RESUMEN
We aimed to investigate the biological role of miR-367 in uveal melanoma cell growth and migration, and the underlying mechanism responsible. Quantitative real-time polymerase chain reaction was performed to evaluate miR-367 expression in uveal melanoma tissue samples and cell lines. A miR-367 mimic, miR-367 inhibitor, and negative control oligonucleotide were transfected into these cells to investigate the function of this microRNA. In addition, the role of PTEN in miR-367-mediated uveal melanoma cell growth and migration was evaluated. miR-367 was significantly upregulated in uveal melanoma cells and tissue samples (both P < 0.01). Its inhibition suppressed the proliferation, cell cycle transition, and migration of such cells, and increased levels had the opposite effect. PTEN was confirmed to be a target gene of miR-367. More importantly, co-transfection with a PTEN construct lacking the 3'-untranslated region mitigated miR-367 mimic-induced promotion of uveal melanoma cell proliferation and migration. In summary, miR-367 was found to be upregulated in this malignancy, and may promote uveal melanoma cell proliferation and migration, at least in part by regulating PTEN.
Asunto(s)
Movimiento Celular , Proliferación Celular , Melanoma/metabolismo , MicroARNs/genética , Fosfohidrolasa PTEN/metabolismo , Neoplasias de la Úvea/metabolismo , Estudios de Casos y Controles , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Melanocitos/metabolismo , Melanocitos/fisiología , Melanoma/genética , Melanoma/patología , MicroARNs/metabolismo , Fosfohidrolasa PTEN/genética , Regulación hacia Arriba , Neoplasias de la Úvea/genética , Neoplasias de la Úvea/patologíaRESUMEN
Cytoplasmic male sterility (CMS) in pepper is a better way to produce hybrid seeds compared to manual production. We used the two sequence characterized amplified region (SCAR) markers (CRF-SCAR and CMS-SCAR130) in CMS pepper, to identify the genotype. We assembled two CMS yellow bud mutants (YBM; YBM12-A and YBM12-B). This mutation in leaf color is controlled by a single dominant nuclear gene. The aim was to create a new hybrid seed production method that reduces the costs and increases F1 hybrid seed purity. The results suggest that the CRF-SCAR and CMS-SCAR130 markers can be used together in multiple generations to screen for restorer or maintainer genes. We found the marker linked to the restorer gene (Rf) in the C-line and F1 hybrids, as well as partially in the F2 generation, whereas it was not found in the sterile YBM12-A or the maintainer line YBM12-B. In the F2 population, sterility and fertility segregated at a 3:1 ratio based on the CRF-SCAR marker. A 130 bp fragment was produced in the YBM12-A, F1, and F2 populations, suggesting that these lines contained sterile cytoplasm. A 140 bp fragment present in the YBM12-B and C-line indicated that these lines contained normal cytoplasm. In addition, we identified some morphological characters distinguishing sterile and fertile buds and flowers that may be linked to the sterility gene. If more restorer lines are identified, CMS expressing the YBM trait can be used in hybrid seed production.
Asunto(s)
Capsicum/genética , Genes de Plantas/genética , Marcadores Genéticos/genética , Mutación , Infertilidad Vegetal/genética , Citoplasma/genética , Fertilidad/genética , Flores/genética , Genotipo , Hibridación Genética , Meristema/genética , Fenotipo , Fitomejoramiento/métodos , Reproducibilidad de los Resultados , Semillas/genéticaRESUMEN
We examined the immunological characteristics of outer membrane protein omp31 of goat Brucella and its monoclonal antibody. Genomic DNA from the M5 strain of goat Brucella was amplified by polymerase chain reaction and cloned into the prokaryotic expression vector pGEX-4T-1. The expression and immunological characteristics of the fusion protein GST-omp31 were subjected to preliminary western blot detection with goat Brucella rabbit immune serum. The Brucella immunized BALB/c mouse serum was detected using purified protein. The high-potency mouse splenocytes and myeloma Sp2/0 cells were fused. Positive clones were screened by enzyme-linked immunosorbent assay to establish a hybridoma cell line. Mice were inoculated intraperitoneally with hybridoma cells to prepare ascites. The mAb was purified using the n-caprylic acid-ammonium sulfate method. The characteristics of mAb were examined using western blotting and enzyme-linked immunosorbent assay. A 680-base pair band was observed after polymerase chain reaction. Enzyme digestion identification and sequencing showed that the pGEX-4T-1-omp31 prokaryotic expression vector was successfully established; a target band of approximately 57 kDa with an apparent molecular weight consistent with the size of the target fusion protein. At 25°C, the expression of soluble expression increased significantly; the fusion protein GST-omp31 was detected by western blotting. Anti-omp31 protein mAb was obtained from 2 strains of Brucella. The antibody showed strong specificity and sensitivity and did not cross-react with Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Mycobacterium tuberculosis, or Bacillus pyocyaneus. The pGEX-4T-1-omp31 prokaryotic expression vector was successfully established and showed good immunogenicity. The antibody also showed strong specificity and good sensitivity.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Brucella/inmunología , Animales , Especificidad de Anticuerpos , Línea Celular Tumoral , Femenino , Cabras , Ratones , Ratones Endogámicos BALB C , ConejosRESUMEN
Many carpal tunnel syndrome (CTS) patients have symptoms in both the median and ulnar digits more frequently than in the median digits alone. This is possibly because of close anatomical contiguity of the carpal tunnel and Guyon's canal, and the high pressure may also affect the latter, causing indirect compression of ulnar nerve fibers. Thus, we evaluated the functional status of the ulnar nerve in patients with CTS in order to investigate the relationship between ulnar nerve impairment and sensory symptoms of the ulnar territory. Electrophysiological studies were conducted in CTS patients and healthy controls. CTS patients were divided into the mild/moderate group and severe group; they were further divided into the symptomatic and asymptomatic subgroups according to the sensory symptom of the fifth digit region. The findings suggest that CTS patients could have coexisting ulnar nerve wrist entrapments that might exacerbate the severity of CTS. Sensory impairment in the ulnar territory was observed more frequently in the mild/moderate stage of CTS, which is associated with ulnar nerve involvement. These findings also suggest that damage to the ulnar nerve fibers caused by compression forces in Guyon's canal may underlie the ulnar spread of symptoms in CTS.
Asunto(s)
Síndrome del Túnel Carpiano/fisiopatología , Nervio Cubital/fisiopatología , Adulto , Anciano , Estudios de Casos y Controles , Femenino , Dedos/inervación , Dedos/fisiopatología , Humanos , Masculino , Persona de Mediana Edad , Conducción NerviosaRESUMEN
Virus-induced gene silencing (VIGS) is an important tool for studying gene function. However, a number of factors highly restrict the application of VIGS, such as unstable efficiency and tissue-specific silencing. We developed a novel evaluation method for improving the applicability of VIGS vectors. In this method, 4 indexes were defined and utilized to evaluate VIGS efficiency by silencing the endogenous phytoene desaturase (PDS) gene with a tobacco rattle virus-based VIGS vector. To illustrate the reliability of this evaluation method, we assessed the silencing efficiency of SpPDS and SpMPK1 in Solanum pimpinellifolium. The silencing results of SpPDS showed that an optical density at 600 nm of 2.0 was more suitable than 1.0 for VIGS in S. pimpinellifolium. This suggests that the proposed evaluation method is a valid technique for optimizing the VIGS system of plants. Moreover, the SpMPK1 gene was highly silenced in the 4th-9th leaves with a 50-95% reduction in transcription levels, further demonstrating that this method can be used to select highly silenced candidates for further experiments, particularly when the target gene shows no phenotypic change after being silenced.
Asunto(s)
Silenciador del Gen , Virus de Plantas/fisiología , Solanum/genética , Solanum/virología , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Hojas de la Planta/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Solanum/enzimología , Transcripción GenéticaRESUMEN
Calmodulin (CALM), a calcium-binding protein, is expressed in the hypothalamic-pituitary-gonadal axis; it plays a pivotal role in the reproductive system by regulating gonadotropin-releasing hormone signaling. Downstream of hypothalamic-pituitary-gonadal signaling pathways, liver receptor homolog-1 (LRH-1) is involved in female gonadal hormone synthesis. In the chicken, although the two genes are known to be associated with reproductive traits, the interaction between gonadotropins and gonadal steroids remains unclear. We used quantitative real-time PCR to quantify the tissular (hypothalamus, pituitary, ovary, liver, kidney, oviduct, heart) and ontogenetic (12, 18, 32, and 45 weeks) mRNA expression profiles of CALM and LRH-1 in Erlang Mountainous chickens to determine their roles in the endocrine control of fertility, and compared these profiles with expression in Roman chickens. We found that the relative expressions of CALM and LRH-1 genes had the highest levels in the pituitary and ovary at 32 weeks. The expression level of CALM mRNA in the pituitary of Roman chickens was significantly higher than that in Erlang Mountainous chickens at 32 and 45 weeks, while the LRH-1 transcript level in the ovaries of Roman chickens was significantly lower than that of Erlang Mountainous chickens at 32 and 45 weeks. In summary, the transcript levels of CALM and LRH-1 genes are associated with chicken reproductive traits; in addition, we found that the CALM gene is the key regulator in the hypothalamic-pituitary-gonadal signaling network.