RESUMEN
The dog bile salt export pump (BSEP; ABCB11) was cloned and expressed in a Sf9 insect cell system. The deduced amino acid sequence encodes a 1325-amino-acid protein, which shows 89.4% and 80.2% homology with human BSEP and rat Bsep, respectively. The transcript of the dog Bsep gene was detected at a high level in liver, but not other tissues, by quantitative RT-PCR. The BSEP-expressing membrane vesicles isolated from Sf9 cells exhibited saturable uptake of [(3)H]taurocholic acid with Michaelis constants (K(m)) of 33.7, 22.2 and 19.9 microM for the dog, rat and human transporters, respectively. The uptake of [(3)H]taurocholic acid by all three transporters was significantly inhibited by troglitazone, glibenclamide, and other several inhibitors, while pravastatin inhibited dog Bsep and human BSEP, but not rat Bsep at 100 microM. The IC(50) of troglitazone for dog Bsep, human BSEP, and rat Bsep were 32, 20, and 60 microM, and those of pravastatin were 441, 240 and >1,000 microM, respectively. In conclusion, while dog Bsep shows similar ATP-dependent bile acid transport characteristics to human BSEP and rat Bsep, there is a species difference in affinity for drugs such as pravastatin and troglitazone.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/fisiología , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP , Transportadoras de Casetes de Unión a ATP/genética , Secuencia de Aminoácidos , Animales , Cromanos/farmacología , Clonación Molecular , Perros , Humanos , Datos de Secuencia Molecular , Especificidad de Órganos , Pravastatina/farmacología , Ratas , Especificidad de la Especie , Spodoptera , Ácido Taurocólico/metabolismo , Tiazolidinedionas/farmacología , TroglitazonaRESUMEN
Bronchial asthma is a complex disease characterized by airway inflammation involving Th2 cytokines. Among Th2 cytokines, the significance of IL-13 in the pathogenesis of bronchial asthma has recently emerged. Particularly, the direct action of IL-13 on bronchial epithelial cells (BECs) is critical for generation of airway hyperresponsiveness. IL-13 has two binding units; the IL-13 receptor alpha1 chain transduces the IL-13 signal comprising a heterodimer with the IL-4R alpha chain, whereas the IL-13 receptor alpha2 chain (IL-13Ralpha2) is thought to act as a decoy receptor. However, it remains obscure how expression of these molecules is regulated in each cell. In this article, we analyzed the expression of these components in BECs. Either IL-4 or IL-13 induced intracellular expression of IL-13Ralpha2 in BECs, which was STAT6-dependent and required de novo protein synthesis. IL-13Ralpha2 expressed on the cell surface as a monomer inhibited the STAT6-dependent IL-13 signal. Furthermore, expression of IL-13Ralpha2 was induced in lung tissues of ovalbumin-induced asthma model mice. Taken together, our results suggested the possibility that IL-13Ralpha2 induced by its ligand is transferred to the cell surface by an unknown mechanism, and it down-regulates the IL-13 signal in BECs, which functions as a unique negative-feedback system for the cytokine signal.
Asunto(s)
Bronquios/metabolismo , Células Epiteliales/metabolismo , Retroalimentación Fisiológica/fisiología , Interleucina-13/metabolismo , Receptores de Interleucina/biosíntesis , Animales , Asma/metabolismo , Modelos Animales de Enfermedad , Humanos , Subunidad alfa1 del Receptor de Interleucina-13 , Interleucina-4/metabolismo , Ratones , ARN Mensajero/metabolismo , Receptores de Interleucina/genética , Receptores de Interleucina-13RESUMEN
Bronchial asthma is a complex disease characterized by airway inflammation involving interleukin (IL)-4 and IL-13. We have applied microarray analyses to human bronchial epithelial cultures to probe for genes regulated by these cytokines and have identified a subset of disease-relevant genes by comparison with cDNA libraries derived from normal and asthmatic bronchial biopsies. Squamous cell carcinoma antigen-1 (SCCA1) and SCCA2, the cysteine and serine protease inhibitors, respectively, showed the highest expression by IL-4 and IL-13, and particularly, SCCA1 was significantly increased in the asthmatic cDNA library. STAT6 was shown to be involved in expression of SCCA1 and SCCA2 in vitro. Furthermore, serum levels of SCCA were also elevated in asthmatic patients. Taken together, it was supposed that SCCA may play some role in the pathogenesis of bronchia asthma, and measuring its serum level may be relevant for diagnosing or monitoring the status of bronchial asthma. In a complex disorder such as asthma, this combination of in vitro and in vivo genomic approaches is a powerful discriminatory method enabling identification of novel disease-related genes and their mechanisms of regulation.
Asunto(s)
Asma/genética , Perfilación de la Expresión Génica , Análisis de Secuencia por Matrices de Oligonucleótidos , Serpinas , Adolescente , Adulto , Antígenos de Neoplasias/biosíntesis , Antígenos de Neoplasias/sangre , Asma/metabolismo , Bronquios/metabolismo , Línea Celular , Células Cultivadas , Niño , Preescolar , Perfilación de la Expresión Génica/métodos , Humanos , Lactante , Interleucina-13/fisiología , Interleucina-4/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Mucosa Respiratoria/metabolismoRESUMEN
BACKGROUND: There are few laboratory tests for evaluating atopic dermatitis (AD) with the exception of IgE levels or the eosinophil count. We attempted to identify new diagnostic markers by screening the genome-wide expression of transcripts in peripheral blood mononuclear cells (PBMC). METHODS: For this study, we enrolled 7 nonatopic healthy volunteers, 5 AD patients who responded well to treatment and 6 who responded poorly. We compared genome-wide transcript levels in PBMC derived from patients with severe AD and healthy volunteers using high-density oligonucleotide arrays (GeneChip, Affymetrix). After the first screening with GeneChip, we employed real-time quantitative PCR to confirm differential expression levels. RESULTS: Screening with GeneChip showed that the levels of a total of 92 transcripts increased at least 3-fold in one population compared to another. After further evaluation of these genes with real-time quantitative PCR, the levels of 4 transcripts were confirmed to be significantly different in PBMC from AD patients compared to controls, namely IFN-gamma, TRAIL (TNF-related apoptosis-inducing ligand), ISGF-3 (STAT1) and defensin-1. With the exception of IFN-gamma, none of these genes has previously been implicated in AD pathology. CONCLUSION: These 4 transcripts in PBMC are expected to be useful markers for evaluating AD.
Asunto(s)
Dermatitis Atópica/sangre , Leucocitos Mononucleares/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , ARN Mensajero/análisis , Células Cultivadas , Defensinas/genética , Humanos , Interferón gamma/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Cathepsin C/dipeptidyl-peptidase I is a papain-like lysosomal cysteine proteinase implicated in the processing of various proenzymes to their active forms. In this study, we identified an alternative splicing variant of cathepsin C in both human and mouse species for the first time. The variant messenger RNA (mRNA) encodes 137 amino acids corresponding to the first and second exons, followed by additional 31 amino acids. The two newly recognized exons are located in the former intron 2. The variant mRNA is distributed ubiquitously, but predominantly in kidney, placenta, and lymph nodes. Furthermore, both interleukin 4 (IL-4) and IL-13, but not a range of cytokines induce expression of the variant in bronchial epithelial cells. These results indicate that the variant may play a role in regulating the biological activities of cathepsin C, involved in the pathogenesis of bronchial asthma.