RESUMEN
Increasing evidence suggests that the cerebrospinal ï¬uid-contacting nucleus (CSF-contacting nucleus) mediates the transduction and regulation of pain signals. However, the precise molecular mechanisms remain unclear. Studies show that release of fractalkine (FKN) from neurons plays a critical role in nerve injury-related pain. We tested the hypothesis that release of FKN from the CSF-contacting nucleus regulates neuropathic pain, in a chronic constriction injury rat model. The results show that FKN is expressed by neurons, via expression of its only receptor CX3CR1 in the microglia. The levels of soluble FKN (sFKN) were markedly upregulated along with the increase in FKN mRNA level in rats subjected to chronic constriction injury. In addition, injection of FKN-neutralizing antibody into the lateral ventricle alleviated neuropathic pain-related behavior followed by reduction in microglial activation in the CSF-contacting nucleus. The results indicate that inhibition of FKN release by the CSF-contacting nucleus may ameliorate neuropathic pain clinically.
Asunto(s)
Núcleo Celular/metabolismo , Líquido Cefalorraquídeo/metabolismo , Quimiocina CX3CL1/metabolismo , Dolor Crónico/metabolismo , Neuralgia/metabolismo , Umbral del Dolor/fisiología , Animales , Modelos Animales de Enfermedad , Inyecciones Intraventriculares , Masculino , Ratas , Ratas Sprague-Dawley , Regulación hacia ArribaRESUMEN
Porcine ear size is an important characteristic for distinguishing among pig breeds. In a previous genome-wide association study of porcine ear size, LEM domain-containing 3 (LEMD3), methionine sulfoxide reductase B3 (MSRB3), high mobility group AT-hook 2 (HMGA2), and Wnt inhibitory factor 1 (WIF1) were implicated as important candidate genes for ear size. This study investigated the expression levels of four candidate genes for ear size in Erhualian and Large White pigs. Ten Erhualian pigs with large ears and eight Large White pigs with small ears at 60 days of age were examined. The mRNA expression levels of the four candidate genes were quantified by real-time polymerase chain reaction. WIF1 mRNA expression was significantly higher in Large White than in Erhualian pigs (P < 0.05), whereas the expression levels of the other three genes were not significantly different between the two breeds. The protein expression levels of the four genes were analyzed using western blot. WIF1 protein expression was significantly higher in Large White than in Erhualian pigs (P < 0.01), whereas MSRB3 protein expression was significantly higher in Erhualian than in Large White pigs (P < 0.05). There were no significant differences between the two breeds in residual protein expression. These results suggest that WIF1 is the main causal gene for ear size in pigs.
Asunto(s)
Oído/crecimiento & desarrollo , Sitios de Carácter Cuantitativo , Porcinos/genética , Animales , Animales Endogámicos , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Perfilación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Metionina Sulfóxido Reductasas/genética , Metionina Sulfóxido Reductasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Mitogen-activated protein kinase kinase kinase 5 (MAP3K5) is essential for apoptosis, proliferation, differentiation, and immune responses, and is a candidate marker for residual feed intake (RFI) in pig. We cloned the full-length cDNA sequence of porcine MAP3K5 by rapid-amplification of cDNA ends. The 5451-bp gene contains a 5'-untranslated region (UTR) (718 bp), a coding region (3738 bp), and a 3'-UTR (995 bp), and encodes a peptide of 1245 amino acids, which shares 97, 99, 97, 93, 91, and 84% sequence identity with cattle, sheep, human, mouse, chicken, and zebrafish MAP3K5, respectively. The deduced MAP3K5 protein sequence contains two conserved domains: a DUF4071 domain and a protein kinase domain. Phylogenetic analysis showed that porcine MAP3K5 forms a separate branch to vicugna and camel MAP3K5. Tissue expression analysis using real-time quantitative polymerase chain reaction (qRT-PCR) revealed that MAP3K5 was expressed in the heart, liver, spleen, lung, kidney, muscle, fat, pancrea, ileum, and stomach tissues. Copy number variation was detected for porcine MAP3K5 and validated by qRT-PCR. Furthermore, a significant increase in average copy number was detected in the low RFI group when compared to the high RFI group in a Duroc pig population. These results provide useful information regarding the influence of MAP3K5 on RFI in pigs.
Asunto(s)
MAP Quinasa Quinasa Quinasa 5/genética , Sus scrofa/genética , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Clonación Molecular , Variaciones en el Número de Copia de ADN , Dosificación de Gen , Expresión Génica , Interacciones Hidrofóbicas e Hidrofílicas , MAP Quinasa Quinasa Quinasa 5/química , MAP Quinasa Quinasa Quinasa 5/metabolismo , Modelos Moleculares , Especificidad de Órganos , Filogenia , Conformación Proteica en Hélice alfa , Sus scrofa/metabolismoRESUMEN
Currently, there is no practical and efficient method for the isolation of bone marrow cells (BMCs) from rat femurs and tibiae. Here, we attempted to develop a rapid, simple, effective, and non-contaminating method for the isolation of BMCs from rat femurs and tibiae. Rat femurs and tibiae were dissected from the ankle to the hip joint; subsequently, a three-step "locate-slide-twist" procedure was performed using scissors and forceps to remove the femurs and tibiae completely, from the surrounding musculature. The bones were flushed with phosphate-buffered saline to harvest BMCs. The femurs and tibiae were dissected in 1.8 ± 0.6 min, and the BMC suspension preparation time was 13.1 ± 2.3 min. The bone marrow cavities did not incur any fractures or injuries during the isolation. Culture of harvested BMCs for 72 h led to a significant increase in cell number from 4.4 ± 0.3 x 106 to 6.9 ± 0.7 x 10(6) (P < 0.01) with no significant decrease in viability (98.1 ± 0.6% vs 96.2 ± 1.1%; P > 0.05). Microscopic examination of the isolated BMCs after the 72-h incubation period revealed the no-microbial or muscle cell contamination. Furthermore, flow cytometry revealed that cultured BMCs (72-h culture) grew well. Here, we have reported a rapid, simple, effective, and non-contaminating method for the isolation of BMCs from rat femurs and tibiae by using retrograde dissection. This method can be used to harvest a large number of viable BMCs without the risk of contamination from muscle and connective tissues.
Asunto(s)
Células de la Médula Ósea/citología , Técnicas de Cultivo de Célula/métodos , Separación Celular/métodos , Supervivencia Celular , Fémur/citología , Animales , Modelos Animales de Enfermedad , Ratas , Tibia/citologíaRESUMEN
The signal peptide CUB EGF-like domain-containing protein 3 (SCUBE3) gene is a member of SCUBE gene family and plays important roles in bone cell biology and the determination of limb bone length. In this study, the full-length transcript of porcine SCUBE3 was cloned using reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends. The full-length sequence of porcine SCUBE3 cDNA was 4131 base pairs and included 21 exons. The SCUBE3 gene contained a 2895-base pair open reading frame that encoded a peptide of 965 amino acids. Comparison of the deduced amino acid sequences of porcine SCUBE3 with those of human, mouse, zebrafish, and rat showed 96, 95, 73, and 95% identities, respectively. Porcine SCUBE3 mRNA expression levels were highest in the backfat, bone marrow, and cartilage tissues. Copy number variation was detected in porcine SCUBE3 and validated by real-time quantitative polymerase chain reaction. Different copy number variations were present in randomly selected individuals and may, therefore, be a good marker for identifying phenotypic traits. Our findings provide a basis for further investigation of the functions and regulatory mechanisms of SCUBE3 in pigs.
Asunto(s)
Clonación Molecular , Variaciones en el Número de Copia de ADN , Expresión Génica , Receptores de Superficie Celular/genética , Sus scrofa/metabolismo , Animales , Médula Ósea/metabolismo , Cartílago/metabolismo , Especificidad de Órganos , Filogenia , ARN Mensajero , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Sus scrofa/genéticaRESUMEN
We conducted a case-control study to assess the relation-ship between rs35767, rs2288377, and rs5742612 insulin-like growth factor-1 (IGF-1) and osteoporosis risk in a Chinese female population. The genotypes of rs35767, rs2288377, and rs5742612 of IGF-1 were determined by polymerase chain reaction-restriction fragment length polymorphism. Patients with osteoporosis were more likely to have drinking and smoking habits and have lower bone mineral density in the L2-L4 vertebrae, femoral neck, and total hip. According to conditional regression analysis, individuals carrying the TT genotype of rs35767 had an increased risk of osteoporosis, with an adjusted odds ratio (95% confidence interval) of 2.29 (1.35-4.97). In conclusion, our results sug-gest that the TT genotype of IGF-I rs35767 was associated with an in-creased risk of osteoporosis, suggesting that this polymorphism can be used as a predictive factor for osteoporosis risk.