/metabolismo , Animales , Células Cultivadas , Citocinas/farmacología , Estabilidad de Enzimas/efectos de los fármacos , Guanilato Ciclasa/genética , Interleucina-1/metabolismo , Interleucina-1/farmacología , Ratones , Ratones Noqueados , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Óxido Nítrico Sintasa/deficiencia , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Subunidades de Proteína , Arteria Pulmonar/citología , Arteria Pulmonar/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Solubilidad , Transcripción Genética/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
OBJECTIVES: To determine the prevalence of anti-heat shock protein 70 (anti-HSP70) antibodies in patients with Meniere's disease and healthy subjects and to probe the relationship between antibody status and clinical features of Meniere's disease. STUDY DESIGN: Prospective cohort study of consecutive consenting patients with Meniere's disease. METHODS: Serum samples were obtained prospectively from 134 patients with Meniere's disease and 124 blood donors. Serial samples were taken at 3-month intervals in 38 of 134 patients with Meniere's disease. Demographic data and clinical characterization of vestibular and auditory status were acquired with each sample. Serum was assayed for anti-HSP70 antibodies by Western blot using bovine renal extract, recombinant bovine HSP70, and recombinant human HSP70 antigens. RESULTS: Immunoreactivity against bovine renal extract HSP70 was found in 38% of patients with Meniere's disease, compared with 25% of blood donors (P < .04). Reactivity with recombinant antigens was not significantly different between patients with Meniere's disease and healthy control subjects. Patients with Meniere's disease who reacted with all three antigens were more likely to have simultaneously active hearing and balance symptoms (P = .03). Neither univariate nor multivariate statistical analysis established any other association between serological findings and clinical features of Meniere's disease. Tests performed on serial samples of patients with Meniere's disease also showed no association of positive or negative test results with changes in clinical course. CONCLUSIONS: Because of the high prevalence of antiHSP70 antibodies in healthy subjects and the very limited association of anti-HSP70 antibody status with clinical features or course of Meniere's disease, we conclude that, at present, the detection of anti-HSP70 antibodies by Western blotting offers little clinically useful information in Meniere's disease.
Asunto(s)
Anticuerpos Antiidiotipos/inmunología , Proteínas HSP70 de Choque Térmico/inmunología , Enfermedad de Meniere/inmunología , Adulto , Anciano , Antígenos/inmunología , Western Blotting , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios ProspectivosRESUMEN
The nuclear body is a multiprotein complex that may have a role in the regulation of gene transcription. This structure is disrupted in a variety of human disorders including acute promyelocytic leukemia and viral infections, suggesting that alterations in the nuclear body may have an important role in the pathogenesis of these diseases. In this study, we identified a cDNA encoding a leukocyte-specific nuclear body component designated Sp110. The N-terminal portion of Sp110 was homologous to two previously characterized components of the nuclear body (Sp100 and Sp140). The C-terminal region of Sp110 was homologous to the transcription intermediary factor 1 (TIF1) family of proteins. High levels of Sp110 mRNA were detected in human peripheral blood leukocytes and spleen but not in other tissues. The levels of Sp110 mRNA and protein in the human promyelocytic leukemia cell line NB4 increased following treatment with all-trans retinoic acid (ATRA), and Sp110 localized to PML-Sp100 nuclear bodies in ATRA-treated NB4 cells. Because of the structural similarities between Sp110 and TIF1 proteins, the effect of Sp110 on gene transcription was examined. An Sp110 DNA-binding domain fusion protein activated transcription of a reporter gene in transfected mammalian cells. In addition, Sp110 produced a marked increase in ATRA-mediated expression of a reporter gene containing a retinoic acid response element. Taken together, the results of this study demonstrate that Sp110 is a member of the Sp100/Sp140 family of nuclear body components and that Sp110 may function as a nuclear hormone receptor transcriptional coactivator. The predominant expression of Sp110 in leukocytes and the enhanced expression of Sp110 in NB4 cells treated with ATRA raise the possibility that Sp110 has a role in inducing differentiation of myeloid cells.
Asunto(s)
Antígenos Nucleares , Autoantígenos/genética , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Receptores Citoplasmáticos y Nucleares/genética , Factores de Transcripción/genética , Activación Transcripcional , Secuencia de Aminoácidos , Autoantígenos/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , ADN Complementario/análisis , ADN Complementario/genética , Células HL-60 , Humanos , Antígenos de Histocompatibilidad Menor , Datos de Secuencia Molecular , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Proteína de la Leucemia Promielocítica , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Alineación de Secuencia , Transducción de Señal/genética , Factores de Transcripción/metabolismo , Proteínas Supresoras de TumorRESUMEN
The nuclear body is a cellular structure that appears to be involved in the pathogenesis of acute promyelocytic leukemia and viral infection. In addition, the nuclear body is a target of autoantibodies in patients with the autoimmune disease primary biliary cirrhosis. Although the precise function of the nuclear body in normal cellular biology is unknown, this structure may have a role in the regulation of gene transcription. In a previous investigation, we identified a leukocyte-specific, gamma interferon (IFN-gamma)-inducible autoantigen designated Sp140. The objectives of the present study were to investigate the cellular location of Sp140 with respect to the nuclear-body components PML and Sp100 and to examine the potential role of Sp140 in the regulation of gene transcription. We used adenovirus-mediated gene transfer to express Sp140 in human cells and observed that the protein colocalized with PML and Sp100 in resting cells and associated with structures containing PML during mitosis. In cells infected with the adenovirus expressing Sp140 and incubated with IFN-gamma, the number of PML-Sp100 nuclear bodies per cell increased but immunoreactive Sp140 was not evenly distributed among the nuclear bodies. Sp140 associated with a subset of IFN-gamma-induced PML-Sp100 nuclear bodies. To examine the potential effect of Sp140 on gene transcription, a plasmid encoding Sp140 fused to the DNA-binding domain of GAL4 was cotransfected into COS cells with a chloramphenicol acetyltransferase (CAT) reporter gene containing five GAL4-binding sites and a simian virus 40 enhancer region. The GAL4-Sp140 fusion protein increased the expression of the reporter gene. In contrast, Sp100 fused to the GAL4 DNA-binding domain inhibited CAT activity in transfected mammalian cells. The results of this study demonstrate that Sp140 associates with a subset of PML-Sp100 nuclear bodies in IFN-gamma-treated cells and that Sp140 may activate gene transcription. Taken together, these observations suggest that the nuclear bodies within a cell may be heterogeneous with respect to both composition and function.
Asunto(s)
Antígenos Nucleares , Núcleo Celular/química , Núcleo Celular/fisiología , Factores de Transcripción/fisiología , Adenoviridae , Animales , Autoantígenos/metabolismo , Autoantígenos/fisiología , Células COS , Cloranfenicol O-Acetiltransferasa/metabolismo , Electroforesis en Gel de Poliacrilamida , Regulación de la Expresión Génica , Células HeLa , Humanos , Immunoblotting , Mitosis , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/fisiología , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiología , Proteína de la Leucemia Promielocítica , Factores de Transcripción/análisis , Factores de Transcripción/metabolismo , Transcripción Genética , Transfección , Proteínas Supresoras de TumorRESUMEN
OBJECTIVE: To investigate the specificity of antibodies to heat-shock protein 70 (HSP70) in patients with idiopathic, progressive, bilateral sensorineural hearing loss (IPBSNHL) and Meniere disease. STUDY DESIGN: Test immunoreactivity of patients' sera using recombinant human (rh) and bovine (rb) HSP70, as well as segments representing different regions of bovine HSP70 as antigen. METHODS: Sera were tested by Western blotting. RESULTS: Of 52 patients with IPB-SNHL, 40 sera reacted only with rbHSP70; 12 reacted with both rbHSP70 and rhHSP70. Sera from 13 patients with IPBSNHL and from 8 with Meniere disease were tested on the panel of bovine HSP70 segments. Eleven and 7 samples, respectively, reacted with amino acid segment 427-461 from the carboxy (C)-terminal region of the molecule. CONCLUSION: In IPBSNHL and Meniere disease, antibodies are directed primarily against an epitope(s) within the C-terminal region of HSP70 where diversity in sequence among different species, including possible pathogens, is greatest. These findings may provide clues to the pathogenesis or specific serodiagnosis (or both) of diseases of the inner ear.
Asunto(s)
Oído Interno/inmunología , Proteínas HSP70 de Choque Térmico/inmunología , Pérdida Auditiva Sensorineural/inmunología , Epítopos Inmunodominantes/inmunología , Enfermedad de Meniere/inmunología , Animales , Especificidad de Anticuerpos/inmunología , Reacciones Antígeno-Anticuerpo/inmunología , Bovinos , ADN Complementario/genética , Proteínas HSP70 de Choque Térmico/sangre , Pérdida Auditiva Sensorineural/sangre , Humanos , Epítopos Inmunodominantes/sangre , Enfermedad de Meniere/sangreRESUMEN
Studies in vitro have underestimated the importance of cGMP-dependent protein kinase (PKG) in the modulation of vascular smooth muscle cell (SMC) proliferation and apoptosis in vivo. This is attributable, in part, to a rapid decline in PKG levels as vascular SMC are passaged in culture. We used a recombinant adenovirus encoding PKG (Ad.PKG) to augment kinase activity in cultured rat pulmonary artery SMC (RPaSMC). Incubation of Ad. PKG-infected RPaSMC (multiplicity of infection = 200) with 8-Br-cGMP decreased serum-stimulated DNA synthesis by 85% and cell proliferation at day 5 by 74%. The effect of 8-Br-cGMP on DNA synthesis in Ad.PKG-infected RPaSMC was blocked by KT5823 (PKG inhibitor), but not by KT5720 (cAMP-dependent protein kinase inhibitor). A nitric oxide (NO) donor compound, S-nitrosoglutathione, at concentrations as low as 100 nM, inhibited DNA synthesis in Ad. PKG-infected RPaSMC, but not in uninfected cells or in cells infected with a control adenovirus. In addition, 8-Br-cGMP and S-nitrosoglutathione induced apoptosis in serum-deprived RPaSMC infected with Ad.PKG, but not in uninfected cells or in cells infected with a control adenovirus. These results demonstrate that modulation of PKG levels in vascular SMC can alter the sensitivity of these cells to NO and cGMP. Moreover, these observations suggest an important role for PKG in the regulation of vascular SMC proliferation and apoptosis by NO and cGMP.
Asunto(s)
Apoptosis/fisiología , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/fisiología , Glutatión/análogos & derivados , Músculo Liso Vascular/citología , Músculo Liso Vascular/fisiología , Donantes de Óxido Nítrico/farmacología , Compuestos Nitrosos/farmacología , Adenoviridae , Animales , Apoptosis/efectos de los fármacos , Encéfalo/enzimología , División Celular/efectos de los fármacos , Células Cultivadas , Clonación Molecular , GMP Cíclico/farmacología , Proteínas Quinasas Dependientes de GMP Cíclico/genética , Técnicas de Transferencia de Gen , Vectores Genéticos , Glutatión/farmacología , Humanos , Músculo Liso Vascular/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Óxido Nítrico/fisiología , Reacción en Cadena de la Polimerasa , Arteria Pulmonar/citología , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/fisiología , Ratas , Proteínas Recombinantes/metabolismo , S-Nitrosoglutatión , TransfecciónRESUMEN
Nitric oxide stimulates soluble guanylate cyclase (sGC) to convert GTP to the intracellular second messenger cGMP. In rat pulmonary artery smooth muscle cells, sGC is an obligate heterodimer composed of alpha1 and beta1 subunits. We investigated the effect of NO donor compounds on sGC subunit gene expression in rat pulmonary artery smooth muscle cells. Sodium nitroprusside and S-nitroso-glutathione decreased sGC subunit mRNA and protein levels, as well as sGC enzyme activity. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an sGC inhibitor, blocked the effect of sodium nitroprusside on sGC subunit gene expression, whereas 8-bromo cGMP decreased subunit mRNA levels, demonstrating that NO-mediated decrease in sGC subunit mRNA levels is cGMP-dependent. sGC subunit mRNA levels decreased more rapidly in rat pulmonary artery smooth muscle cells exposed to NO than in cells exposed to actinomycin D, suggesting that NO decreases sGC subunit mRNA stability. Actinomycin D and cycloheximide blocked the ability of NO to decrease sGC subunit mRNA levels. These results demonstrate that NO decreases sGC subunit mRNA stability via a transcription- and translation-dependent mechanism.
Asunto(s)
Regulación de la Expresión Génica , Guanilato Ciclasa/genética , Músculo Liso Vascular/fisiología , Óxido Nítrico/fisiología , ARN Mensajero/genética , Animales , Células Cultivadas , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacología , Cicloheximida/farmacología , Dactinomicina/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Glutatión/análogos & derivados , Glutatión/farmacología , Guanilato Ciclasa/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/enzimología , Nitroprusiato/farmacología , Compuestos Nitrosos/farmacología , Oxadiazoles/farmacología , Quinoxalinas/farmacología , ARN Mensajero/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , S-Nitrosoglutatión , Factores de TiempoRESUMEN
The nuclear body (NB) is a cellular organelle that is involved in the pathogenesis of acute promyelocytic leukemia and viral infection. The NB is also a target of antibodies in the serum of patients with the autoimmune disease primary biliary cirrhosis. In this study, serum from a patient with primary biliary cirrhosis was used to identify a cDNA encoding a novel component of the NB, a 140-kDa protein designated Sp140. The predicted amino acid sequence of the amino-terminal portion of Sp140 was similar to Sp100, a previously identified NB protein. The carboxyl portion of Sp140 contained a zinc-finger domain and a bromodomain, motifs that are present in proteins regulating gene transcription. High levels of Sp140 mRNA were detected in human spleen and peripheral blood leukocytes, but not other human tissues. The level of SP140 mRNA in myeloid precursor cell lines HL60 and NB4 markedly increased in response to chemically induced cellular differentiation. Immunohistochemical techniques were used to demonstrate that SP140 localized to the NB in differentiated HL60 and NB4 cells. The location of Sp140 in the NB, and expression of this gene in cells involved in host defense, suggest that Sp140 may be involved in the pathogenesis of acute promyelocytic leukemia and viral infection.
Asunto(s)
Antígenos Nucleares , Leucocitos/metabolismo , Proteínas de Neoplasias , Proteínas Nucleares , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Complementario , Regulación de la Expresión Génica/efectos de los fármacos , Células HL-60 , Humanos , Inmunohistoquímica , Interferones/farmacología , Cirrosis Hepática Biliar/metabolismo , Datos de Secuencia Molecular , Proteína de la Leucemia Promielocítica , Homología de Secuencia de Aminoácido , Factores de Transcripción/genética , Proteínas Supresoras de TumorRESUMEN
OBJECTIVE: To identify the 68-kd target of antibody in serum samples from patients with idiopathic, progressive, bilateral sensorineural hearing loss. DESIGN: To purify target protein from renal extracts using gel filtration, ion-exchange chromatography, and polyacrylamide gel electrophoresis and to transfer to nitrocellulose membranes. The purified protein was digested with trypsin, and peptide fragments were separated by high-pressure liquid chromatography. RESULTS: One fraction obtained by high-performance liquid chromatography contained a peptide of 2776 molecular weight. The sequence of a stretch of 22 amino acids within this peptide was identical to that of amino acids 424 through 445 of heat shock protein 70 (HSP70). On Western blotting, monoclonal antibody directed against HSP70 (but capable of recognizing both constitutive HSP70 [HSC70] and stress-inducible HSP70) reacted with the purified 68-kd protein. We compared the reactivity of serum samples from six patients with idiopathic, progressive, bilateral sensorineural hearing loss, as well as monoclonal antibody to HSC70, and monoclonal antibody to HSP70 with renal extract. The pattern obtained suggested that patient antibodies are preferentially directed at HSP70. CONCLUSION: The target of antibody in serum samples from patients with idiopathic, progressive, bilateral sensorineural hearing loss is HSP70.
Asunto(s)
Autoanticuerpos/sangre , Proteínas HSP70 de Choque Térmico/inmunología , Pérdida Auditiva Sensorineural/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Western Blotting , Bovinos , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Colodión , Electroforesis en Gel de Poliacrilamida , Proteínas HSP70 de Choque Térmico/análisis , Pérdida Auditiva Bilateral/sangre , Pérdida Auditiva Bilateral/inmunología , Pérdida Auditiva Sensorineural/sangre , Humanos , Immunoblotting , Riñón/química , Datos de Secuencia Molecular , Peso Molecular , Fragmentos de Péptidos/análisis , Extractos de Tejidos/análisisRESUMEN
BACKGROUND: Ab in the serum of patients with autoimmune diseases have been used to identify, characterize, and purify many autoantigens. EXPERIMENTAL DESIGN: Serum from a patient (Ge) with Sjögren's syndrome was used to identify cDNA clones encoding novel autoantigens. This patient's serum was chosen for study because it contained antinuclear Ab that were different from those frequently detected in patients with autoimmune diseases. RESULTS: Ge serum identified a cDNA clone encoding part of protein Ki-67, a cell proliferation-associated protein. The Ki-67 protein (pKi-67) was not previously known to be a target of autoantibodies. To investigate the association between Ab directed against pKi-67 and autoimmune diseases, sera from autoimmune mice were tested for reactivity with a recombinant fragment of pKi-67. Ab were detected in serum from MRL/MpJ(-)+/+ and MRL/MpJ-lpr/lpr mice but not in serum from other autoimmune mice or control animals. CONCLUSIONS: Protein Ki-67 joins the proliferating cell nuclear Ag (PCNA) as an example of a cell proliferation-associated protein that is a target of autoantibodies. The presence of anti-pKi-67 Ab in MRL mice, but not other autoimmune mice, suggests that anti-pKi-67 Ab may be specific markers for the systemic lupus erythematosus-like illness that develops in these animals. Further characterization of the immune response directed against this Ag may provide clues to the etiology and pathogenesis of autoimmune disease in these animals.
Asunto(s)
Autoanticuerpos/sangre , Autoanticuerpos/inmunología , Ratones Mutantes/sangre , Proteínas de Neoplasias/inmunología , Proteínas Nucleares/inmunología , Animales , Femenino , Biblioteca de Genes , Humanos , Immunoblotting , Antígeno Ki-67 , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NZB , Proteínas Recombinantes/inmunología , Síndrome de Sjögren/inmunología , Células Tumorales CultivadasRESUMEN
Nitric oxide synthases (NOSs) are a family of enzymes responsible for the synthesis of nitric oxide from L-arginine and molecular oxygen. Three human NOS enzymes (I, II, and III) with differing cellular distribution and regulatory mechanisms have been identified. To determine whether additional NOSs are encoded in the human genome, a bovine NOS II-related cDNA was used to screen two human genomic libraries. Clones containing three independent genes were isolated. One clone encoded the previously identified NOS II gene (NOS2A). The two other genes specified amino acids homologous, but not identical, to human NOS II (NOS2B and NOS2C). Southern blot hybridization demonstrated that all three genes are present in the human genome. DNA from human-mouse somatic cell hybrids were used to determine the chromosomal location of the NOS II-related genes. All three NOS II-related genes colocalized to human chromosome 17 between bands p13.1 and q25. These observations suggest that there is more than one NOS II-related gene in the human genome. This finding may have important implications for the design of NOS isoform-specific inhibitors.
Asunto(s)
Mapeo Cromosómico , Cromosomas Humanos Par 17 , Familia de Multigenes , Óxido Nítrico Sintasa/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Clonación Molecular , ADN Complementario/genética , Humanos , Células Híbridas , Ratones , Datos de Secuencia MolecularRESUMEN
Nitric oxide (NO), a potent vasodilator, is a free-radical gas synthesized from L-arginine by nitric oxide synthases (NOS). NO appears to have an important role in perinatal changes in pulmonary vascular resistance. We previously identified mRNA encoding the constitutive endothelial NOS (ceNOS) isoform in human pulmonary tissue. To begin investigating functions of this enzyme in perinatal pulmonary development, we measured ceNOS mRNA and immunoreactivity in the developing rat lung. With the use of RNA blot hybridization, abundant pulmonary ceNOS mRNA was detected during the late fetal and postnatal period. The highest levels were detected within 24 h after birth, and elevated mRNA levels persisted for 16 days. In contrast, much lower levels of ceNOS mRNA were found in adult rat lung. With the use of immunoblot techniques, ceNOS protein levels were found to be correlated with mRNA levels. To identify the pulmonary cell types expressing the ceNOS gene, in situ hybridization with a digoxigenin-labeled cRNA probe was performed on sections from lungs of 1-day-old and adult rats. In lungs from 1-day-old rats, ceNOS mRNA was detected in alveolar and serosal epithelial cells as well as in endothelial cells lining small and medium-sized blood vessels. In contrast, in adult lungs, ceNOS gene transcripts were detected in rare endothelial cells. These observations suggest that ceNOS gene expression is regulated during lung development and that ceNOS is available to participate in the postnatal reduction of pulmonary vascular resistance. ceNOS gene expression in nonendothelial cells in the neonatal rat lung suggests that NO may also contribute to nonvascular functions in the developing lung.
Asunto(s)
Aminoácido Oxidorreductasas/genética , Animales Recién Nacidos/crecimiento & desarrollo , Animales Recién Nacidos/fisiología , Regulación de la Expresión Génica , Pulmón/enzimología , Pulmón/fisiología , Aminoácido Oxidorreductasas/metabolismo , Animales , Secuencia de Bases , Endotelio/enzimología , Hibridación in Situ , Isoenzimas/genética , Pulmón/crecimiento & desarrollo , Datos de Secuencia Molecular , Óxido Nítrico Sintasa , Sondas de Oligonucleótidos/genética , ARN Mensajero/metabolismo , RatasRESUMEN
Antibodies in the serum of patients with autoimmune diseases have been used to identify human autoantigens. Because autoantibodies often recognize active sites within corresponding protein antigens, autoantibodies have facilitated the functional characterization of these polypeptides. In the present study, serum from a patient with Sjögren's syndrome was used to identify a novel autoantigen which was designated Ge-1. Using the patient's serum, a 4.8-kb cDNA encoding Ge-1 was identified. Fragments of the cDNA were ligated into prokaryotic expression vectors, expressed in Escherichia coli, and used to produce recombinant Ge-1 fusion proteins. Fusion proteins containing different portions of Ge-1 were used to identify a 58 amino acid immunoreactive region within the protein. This immunoreactive region contained the protein's putative nuclear localization sequence (NLS). To demonstrate that the immunoreactive region was capable of functioning as a NLS, a eukaryotic expression plasmid was constructed to encode the immunoreactive region fused to the cytoplasmic protein, chicken muscle pyruvate kinase. After transfection of this plasmid into COS-1 cells, the fusion protein was detected in the nucleus. The presence of the NLS motif within the immunoreactive region of Ge-1 and other nuclear autoantigens suggests that the NLS may be a target of human autoantibodies.
Asunto(s)
Autoantígenos/inmunología , Epítopos/inmunología , Proteínas Nucleares/inmunología , Secuencia de Aminoácidos , Animales , Autoantígenos/química , Secuencia de Bases , Línea Celular , Electroforesis en Gel de Poliacrilamida , Técnica del Anticuerpo Fluorescente , Biblioteca Genómica , Humanos , Sueros Inmunes/inmunología , Masculino , Datos de Secuencia Molecular , Proteínas Nucleares/química , Proteínas , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/inmunología , Síndrome de Sjögren/inmunologíaRESUMEN
Endothelium-derived relaxing factor (EDRF)/nitric oxide (NO) activates soluble guanylate cyclase, thereby stimulating the synthesis of guanosine 3',5'-cyclic monophosphate (cGMP). To investigate the regulation of this important EDRF/NO receptor, we studied soluble guanylate cyclase gene expression in a rat fetal lung fibroblast cell line (RFL-6). 3-Isobutyl-1-methylxanthine, forskolin, and dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP), agents which increase intracellular cAMP, decreased the concentration of mRNA encoding the beta 1-subunit of soluble guanylate cyclase in RFL-6 cells. To investigate whether a decrease in beta 1-subunit mRNA concentration was reflected in diminished capacity to produce cGMP, forskolin-treated RFL-6 cells were exposed to the NO-donor compound sodium nitroprusside. Exposure to forskolin reversibly reduced the ability of RFL-6 cells to increase cGMP in response to NO. These observations suggest that cAMP can modulate the cellular response to EDRF/NO by decreasing the expression of one of the subunits of soluble guanylate cyclase.
Asunto(s)
AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Regulación Enzimológica de la Expresión Génica/fisiología , Guanilato Ciclasa/biosíntesis , ARN Mensajero/biosíntesis , 1-Metil-3-Isobutilxantina/farmacología , Animales , Bucladesina/farmacología , Línea Celular , Colforsina/farmacología , Relación Dosis-Respuesta a Droga , Fibroblastos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Cinética , Pulmón , Sustancias Macromoleculares , Nitroprusiato/farmacología , ARN Mensajero/aislamiento & purificación , ARN Mensajero/metabolismo , RatasRESUMEN
Nitric oxide, which accounts for the biological activity of endothelium-derived relaxing factor (EDRF), is synthesized in endothelial cells from L-arginine by nitric oxide synthase (NOS). We report here the cloning and functional expression of a cDNA encoding human endothelial NOS. Oligonucleotides corresponding to amino acid sequences shared by cytochrome P450 reductase and the recently identified brain NOS were synthesized to amplify a partial cDNA encoding a bovine endothelial cell NOS-related protein. This partial cDNA was used to isolate a cDNA encoding a human vascular endothelial NOS. The translated human protein is 1294 amino acids long and shares 52% of its amino acid sequence with brain NOS. Using RNA blot hybridization, abundant endothelial NOS mRNA was detected in unstimulated human umbilical vein endothelial cells. To determine the functional activity of the endothelial protein, we ligated the cDNA into an expression vector and transfected it into NIH3T3 cells. Cells expressing this cDNA contained abundant NADPH diaphorase activity, a histochemical marker for NOS. In co-culture assays, nitric oxide production by transfected cells increased guanylate cyclase activity in reporter rat fetal lung fibroblasts. In addition, NOS-catalyzed conversion of arginine to citrulline in transfected cells was significantly increased by A23187, a calcium ionophore. Isolation of a cDNA encoding a calcium-regulated, constitutively expressed human endothelial NOS, capable of producing EDRF in blood vessels, will accelerate the characterization of the role of this enzyme in normal and abnormal endothelial regulation of vascular tone.
Asunto(s)
Aminoácido Oxidorreductasas/genética , ADN , Óxido Nítrico/genética , Células 3T3 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encéfalo/enzimología , Bovinos , Clonación Molecular , Expresión Génica , Humanos , Inmunohistoquímica , Ratones , Datos de Secuencia Molecular , NADPH Deshidrogenasa/metabolismo , Óxido Nítrico Sintasa , Arteria Pulmonar/enzimología , ARN Mensajero/metabolismo , Ratas , Alineación de SecuenciaRESUMEN
Cellular receptors for many hormones, neurotransmitters, and growth factors are coupled to intracellular effector enzymes or ion channels through a set of heterotrimeric G proteins. In order to determine whether isoforms of G protein alpha subunits contribute differentially to mitogenic responses, we introduced an alpha subunit isoform, alpha i-1, into Balb/c 3T3 cells that normally lack this subtype. Balb/c 3T3 cells transfected with a plasmid containing cDNA encoding alpha i-1 expressed the alpha i-1 protein as judged both by the appearance of immunoreactive alpha i-1 protein on Western blots and by two-dimensional analysis of the proteins [32P]ADP-ribosylated by pertussis toxin. The amount of alpha i-1 expressed is less than the amount of alpha subunits endogenously present in these cells. Expression of alpha i-1 in the transfected cells slightly blunts stimulation of adenylylcyclase by GTP, guanosine 5'-3-O-(thio)triphosphate, or forskolin, but has no major effect on the ability of thrombin to inhibit the enzyme. In contrast, the expression of alpha i-1 has significant effects on cell growth and on the mitogenic response to thrombin. The alpha i-1-transfected cells have a doubling time that is twice as long as control cells transfected with the same plasmid without a cDNA insert. Despite their slower growth, thymidine incorporation in response to thrombin is greater in transfected than in control cells. Thrombin-stimulated DNA synthesis is sensitive to inhibition by pertussis toxin and is 5-fold more sensitive to inhibition by pertussis toxin in transfected cells than in control cells. The changes are receptor-specific since the mitogenic response to platelet-derived growth factor is indistinguishable between control and transfected cells. These studies suggest that the alpha i subunit composition of the cell may have profound effects on its growth and its response to stimulation through a specific cell surface receptor.
Asunto(s)
Proteínas de Unión al GTP/genética , Mitógenos , Células 3T3/efectos de los fármacos , Adenilil Ciclasas/metabolismo , Animales , Western Blotting , Ciclo Celular/efectos de los fármacos , ADN/biosíntesis , ADN/efectos de los fármacos , ADN/genética , Electroforesis en Gel Bidimensional , Proteínas de Unión al GTP/metabolismo , Vectores Genéticos , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Ratones , Ratones Endogámicos BALB C , Toxina del Pertussis , Factor de Crecimiento Derivado de Plaquetas/farmacología , Trombina/farmacología , Transfección , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
The physiological role of the alpha o subunit of guanine nucleotide-binding (G) protein was investigated with a murine adrenal cell line (Y1) transfected with a rat alpha o cDNA cloned in a retroviral expression vector. The parental cell line lacked detectable alpha o subunit. Expression of the alpha o cDNA in transfected cell lines was confirmed by Western blot (immunoblot) analysis. The rat alpha o subunit interacted with murine beta and gamma subunits and associated with cell membranes. Y1 cells containing large amounts of alpha o subunit had altered cellular morphology and reduced rate of cell division. In addition, GTP-gamma S-stimulated release of arachidonic acid from these cells was significantly increased compared with that in control cells. The alpha o subunit appears directly or indirectly to regulate cellular proliferation, morphology, and phospholipid metabolism.
Asunto(s)
División Celular , Proteínas de Unión al GTP/fisiología , Fosfolípidos/metabolismo , Transfección , Animales , Ácidos Araquidónicos/metabolismo , Línea Celular , Clonación Molecular , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/aislamiento & purificación , Genes , Vectores Genéticos , Cinética , Sustancias Macromoleculares , Ratones , RatasRESUMEN
The generation of an appropriate, specific immune response to an antigen is a remarkable biological phenomenon. An examination of both allelic exclusion and lymphocyte development is critical for an understanding of this response. Over the last several years, studies using transgenic mice that carry immunoglobulin or T cell receptor transgenes have provided a more detailed understanding of the mechanism of allelic exclusion. Recently, these mice have been used to examine lymphocyte development. In the future, these mice may be used to study the role of lymphocytes in autoimmune diseases.
Asunto(s)
Alelos , Linfocitos/inmunología , Ratones Transgénicos/genética , Animales , Ratones , Receptores de Superficie Celular/genéticaRESUMEN
The guanine nucleotide-binding proteins (G proteins), which mediate hormonal regulation of many membrane functions, are composed of alpha, beta, and gamma subunits. We have cloned and characterized cDNA from a human T-cell library encoding a form of alpha i that is different from the human alpha i subtypes previously reported [Didsbury, J. R., Ho, Y.-S. & Snyderman, R. (1987) FEBS Lett. 211, 160-164 and Bray, P., Carter, A., Guo, V., Puckett, C., Kamholz, J., Spiegel, A. & Nirenberg, M. (1987) Proc. Natl. Acad. Sci. USA 84, 5115-5119]. alpha i is the alpha subunit of a class of G proteins that inhibits adenylate cyclase and regulates other enzymes and ion channels. This cDNA encodes a polypeptide of 354 amino acids and is assigned to encode the alpha i-3 subtype of G proteins on the basis of its similarity to other alpha i-like cDNAs and the presence of a predicted site for ADP ribosylation by pertussis toxin. We have determined the expression of mRNA for this and two other subtypes of human alpha i (alpha i-1 and alpha i-2) in a variety of human fetal tissues and in human cell lines. All three alpha i subtypes were present in the tissues tested. However, analysis of individual cell types reveals specificity of alpha i-1 expression. mRNA for alpha i-1 is absent in T cells, B cells, and monocytes but is present in other cell lines. The finding of differential expression of alpha i-1 genes may permit characterization of distinct physiological roles for this alpha i subunit. mRNA for alpha i-2 and alpha i-3 was found in all the primary and transformed cell lines tested. Thus, some cells contain all three alpha i subtypes. This observation raises the question of how cells prevent cross talk among receptors that are coupled to effectors through such similar alpha proteins.