Molecular cloning of Japanese eel Anguilla japonica TNF-α and characterization of its expression in response to LPS, poly I:C and Aeromonas hydrophila infection

As a potent pleiotropic cytokine, tumor necrosis factor-alpha （TNF-ct） plays an important role in innate immune responses. The cDNA sequence and genomic structure of the TNF-α gene （AjTNF-c0 in the Japanese eel （Anguilla japonica） were identified and characterized. The full-length AjTNF-α cDNA was 1 546 bp, including a 5＇-untranslated region （UTR） of 13 bp, a 3＇-UTR of 879 bp and an open reading frame of 654 bp encoding a protein of 218 amino acids. The full-length genomic sequence of AjTNF-ct was 2 392 bp and included four exons and three introns. The putative AjTNF-α protein contained TNF family signature motifs, including a protease cleavage site, a transmembrane domain and two conserved cysteine residues. Quantitative real-time reverse transcription PCR analysis revealed AjTNF-α expression in a wide range of tissues, with predominant expression in blood and liver. Lower levels of expression were seen in spleen, gills, kidney, intestine, heart, and skin, with very low levels in muscle. The modulation of AjTNF-ct expression after injection of eels with lipopolysaccharide （LPS）, the viral mimic, poly I：C, or Aeromonas hydrophila was assessed in blood, liver, and kidney. In blood, TNF-α mRNA levels increased rapidly and then rapidly decreased after stimulation with LPS, poly I：C or A. hydrophila. However, the response to LPS and A. hydrophila peaked at 6 h while for poly I：C the peak was at 12 h. In liver, after injection with A. hydrophila, an up- and down-regulation of AjTNF-ct expression occurred twice, peaking at 6 h and 24 h, respectively. No remarkable increase of AjTNF-α expression appeared in liver until 72 h after LPS or poly I：C treatment. In kidney, ApiNF-α expression increased significantly only at 72 h post-stimulation with LPS orA. hydrophila. Our results suggest that AjTNF-α plays an important role in fish in the defense against viral and bacterial infection.

A Sensitive Reversed-Phase High-Performance Liquid Chromatography Method for the Quantitative Determination of Milk Xanthine Oxidase Activity

A new reversed-phase high performance liquid chromatography method was developed to quantitate the activity of xanthine oxidase involved in milk fat globule membrane with xanthine as the substrate and the separation of product (uric acid). The increment of uric acid in the reaction system was used to calculate the total activity of XO. The optimized assay conditions, linearity of detection, recovery of uric acid and chromatogram were developed in text, indicating this method is simple, rapid and efficient. It is an alternative potential method for the determination of the activity of XO in milk.

Molecular characterization,expression patterns,and subcellular localization of a classical and a novel nonclassical MHC class Iαmolecules from Japanese eel Anguilla japonica

Major histocompatibility complex(MHC)molecules play a critical role in the immune response of vertebrate animals by presenting foreign antigens to T lymphocytes.In this study,we first cloned and identified a classical and a novel nonclassical MHC Iαmolecules from Japanese eel(Anguilla japonica),named as AjMHC I-UBA and AjMHC I-L,respectively.The full-length cDNA of AjMHC I-UBA contains an open reading frame(ORF)of 1047 bp encoding a predicted protein of 348 amino acids,while AjMHC I-L 1089 bp encodes 362 amino acids.The multiple alignment of the amino acid sequence showed that AjMHC I-UBA and AjMHC I-L consist of an N-terminal MHC I superfamily domain withinα1 andα2 helices,an IgC-MHC Iα3 domain,and a transmembrane region close to the C-terminal,which are similar to other fish and mammal species.Molecular polymorphism analysis showed that eight different major alleles of AjMHC I-UBA,named as AjMHC I-UBA*0101~1001,were identified from six Japanese eel individuals.Furthermore,a distinguishing signature of the nonclassical L-lineage genes specific motif“HINMTL”,including an N-glycosylation site(NXS/T),was present in theα3 domain of AjMHC I-L sequences.Although the predicted three-dimensional structures of AjMHC I-UBA and AjMHC I-L are similar to that of human MHC Iα,phylogenetic analysis showed that these two protein molecules belong to classical MHC I UBA gene of U-lineage and nonclassical MHC I gene of L-lineage,respectively.Quantitative real-time polymerase chain reaction(qRT-PCR)analysis revealed that the highest expression of AjMHC I-UBA and AjMHC I-L was found in the intestine and the expression level of AjMHC I-UBA in all of the examined tissues was significantly higher than that of AjMHC I-L.The expressions of AjMHC I-UBA and AjMHC I-L in liver,kidney and spleen were significantly induced following injection with the viral mimic poly I:C,LPS,and Aeromonas hydrophila infection.In vitro,the AjMHC I-UBA and AjMHC I-L transcripts of Japanese eel liver cells were significantly enhanced by the treatment of poly I:C or the stimulation of the high concentration of A.hydrophila.Subcellular localization showed that under natural conditions,AjMHC I-UBA and AjMHC I-L were uniformly distributed in the cytoplasm,and aggregated into spots or flakes in the cytoplasm after the stimulation of poly I:C or LPS.These results collectively suggested that AjMHC I-UBA and AjMHC I-L are important components possibly involved in Japanese eel defense against viral and bacterial infection.Taken together,these findings provide valuable insight into the immune function of MHC class I in the immune system of teleost.