RESUMEN
RNA extraction from the nucleus pulposus of intervertebral discs has been extensively used in orthopedic studies. We compared two methods for extracting RNA from the nucleus pulposus: liquid nitrogen grinding and enzyme digestion. The RNA was detected by agarose gel electrophoresis, and the purity was evaluated by absorbance ratio using a spectrophotometer. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression was assayed by reverse transcription-polymerase chain reaction (RT-PCR). Thirty human lumbar intervertebral discs were used in this study. The liquid nitrogen-grinding method was used for RNA extraction from 15 samples, and the mean RNA concentration was 491.04 ± 44.16 ng/mL. The enzyme digestion method was used on 15 samples, and the mean RNA concentration was 898.42 ± 38.64 ng/mL. The statistical analysis revealed that there was a significant difference in concentration between the different methods. Apparent 28S, 18S, and 5S bands were detectable in RNA extracted using the enzyme digestion method, whereas no 28S or 18S bands were detected in RNA extracted using the liquid nitrogen-grinding method. The GAPDH band was visible, and no non-specific band was detected in the RT-PCR assay by the enzyme digestion method. Therefore, the enzyme digestion method is an efficient and easy method for RNA extraction from the nucleus pulposus of intervertebral discs for further intervertebral disc degeneration-related studies.
Asunto(s)
Degeneración del Disco Intervertebral/genética , Disco Intervertebral/metabolismo , Núcleo Pulposo/metabolismo , ARN/aislamiento & purificación , Gliceraldehído-3-Fosfato Deshidrogenasas/biosíntesis , Humanos , ARN/genéticaRESUMEN
Rhodiola alsia, which has been used widely in traditional Chinese medicine for a considerable time, grows on moist habitats at high altitude near the snow line. Microsatellite loci were developed for R. alsia to investigate its population genetics. In total, 17 polymorphic microsatellites were developed based on ESTs from the Illumina HiSeq(TM) 2000 platform. The microsatellite loci were checked for variability using 80 individuals of R. alsia sampled from four locations on the Qinghai-Tibet Plateau. The total number of alleles per locus ranged from 10 to 20, and the observed heterozygosity ranged from 0.000 to 1.000. The null allele frequency ranged from 0.000 to 0.324. These microsatellites are expected to be helpful in future studies of population genetics in R. alsia and related species.
Asunto(s)
Repeticiones de Microsatélite/genética , Plantas Medicinales/genética , Rhodiola/genética , Alelos , Humanos , Medicina Tradicional Tibetana , Polimorfismo Genético , TibetRESUMEN
Tumor necrosis factor receptor-associated factor 3 (TRAF3) is a crucial regulator that suppresses c-Jun N-terminal kinase and non-canonical nuclear factor-kB signaling, but facilitates type I interferon production. To determine TRAF3 function in innate immune responses among birds, particularly chicken, we cloned and characterized the chicken TRAF3 gene (chTRAF3) and detected its tissue expression profile in chicken. We also detected the differential expression of chTRAF3 and its downstream gene interferon-ß (IFN-ß) upon different stimuli in primary chicken embryo fibroblast cells. Two chTRAF3 gene products, chTRAF3-1 and chTRAF3-2, can be produced by alternative splicing. The full-length coding sequence of chTRAF3 (chTRAF3-1) was 1704 base pairs and encoded a protein of 567 amino acids with high identity to TRAF3 homologs from mammals and other birds. The deduced amino acid sequence showed typical characteristics of TRAFs, with a RING finger domain, 2 zf-TRAF motifs, and a MATH domain. Quantitative real-time polymerase chain reaction analysis revealed broad expression of chTRAF3 in all detected tissues, with abundant expression in the spleen, thymus, lung, and small intestine. Expression of chTRAF3 was significantly upregulated in a time- and concentration-dependent manner in chicken embryo fibroblast cells challenged with poly I:C or poly dA-dT. Furthermore, chTRAF3 and IFN-ß mRNA expression from chicken embryo fibroblast cells challenged with Newcastle disease virus F48E9 suffered intense suppression compared with Newcastle disease virus Mukteswar infection. Our results indicate that chTRAF3 plays important roles in defending against both RNA and DNA virus infection.
Asunto(s)
Proteínas Aviares/genética , Pollos/genética , Regulación de la Expresión Génica , Factor 3 Asociado a Receptor de TNF/genética , Empalme Alternativo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Proteínas Aviares/metabolismo , Secuencia de Bases , Embrión de Pollo , Pollos/inmunología , Pollos/metabolismo , Clonación Molecular , Evolución Molecular , Inmunidad Innata , Datos de Secuencia Molecular , Enfermedad de Newcastle/inmunología , Especificidad de Órganos , Alineación de Secuencia , Factor 3 Asociado a Receptor de TNF/metabolismoRESUMEN
Evodiamine, the major alkaloid component isolated from the fruit of dried, unripened Evodia rutaecarpa Bentham, affects the plasma levels of cholecystokinin and various biological events such as gastric emptying and gastrointestinal transit; these effects of evodiamine were previously investigated in male rats. In this study, we aimed to investigate the effects of evodiamine on average daily weight gain, rectal temperature, and expressions of genes involved in lipid metabolism in liver and adipose tissues. Evodiamine was added as a supplement, comprising 0.02, 0.04, and 0.06% of the diet fed to mice for 1, 2, 3, and 4 weeks. Results showed that average daily weight gain and rectal temperature decreased significantly over time in a dose-dependent manner. Evodiamine changed expressions of the peroxisome proliferator-activated receptor-g (PPARg) in mouse adipose and liver tissues in time- and dose-dependent manners. We found that evodiamine decreased mRNA expression of the sterol-regulatory element binding protein (SREBP-1c) and fatty acid synthase in adipose tissue. In addition, evodiamine increased expressions of hormone-sensitive lipase in both liver and adipose tissues. Interestingly, evodiamine increased the expression of triglyceride hydrolase only in adipose tissue. In conclusion, evodiamine could influence lipid metabolism through regulation of the expressions of its key genes, as well as reduce body heat and body weight.
Asunto(s)
Tejido Adiposo/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Lipogénesis/efectos de los fármacos , Lipólisis/genética , Hígado/metabolismo , Quinazolinas/administración & dosificación , Tejido Adiposo/metabolismo , Animales , Temperatura Corporal/efectos de los fármacos , Dieta , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Lipogénesis/genética , Lipólisis/efectos de los fármacos , Hígado/efectos de los fármacos , Masculino , Ratones , Quinazolinas/farmacología , Aumento de Peso/efectos de los fármacosRESUMEN
Evodiamine is the main active alkali of Wu Zhuyu, a widely distributed Chinese herb. It plays an important role in the regulation of body fat deposition. In this study, we aimed to investigate the effect of evodiamine administration on the expression of genes involved in lipid metabolism in the liver and adipose tissue. Fasted mice were subcutaneously injected with evodiamine (37 °C, 20 mg/kg), and the core body temperature change and expression levels of lipid metabolism-related genes were evaluated at baseline, 0.5, 1, and 2 h. We detected the mRNA expression of fatty acid synthesis enzyme (FAS), peroxisome proliferator-activated receptor gamma (PPAR-γ), sterol regulatory element binding protein 1c (SREBP-1c), triglyceride hydrolase (TGH), and hormone-sensitive lipase (HSL) by real-time PCR and analyzed their correlation with core body temperature. Our results showed that the core body temperature was reduced greater than 1 °C with evodiamine treatment at 1 and 2 h (P < 0.01). In mouse livers, SREBP-1c, HSL, and TGH mRNA expression was significantly increased, and they reached the highest levels 1 h after injection (P < 0.01). However, PPAR-γ mRNA expression was decreased and reached a significant level at 0.5 h (P < 0.01) and FAS mRNA expression was not significantly different; FAS and SREBP-1c mRNA expression were reduced and reached significant levels at 1 h (P < 0.01). Of note, other genes demonstrated opposite changes in adipose tissue, and HSL mRNA expression was significantly reduced at 0.5 h (P < 0.01). The decreasing core temperature had a significant negative correlation with the expression of TGH, HSL, FAS, and SREBP- 1c mRNA in the liver (P < 0.01), but had significant positive correlation with levels of FAS and SREBP-1c mRNA in adipose tissue (P < 0.01). In light of these results, the main mechanism of the regulation of body fat deposition by evodiamine is raising energy consumption through reducing body temperature and promoting fat decomposition.
Asunto(s)
Tejido Adiposo/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Metabolismo de los Lípidos/genética , Hígado/metabolismo , Quinazolinas/farmacología , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/enzimología , Animales , Temperatura Corporal/efectos de los fármacos , Temperatura Corporal/genética , Ácido Graso Sintasas/genética , Ácido Graso Sintasas/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Lipólisis/efectos de los fármacos , Lipólisis/genética , Hígado/efectos de los fármacos , Hígado/enzimología , Masculino , Ratones , PPAR gamma/genética , PPAR gamma/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismoRESUMEN
We investigated the effect of overexpression suppressor of cytokine signaling 2 (SOCS2) on lipolysis in swine primary adipocytes (pAd) induced by growth hormone (GH). We constructed pAd-SOCS2 adenoviral overexpression vectors to infect HEK293 cells for virus packaging and propagation. Cultured swine primary adipocytes were infected with virus particles; after 48 h the infected adipocytes were treated with 500 ng GH/mL in the growth medium. Lipometabolism-related gene expressions were detected at 0, 0.25, 0.5, 1, 2, and 4 h, by measuring mRNA and protein levels. The pAd-SOCS2 overexpression vector was successfully constructed and the concentration of titrated virus was 1.2 x 10(9) PFU/mL. We found that virus infection significantly increased SOCS2 mRNA and protein levels in swine primary adipocytes. Overexpression of SOCS2 significantly inhibited the increase in fatty acid synthase, adipose triglyceride lipase mRNA, and protein expression at 0.5 h. However, after 0.5 h, this inhibition was not significant. We concluded that overexpression of SOCS2 inhibited the increase in lipolysis induced by GH in swine primary adipocytes; this could provide a basis for studies of lipometabolism.