RESUMEN
Renal macrophages (RMs) participate in tissue homeostasis, inflammation and repair. RMs consist of embryo-derived (EMRMs) and bone marrow-derived RMs (BMRMs), but the fate, dynamics, replenishment, functions and metabolic states of these two RM populations remain unclear. Here we investigate and characterize RMs at different ages by conditionally labeling and ablating RMs populations in several transgenic lines. We find that RMs expand and mature in parallel with renal growth after birth, and are mainly derived from fetal liver monocytes before birth, but self-maintain through adulthood with contribution from peripheral monocytes. Moreover, after the RMs niche is emptied, peripheral monocytes rapidly differentiate into BMRMs, with the CX3CR1/CX3CL1 signaling axis being essential for the maintenance and regeneration of both EMRMs and BMRMs. Lastly, we show that EMRMs have a higher capacity for scavenging immune complex, and are more sensitive to immune challenge than BMRMs, with this difference associated with their distinct glycolytic capacities.
Asunto(s)
Células de la Médula Ósea/citología , Linaje de la Célula , Riñón/embriología , Macrófagos/citología , Animales , Receptor 1 de Quimiocinas CX3C/metabolismo , Quimiocina CX3CL1/sangre , Quimiocina CX3CL1/metabolismo , Femenino , Feto/citología , Hígado/embriología , Masculino , Ratones , Monocitos/citologíaRESUMEN
BACKGROUND: Exposure to aminoglycoside antibiotics can induce ototoxicity in genetically susceptible individuals carrying certain mitochondrial DNA (mtDNA) mutations (C1494T and A1555G), resulting in hearing loss. So, a rapid diagnostic approach is needed to accurately identify subjects carrying such gene mutations. METHODS: In the present study, we describe a rapid and reliable four-color, real-time quantitative polymerase chain reaction (qPCR) assay for simultaneously detecting two mtDNA 12S rRNA gene variants, A1555G and C1494T, which are prevalent in the Han Chinese population. This multiplex assay incorporates three allele-specific TaqMan probes labeled with different fluorophores in a single reaction, providing high genotyping accuracy for clinical blood samples. RESULTS: Tests with C1494T, A1555G and wild-type DNA exhibited high sensitivity, specificity, reproducibility and accuracy of discriminating mutations from wild-type. CONCLUSIONS: This study shows that this simple and inexpensive method can be used for routine molecular diagnostics and potentially for large-scale genetic screening.
Asunto(s)
ADN Mitocondrial/genética , Pruebas Genéticas/métodos , Pérdida Auditiva Sensorineural/inducido químicamente , Reacción en Cadena de la Polimerasa Multiplex , Mutación , ARN Ribosómico/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Marcadores Genéticos , Predisposición Genética a la Enfermedad , Pérdida Auditiva Sensorineural/genética , Humanos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Diagnostic testing for the fragile X syndrome is designed to detect the most common mutation, a CGG expansion in the 5'-untranslated region of the fragile X mental retardation (FMRI) gene. PCR can determine the number of CGG repeats less than 100, whereas Southern analysis can detect large premutations, full mutations, and their methylation status. Bands larger than 5.8 kb observed via Southern analysis are usually considered a methylated full mutation, causing fragile X syndrome in males and varied clinical presentations in females. We observed a 10.9-kb band on a Southern blot assay from an autistic girl with language delay. Further investigation identified a novel G-to-A transition at an EcoRI cleavage site, upstream of the CGG repeat region of the FMRI gene. This base change abolished the EcoRI restriction site, resulting in a 10.9-kb pseudo-full mutation. This G-to-A base change has not been previously reported and was not identified in a subsequent analysis of 105 male and 30 female patient samples. The clear 10.9-kb band detected on a Southern blot assay for fragile X syndrome mimics a large, methylated full mutation, which could result in a misdiagnosis without the benefit of family studies and further testing.
Asunto(s)
Adenosina/genética , Enzimas de Restricción-Modificación del ADN/genética , Síndrome del Cromosoma X Frágil/diagnóstico , Síndrome del Cromosoma X Frágil/genética , Mutación , Secuencia de Bases , Southern Blotting , Preescolar , Análisis Mutacional de ADN , Femenino , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Ácido Nucleico , Expansión de Repetición de Trinucleótido/genéticaRESUMEN
Primary human renal proximal tubule epithelial cells (RPTECs) are of limited use for basic research and for clinical applications due to their limited lifespan in culture. Here we used two lentivirus vectors carrying the human telomerase (hTERT) and the SV40T antigen (Tag) flanked by loxP sites to reversibly immortalize RPTECs. Transduced RPTEC clones continued to proliferate while retaining biochemical and functional characteristics of primary cells. The clones exhibited contact-inhibited, anchorage- and growth factor-dependent growth and did not form tumors in nude mice, suggesting that the cells were not transformed. Transient Cre expression in these cells led to efficient proviral deletion, upregulation of some renal specific activities, and decreased growth rates. Ultimately, the cells underwent replicative senescence, indicating intact cell cycle control. Thus, reversible immortalization allows the expansion of human RPTECs, leading to large production of RPTECs that retain most tissue-specific properties.
Asunto(s)
Integrasas/fisiología , Túbulos Renales Proximales/citología , Proteínas Virales/fisiología , Antígenos Transformadores de Poliomavirus/genética , Línea Celular Tumoral , Proteínas de Unión al ADN , Células Epiteliales/citología , Vectores Genéticos , VIH/genética , Humanos , Integrasas/genética , Recombinación Genética , Telomerasa/genética , Proteínas Virales/genéticaRESUMEN
The activation of phosphodiesterase 3B (PDE3B) reduces free fatty acid output from adipocytes. A reduced PDE3B gene expression could lead to insulin resistance. To determine whether there are polymorphisms associated with type 2 diabetes in PDE3B gene promoter, this 5(') flanking region was isolated. The transcription initiation site was located 206bp upstream from the translation start site. Sequences of 2kb of the 5(') flanking region for 24 type 2 diabetic Japanese subjects were initially analyzed using PCR direct sequencing, and the regions including the identified polymorphisms were then examined. In 98 controls and 98 type 2 diabetic subjects, -1947T>C, -567G>A, -465G>T, -458T>C, and -1727_-1726insTCAATT were found. Only -465G>T and this insertion had more than 5% frequencies. Since a complete linkage disequilibrium existed between them, -465G>T was further analyzed, along with a previously identified +1389G>A in the coding region, in a total of 200 controls and 207 type 2 diabetic subjects. These allele frequencies were not significantly different between these two groups (controls vs. cases; -465G>T, 12.0% vs. 10.1%, P=0.435; +1389G>A, 30.3% vs. 33.3%, P=0.408). These genotype distributions were not significantly different between these two groups. The T/T genotype at -465 was rare although this frequency could be higher in type 2 diabetes (4/207 subjects) than controls (0/200 subjects). The linkage disequilibrium existed between -465G>T and +1389G>A, and the estimated haplotype frequencies defined by these SNPs were not significantly different between the cases and controls. Thus, the identified polymorphisms are unlikely to have major effects on susceptibility to Japanese type 2 diabetes.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/genética , Región de Flanqueo 5'/genética , Diabetes Mellitus Tipo 2/genética , Polimorfismo de Nucleótido Simple/genética , Adulto , Edad de Inicio , Estudios de Casos y Controles , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3 , Susceptibilidad a Enfermedades , Femenino , Frecuencia de los Genes , Haplotipos , Humanos , Resistencia a la Insulina , Japón , Desequilibrio de Ligamiento , Masculino , Persona de Mediana Edad , Reacción en Cadena de la PolimerasaRESUMEN
Towards understanding the IGF system during cancer growth and progression, progressive prostate cancer models, such as SV40 large T antigen immortalized human prostate epithelial cells (P69, M2182, M2205, and M12) and LNCaP sublines (C4, C4-2, and C4-2B4), were used. IGF-II mRNA levels progressively increase as prostate cancer cells become more tumorigenic and metastatic, suggesting that IGF-II contributes in part to prostate cancer progression. The role of IGF-II in cancer cell growth was evaluated in LNCaP, PC3, and M12 prostate cancer cell lines and MCF-7 breast cancer cell line by ribozyme/antisense strategies which were previously shown to suppress endogenous IGF-II expression and cell growth in PC-3 cells [Xu et al., Endocrinol 140 (1999) 2134]. Retroviral mediated transient expression of IGF-II-specific ribozyme (RZ) caused extensive cell death. In stably cloned cell lines, both RZ and mutant ribozyme (MRZ) inhibited cancer cell growth, suggesting that antisense effects of MRZ may be sufficient for cell growth inhibition. These results confirm an important role of IGF-II in cancer cell growth and progression, and support further development of gene therapy targeting IGF-II.