RESUMEN
Different morphologies have been detected in teleost macrophages. In this study, two macrophage cell lines were sub-cloned from a large yellow croaker head kidney cell line, LYCK. One type of sub-cloned cells was fusiform but the other was round, named LYC-FM and LYC-RM cells respectively, based on their morphologies. Both types showed the characteristics of macrophages, including expression of macrophage-specific marker genes, possession of phagocytic and bactericidal activities, and production of reactive oxygen species (ROS) and nitric oxide (NO). The transcription factor PU.1, crucial for the development of macrophages in mammals, was found to exist in two transcripts, PU.1a and PU.1b, in large yellow croaker, and constitutively expressed in LYC-FM and LYC-RM cells. The expression levels of PU.1a and PU.1b could be upregulated by recombinant large yellow croaker IFN-γ protein (rLcIFN-γ). Further studies showed that both PU.1a and PU.1b increased the expression of cathepsin S (CTSS) by binding to different E26-transformation-specific (Ets) motifs of the CTSS promoter. Additionally, we demonstrated that all three domains of PU.1a and PU.1b were essential for initiating CTSS expression by truncated mutation experiments. Our results therefore provide the first evidence that teleost PU.1 has a role in regulating the expression of CTSS.
Asunto(s)
Catepsinas/genética , Regulación de la Expresión Génica , Macrófagos/inmunología , Macrófagos/metabolismo , Perciformes/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismo , Animales , Línea Celular , Enfermedades de los Peces/genética , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/microbiología , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Fagocitosis , Regiones Promotoras Genéticas , Unión Proteica , Especies Reactivas de Oxígeno/metabolismoRESUMEN
An 8-week feeding trial was conducted to evaluate the effects of dietary xylanase supplementation on growth performance, digestive enzyme activity, intestinal morphology parameter, intestinal microbiome diversity, and carbohydrate metabolism for juvenile large yellow croaker (Larimichthys crocea). Four levels of xylanase were added to basal diets (0, 600, 1200, and 1800 U kg-1). The results indicated that fish fed the 1200 U kg-1 xylanase diet had higher weight gain than those fed the 0 and 600 U kg-1 xylanase diet. The highest intestinal folds and microvillous height were observed at fish fed the 1200 U kg-1 xylanase diet. High-throughput sequencing revealed that the majority of reads derived from the large yellow croaker digesta belonged to members of Proteobacteria followed by Chloroflex, Bacteroidetes, Spirochaetae, and Firmicute. Supplementation of xylanase in diets increased the relative abundance of Bacteroides and Gemmatimonadete. The higher hepatic glucokinase (GK) and glucose-6-phosphate dehydrogenase (G6PD) activities were observed in fish fed the xylanase supplementation diet. Accordingly, dietary xylanase supplementation upgraded the relative expressions of gk and g6pd genes in liver. In conclusion, optimum dietary xylanase supplementation (600-1200 U kg-1) could improve the growth performance, optimize the intestinal morphology structure and microbiota constitution, and enhance the ability of carbohydrate utilization of juvenile large yellow croaker.
Asunto(s)
Metabolismo de los Hidratos de Carbono/efectos de los fármacos , Suplementos Dietéticos , Endo-1,4-beta Xilanasas/farmacología , Mucosa Intestinal/efectos de los fármacos , Hígado/efectos de los fármacos , Perciformes , Alimentación Animal , Animales , Dieta/veterinaria , Glucoquinasa/genética , Glucoquinasa/metabolismo , Glucosafosfato Deshidrogenasa/genética , Glucosafosfato Deshidrogenasa/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Intestinos , Lipasa/metabolismo , Hígado/metabolismo , Pepsina A/metabolismo , Perciformes/sangre , Perciformes/genética , Perciformes/crecimiento & desarrollo , Perciformes/metabolismo , Triglicéridos/sangreRESUMEN
In this study, three nanos gene subtypes (Lcnanos1, Lcnanos2 and Lcnanos3) from Larimichthys crocea, were cloned and characterized. We determined the spatio-temporal expression patterns of each subtype in tissues as well as the cellular localization of mRNA in embryos. Results showed that deduced Nanos proteins have two main homology domains: N-terminal CCR4/NOT1 deadenylase interaction domain and highly conserved carboxy-terminal region bearing two conserved CCHC zinc-finger motifs. The expression levels of Lcnanos1 in testis were significantly higher than other tissues, followed by heart, brain, eye, and ovary. Nevertheless, both Lcnanos2 and Lcnanos3 were restrictedly expressed in testis and ovary, respectively. No signals of Lcnanos1 and Lcnanos2 expression were detected at any developmental stages during embryogenesis. On the contrary, the signals of Lcnanos3 were detected in all stages examined. Lcnanos3 transcripts were firstly localized to the distal end of cleavage furrow at the 2-cell stage. Subsequently, mounting positive signals started to appear in a small number of cells as the embryo developed to blastula stage and early-gastrula stage. As development proceeded, positive signals were found in the primitive gonadal ridge. These cells of Lcnanos3 positive signals implied the specification of the future PGCs at this stage. It also suggested that PGCs of croaker originate from four clusters of cells which inherit maternal germ plasm at blastula stage. Furthermore, we preliminarily analyzed the migration route of PGCs in embryos of L. crocea. In short, this study laid the foundation for studies on specification and development of germ cell from L. crocea during embryogenesis.
Asunto(s)
Desarrollo Embrionario/genética , Proteínas de Peces/genética , Óvulo/metabolismo , Perciformes/embriología , Perciformes/genética , Espermatozoides/metabolismo , Secuencia de Aminoácidos , Animales , Femenino , Regulación del Desarrollo de la Expresión Génica , Humanos , Masculino , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
This study was conducted to clone and functionally characterize a full-length cDNA encoding arachidonate 5-lipoxygenase (Alox5) from large yellow croaker (Larmichthys crocea) and investigate its gene expression in response to graded dietary ratio of linolenic acid (ALA) to linoleic acid (LNA) (0.03, 0.06, 0.45, 0.90 and 1.51). An isolated 2372bp cDNA clone of Alox5 contained an open reading frame spanning 2025bp encoding a protein with the ability to modify arachidonate acid (AA) to 5-hydroxyeicosatetraenoic (5-HETE). In the liver, the Alox5 mRNA expression levels significantly increased to the maximum when the dietary ALA/LNA increased from 0.03 to 0.06, and then significantly decreased with dietary ALA/LNA increased to 1.51 (P<0.05). In the kidney, the expression levels of Alox5 of fish fed diets with low dietary ALA/LNA (0.03-0.06) were significantly higher than those of fish fed diets with high dietary ALA/LNA (0.45-1.51) (P<0.05). The dual-luciferase reporter assays showed that the nuclear factor kappa B (NF-κB) could act on cognate cis-acting elements in the promoter of Alox5 and increased the transcription of Alox5. Results of the present study suggested that the expression of Alox5 is higher in croakers fed high concentrations of LNA compared to those fed high concentrations of ALA, which might be regulated by NF-κB and contribute to the inflammation process by catalyzing the dioxygenation of AA.
Asunto(s)
Araquidonato 5-Lipooxigenasa/genética , Dieta , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Ácido Linoleico/farmacología , Perciformes/genética , Ácido alfa-Linolénico/farmacología , Animales , Clonación Molecular , Células HEK293 , Humanos , Riñón/efectos de los fármacos , Riñón/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Filogenia , Alineación de Secuencia , Transcripción Genética/efectos de los fármacosRESUMEN
This study was conducted to elucidate the effects of oxidised dietary lipids and high-dose vitamin E (VE) on growth performance and immune responses of large yellow croaker. Juvenile fish (initial average body weight of 7·82 (sem 0·68) g) were fed diets containing either fresh fish oil (fresh diet, peroxide value (POV)=1·72 mEq/kg) or fish oil oxidised to varying degrees (oxidised diets, POV=28·29-104·21 mEq/kg), with or without supplementary 600 mg VE/kg diet, for 10 weeks in floating cages. Growth was significantly lower and feed intake (g/100 g body weight per d) was higher in fish fed the oxidised diet. Supplementation with VE increased the growth of fish fed the oxidised diets, but significantly decreased the growth of fish fed the fresh diet. Hepatosomatic index increased with increasing dietary POV and decreased with VE supplementation. Hepatic catalase activity, superoxide dismutase (SOD) activity and malondialdehyde content were significantly higher in fish fed the oxidised diets, and these values decreased significantly following VE supplementation. However, hepatic SOD activity was enhanced by VE supplementation in fish fed the fresh diet. Air-exposure mortality was significantly increased by dietary POV, and this effect was inhibited by VE supplementation. These results suggest that dietary oxidised fish oil could stimulate the activities of antioxidant defence enzymes in stressed large yellow croaker. High-dose VE supplementation can alleviate oxidative stress of large yellow croaker fed oxidised fish oil, but can exert deleterious effects on fish in the absence of oxidative stress.
Asunto(s)
Antioxidantes/farmacología , Dieta , Suplementos Dietéticos , Aceites de Pescado/farmacología , Estrés Oxidativo/efectos de los fármacos , Perciformes/crecimiento & desarrollo , Vitamina E/farmacología , Alimentación Animal , Animales , Antioxidantes/efectos adversos , Antioxidantes/metabolismo , Peso Corporal/efectos de los fármacos , Catalasa/metabolismo , Grasas de la Dieta/efectos adversos , Grasas de la Dieta/metabolismo , Grasas de la Dieta/farmacología , Ingestión de Alimentos/efectos de los fármacos , Ingestión de Energía/efectos de los fármacos , Aceites de Pescado/efectos adversos , Aceites de Pescado/metabolismo , Peroxidación de Lípido , Hígado/efectos de los fármacos , Hígado/enzimología , Hígado/metabolismo , Malondialdehído/metabolismo , Oxidación-Reducción , Superóxido Dismutasa/metabolismo , Vitamina E/efectos adversosRESUMEN
Comparative effects of different concentrations of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on immune responses of head-kidney macrophages isolated from large yellow croaker were studied in vitro. After exposing to serum-free medium for 1 day, cultured cells were incubated in medium supplemented with graded levels of EPA or DHA (0, 5, 25, 100, 200 and 1000 µM, respectively) in the form of fatty acid bovine serum albumin (FA-BSA) complex for 12 h, 24 h and 36 h, respectively. Control samples were incubated in the absence of EPA or DHA (2% bovine serum albumin, BSA). Following stimulation, cell viability, lipid peroxidation, secretary phopholipase A2 (sPLA2) and prostaglandin E2 (PGE2) production as well as some immune parameters including phagocytosis, respiratory burst activity and interleukin 1ß (IL-1ß) production were determined. Results showed that EPA and DHA affected cell viability in dose-dependent and time-dependent manners. In particular, cell viability was significantly decreased after 24 h and 36 h incubation with 1000 µM EPA or DHA (P < 0.05). Higher levels of EPA (200 and 1000 µM) caused a significant increase in the production of malondialdehyde (MDA) (P < 0.05), while DHA did not significantly affect the MDA production. EPA significantly increased the intracellular superoxide anion synthesis which, on the contrary, was significantly reduced by DHA. Phagocytosis percentage (PP) values were significantly higher in treatments with 5 µM DHA (P < 0.05), but significantly decreased by 200 and 1000 µM EPA and DHA compared to the control group (P < 0.05). Decreased PGE2 production was produced by cells treated with relatively low doses of EPA or DHA. When high levels of stimulants (1000 µM EPA or DHA) were used, PGE2 levels were elevated and reached a significant level (P < 0.05). Both EPA and DHA significantly inhibited the production of sPLA2, where DHA exerted the more potent inhibitory effects than EPA. No pronounced effect was observed on IL-1ß production among all the treatments, and IL-1ß level in cell culture supernatant was fairly low (only approximately 6 pg/ml). Those findings suggested that EPA and DHA could influence the immunity and physiological conditions of macrophages from head kidney of large yellow croaker in vitro.