RESUMEN
AIM: Slc26a9 is a member of the Slc26 multifunctional anion transporter family. Polymorphisms in Slc26a9 are associated with an increased incidence of meconium ileus and diabetes in cystic fibrosis patients. We investigated the expression of Slc26a9 in the murine pancreatic ducts, islets and parenchyma, and elucidated its role in pancreatic ductal electrolyte and fluid secretion and endocrine function. METHODS: Pancreatic Slc26a9 and CFTR mRNA expression, fluid and bicarbonate secretion were assessed in slc26a9-/- mice and their age- and sex-matched wild-type (wt) littermates. Glucose and insulin tolerance tests were performed. RESULTS: Compared with stomach, the mRNA expression of Slc26a9 was low in pancreatic parenchyma, 20-fold higher in microdissected pancreatic ducts than parenchyma, and very low in islets. CFTR mRNA was ~10 fold higher than Slc26a9 mRNA expression in each pancreatic cell type. Significantly reduced pancreatic fluid secretory rates and impaired glucose tolerance were observed in female slc26a9-/- mice, whereas alterations in male mice did not reach statistical significance. No significant difference was observed in peripheral insulin resistance in slc26a9-/- compared to sex- and aged-matched wt controls. In contrast, isolated slc26a9-/- islets in short term culture displayed no difference in insulin content, but a significantly reduced glucose-stimulated insulin secretion compared to age- and sex-matched wt islets, suggesting that the impaired glucose tolerance in the absence of Slc26a9 expression these is a pancreatic defect. CONCLUSIONS: Deletion of Slc26a9 is associated with a reduction in pancreatic fluid secretion and impaired glucose tolerance in female mice. The results underline the importance of Slc26a9 in pancreatic physiology.
Asunto(s)
Antiportadores , Secreción de Insulina , Páncreas/fisiología , Transportadores de Sulfato , Anciano , Animales , Antiportadores/genética , Antiportadores/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Femenino , Humanos , Insulina , Masculino , Ratones , Transportadores de Sulfato/genética , Transportadores de Sulfato/metabolismoRESUMEN
MicroRNAs (miRNAs) represent a new class of small non-coding RNAs regulating gene expression by inducing RNA degradation or interfering with translation. Aberrant miRNA expression has been described for several human malignancies and tumour suppressor functions have been ascribed to this new class of small regulatory RNAs. Accordingly, inactivation due to deletion or mutation has been found in human malignancies. Here, we describe the role of aberrant hypermethylation as an additional mechanism for miRNA gene inactivation in human breast cancer. Aberrant hypermethylation was shown for mir-9-1, mir-124a3, mir-148, mir-152, and mir-663 in 34-86% of cases in a series of 71 primary human breast cancer specimens. For comprehensive methylation analysis, combined bisulphite restriction analysis, bisulphite sequencing, and Pyrosequencing were employed. miRNA gene hypermethylation correlated strongly with methylation of known tumour suppressor genes (p = 0.003). After treatment of various breast cancer cell lines with the demethylating agent 5-aza-2'-deoxycytidine, reduction of mir-9-1 gene methylation and concomitant reactivation of expression could be observed. For the mir-9-1 gene, which is already hypermethylated in pre-invasive intraductal lesions, a good correlation between quantitative methylation level and reduction of expression could be demonstrated in a subset of primary human breast cancer specimen (r = 0.8). In conclusion, this study demonstrates that various microRNA genes are also affected by epigenetic inactivation due to aberrant hypermethylation and that this is an early and frequent event in breast cancer development.
Asunto(s)
Neoplasias de la Mama/genética , Silenciador del Gen , MicroARNs/genética , ARN Neoplásico/genética , Azacitidina/análogos & derivados , Azacitidina/farmacología , Carcinoma Ductal de Mama/genética , Carcinoma Lobular/genética , Islas de CpG/genética , Metilación de ADN , Metilasas de Modificación del ADN/antagonistas & inhibidores , Decitabina , Inhibidores Enzimáticos/farmacología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Genes Supresores de Tumor , Humanos , Células Tumorales CultivadasRESUMEN
AIMS: This study addresses the question whether microRNA genes are affected by aberrant hypermethylation and subsequent transcriptional repression in human breast cancer. METHODS: All known human microRNA genes were screened for the association with CpG islands using different algorithms. Methylation status of candidate genes was assessed in a panel of breast cancer cell lines, various normal tissue samples and primary breast cancer specimen using COBRA, bisulfite sequencing and pyrosequencing. Transcriptional silencing was measured by real-time RT-PCR. The effect of demethylation on microRNA gene expression was analysed in breast cancer cell lines after treatment with the DNMT inhibitor 5'-deoxy-2-azacytidine. RESULTS: Aberrant hypermethylation was shown for mir-9-1, mir-124a3, mir-148, mir-152, and mir-663 in 34-86% of cases in a series of 71 primary human breast cancer specimens. miRNA gene hypermethylation correlated strongly with methylation of known tumour suppressor genes (p = 0.003). After treatment of various breast cancer cell lines with the demethylating agent 5-aza-2'deoxycytidine, reduction of mir-9-1 gene methylation and concomitant reactivation of expression could be observed. For the mir-9-1 gene, which is already hypermethylated in preinvasive intraductal lesions, a good correlation between quantitative methylation level and reduction of expression could be demonstrated in a subset of primary human breast cancer specimen (r = 0.8). CONCLUSIONS: In addition to deletion and mutation, microRNA genes are also affected by aberrant hypermethylation in human breast cancer. This epigenetic inactivation is frequent and occurs early during breast cancer progression.
Asunto(s)
Neoplasias de la Mama/genética , Silenciador del Gen , MicroARNs/genética , Secuencia de Bases , ADN de Neoplasias/genética , ADN de Neoplasias/aislamiento & purificación , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , ARN Neoplásico/genética , ARN Neoplásico/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
OBJECTIVES: This study analyzes changes in the degree of global methylationlevel in myelodysplastic syndrome during progression of the disease. METHODS: Methylation status was analyzed in 127 patients with histologically confirmed MDS and 26 reactive controls. We employed Pyrosequencing, Luminometric Methylation Assay (LUMA) and a realtime PCR-based quantitative assay. RESULTS: We detected an increase of methylation level of LINE-1 sequences using pyrosequencing and an increase of methylation in the HpaII recognition site employing LUMA during the progression of MDS. Methylation sensitive quantitative PCR showed no statistically significant differences, only a trend. CONCLUSIONS: LINE-1 and methylation sensitive cleavage of DNA can act as a surrogatmarker for global DNA methylation. The genome wide hypermethylation of MDS is a distinct feature of this disease. It discriminates MDS from other neoplasia and may explains the success of hypomethylation inducing reagents like azadeoxycytidine in MDS therapy.
Asunto(s)
Metilación de ADN , Elementos de Nucleótido Esparcido Largo/genética , Síndromes Mielodisplásicos/genética , Humanos , Leucemia Mieloide Aguda/genéticaRESUMEN
To study the influence of antigen density on the efficiency of negative selection in the thymus, MHC class I (H-2K(b), K(b)) transgenic mice were generated, which expressed a K(b) transgene under the control of its natural promoter at 33% (K(b-lo)) or 150% (K(b-hi)) the surface density of Kb in C57BL/6 (B6, H-2(b)) mice. These mice were crossed to anti-K(b) T-cell receptor (Des-TCR) transgenic mice. In Des-TCRxK(b-hi) double transgenic mice, Des-TCR bearing T cells were completely eliminated during thymocyte maturation. In contrast, in Des-TCRxK(b-lo) double transgenic mice, two populations of Des-TCR T cells were evident, which either expressed the Des-TCR at intermediate density in the absence of CD8 (Des-TCR(int)CD8(-)) or expressed both the Des-TCR and CD8 at low density (Des-TCRloCD8lo). In the thymus of both types of double transgenic mice, no Des-TCR(+)CD4(+)CD8(+) thymocytes were detected, suggesting that deletion of Des-TCR cells occurred before the CD4(+)CD8(+) stage. Because only very few Des-TCR(+) thymocytes were found in Des-TCRxK(b-hi) transgenic mice, deletion of these T cells apparently occurred upon expression of the Des-TCR. By contrast, Des-TCRxK(b-lo) transgenic mice showed distinct populations of Des-TCR(int)CD4-8- and Des-TCR(lo)CD8(lo) thymocytes, suggesting that expression of the CD8 coreceptor was required to allow negative selection to proceed. Functional analyses showed that sublethally irradiated Des-TCRxK(b-lo) double transgenic mice were protected from lethal graft-versus-host disease by injected Des-TCR lymph node cells.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T/análisis , Supervivencia de Injerto/inmunología , Enfermedad Injerto contra Huésped/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Trasplante de Piel/inmunología , Linfocitos T/inmunología , Timo/inmunología , Animales , Antígenos CD4/análisis , Antígenos CD8/análisis , Cruzamientos Genéticos , Antígenos H-2/genética , Antígenos H-2/inmunología , Ligandos , Ganglios Linfáticos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Endogámicos , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/genética , Factores de TiempoRESUMEN
The nucleotide sequence of the rpoN gene, formerly designated hno, and flanking DNA regions of the aerobic hydrogen bacterium Alcaligenes eutrophus has been determined; rpoN codes for the RNA polymerase sigma factor sigma 54 involved in nitrogen regulation and diverse physiological functions of gram-negative bacteria. In A. eutrophus hydrogen metabolism is under control of rpoN. The Tn5-Mob insertion in a previously isolated pleiotropic mutant was mapped within the rpoN gene. The derived amino acid sequence of the A. eutrophus RpoN protein shows extensive homology to the RpoN proteins of other organisms. Sequencing revealed four other open reading frames: one upstream (ORF280) and three downstream (ORF130, ORF99 and ORF greater than 54) of the rpoN gene. A similar arrangement of homologous ORFs is found in the rpoN regions of other bacteria and is indicative of a conserved gene cluster.
Asunto(s)
Alcaligenes/genética , Proteínas de Unión al ADN , ARN Polimerasas Dirigidas por ADN , Genes Bacterianos/genética , Familia de Multigenes/genética , Factor sigma/genética , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Regulación Bacteriana de la Expresión Génica , Variación Genética , Hidrógeno/metabolismo , Datos de Secuencia Molecular , Mutagénesis Insercional , ARN Polimerasa Sigma 54 , Sistemas de Lectura/genética , Homología de SecuenciaRESUMEN
Pleiotropic mutants of Alcaligenes eutrophus with the phenotype Hno- have been characterized previously. They are deficient in several diverse metabolic activities, including hydrogen oxidation, nitrate and urea assimilation, denitrification, and various substrate transport systems. Phenotypically similar mutants were identified among hydrogenase-deficient strains of Pseudomonas facilis. The Tn5-labeled hno gene was cloned from a genomic DNA library of A. eutrophus and used to identify the corresponding unimpaired wild-type DNA sequence. The recombinant plasmid pCH148 contained an insert of 12.3 kilobase pairs and was shown to restore the Hno+ phenotype to mutants of A. eutrophus and P. facilis. A cosmid isolated from a DNA library of P. facilis also exhibited intergeneric Hno-complementing activity. The cloned hno loci from both organisms showed DNA homology by Southern blot hybridization. A subclone of pCH148 which contained a 6.5-kilobase-pair insert was constructed. The resulting hybrid, pCH170, not only was able to complement Hno- mutants but also relieved glutamine auxotrophy in NtrA- mutants of enteric bacteria. This suggests that the hno gene product from A. eutrophus is functionally similar to the NtrA protein, which has been identified as a novel sigma factor (sigma 54) of RNA polymerase.
Asunto(s)
Alcaligenes/genética , Genes Bacterianos , Pseudomonas/genética , Alcaligenes/metabolismo , Elementos Transponibles de ADN , Genes , Hidrógeno/metabolismo , Mutación , Fijación del Nitrógeno/genética , Nitrogenasa/genética , Oxidación-Reducción , Fenotipo , Plásmidos , Regiones Promotoras Genéticas , Pseudomonas/metabolismo , Mapeo RestrictivoRESUMEN
Curing of the hydrogenase-specifying megaplasmid pHG indigenous to strains of the facultative lithoautotrophic bacterium Alcaligenes eutrophus was correlated with a loss of denitrifying ability (Nitd). The retransfer of plasmid pHG1 reconstituted the Nitd phenotype. Plasmid-free mutants were still capable of converting some nitrate to nitrite, but they did not metabolize nitrite under anaerobic conditions.
Asunto(s)
Alcaligenes/metabolismo , Nitratos/metabolismo , Alcaligenes/genética , Anaerobiosis , Hidrogenasas/genética , Nitritos/metabolismo , PlásmidosRESUMEN
Mutants of Alcaligenes eutrophus H16 lacking catalytically active soluble hydrogenase (Hos-) grew very slowly lithoautotrophically with hydrogen. Mutants devoid of particulate hydrogenase activity (Hop-) were not affected in growth with hydrogen. The use of Hos- and Hop- mutants as donors of hydrogen-oxidizing ability in crosses with plasmid-free recipients impaired in both hydrogenases (Hox-) resulted in transconjugants which had inherited the plasmid and the phenotype of the donor. This indicates that the structural genes which code for the hydrogenases reside on plasmid pHG1. The Hox function of one class of Hox- mutants could not be restored by conjugation. These mutants exhibited a pleiotropic phenotype since they were unable to grow with hydrogen and also failed to grow heterotrophically with nitrate (Hox- Nit-). Nitrate was scarcely utilized as electron acceptor or as nitrogen source. Hox- Nit- mutants did not act as recipients but could act as donors of the Hox character. Transconjugants derived from those crosses were Hox+ Nit+, indicating that the mutation which leads to the Hox- Nit- phenotype maps on the chromosome. Apparently, the product of a chromosomal gene is involved in the expression of plasmid-encoded Hox genes. We observed that the elimination of plasmid pHG1 coincided with the occurrence of multiple resistances to various antibiotics. Since Hox+ transconjugate retained the antibiotic-resistant phenotype, we conclude that this property is not directly plasmid associated.