cDNA cloning, characterization and expression analysis of peroxiredoxin 5 gene in the ridgetail white prawn Exopalaemon carinicauda

Peroxiredoxin is a superfamily of antioxidative proteins that play important roles in protecting organisms against the toxicity of reactive oxygen species. In this study, a full-length of peroxiredoxin 5 (designated EcPrx5) cDNA was cloned from the ridgetail white prawn Exopalaemon carinicauda by using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of the EcPrx5 was of 827 bp, containing a 5′ untranslated region (UTR) of 14 bp, a 3′ UTR of 228 bp with a poly (A) tail, and an open reading frame of 585 bp encoding a polypeptide of 194 amino acids with the predicted molecular weight of 20.83 kDa and estimated isoelectric point of 7.62. BLAST analysis revealed that amino acids of EcPrx5 shared 89, 68, 66, 65, 53 and 51 % identity with that of Macrobrachium rosenbergii, Megachile rotundata, Harpegnathos saltator, Acromyrmex echinatior, Danio rerio, and Homo sapiens counterparts, respectively. The conserved Prx domain and the signature of peroxiredoxin catalytic center identified in EcPrx5 suggested that EcPrx5 belonged to the atypical 2-Cys Prx subgroup. Real time quantitative RT-PCR analysis indicated that EcPrx5 could be detected in all the tested tissues with highest expression level in hepatopancreas. As time progressed, the expression level of EcPrx5 both in hemocytes and hepatopancreas increased in the first 6 h after Vibrio anguillarum and white spot syndrome virus challenge, and showed different expression profiles. The results indicated that EcPrx5 involved in immune response against bacterial and viral infection in E. carinicauda.     

Relationship between TRAF6 and deterioration of HCC: an immunohistochemical and in vitro study

Objective

To explore the relationship between tumor necrosis factor receptor-associated factor 6 (TRAF6) and the clinicopathological features in HCC as well as its biological function.

Methods

Totally, 412 liver tissues were collected, including 171 hepatocellular carcinoma (HCC) and their corresponding non-tumor tissues, 37 cirrhosis and 33 normal liver tissues. The expression of TRAF6 was assessed by immunohistochemistry. Then, analysis of the correlations between TRAF6 expression and clinicopathological parameters in HCC was conducted. Furtherer, in vitro experiments on HepG2 and Hep3B cells were performed to validate the biological function of TRAF6 on HCC cells. TRAF6 siRNA was transfected into HepG2 and Hep3B cell lines and TRAF6 expression was evaluated with RT-qPCR and western blot. The assays of cell viability, proliferation, apoptosis and caspase-3/7 activity were carried out to investigate the effects of TRAF6 on HCC cells with RNA interference. Cell viability was assessed with Cell Titer-Blue kit. Cell proliferation was tested with MTS kit. Cell apoptosis was checked through morphologic detection with fluorescence microscope, as well as caspase-3/7 activity was measured with fluorogenic substrate detection.

Results

The positive expression rate of TRAF6 protein was 49.7 % in HCC, significantly higher than that of normal liver (12.1 %), cirrhosis (21.6 %) and adjacent non-cancerous tissues (36.3 %, all P < 0.05). Upregulated TRAF6 was detected in groups with metastasis (Z = ?2.058, P = 0.04) and with low micro-vessel density (MVD) expression (Z = ?2.813, P = 0.005). Spearman correlation analysis further showed that the expression of TRAF6 was positively correlated with distant metastasis (r = 0.158, P = 0.039) and negatively associated with MVD (r = ?0.249, P = 0.004). Besides, knock-down of TRAF6 mRNA in HCC cell lines HepG2 and Hep3B both resulted in cell viability and proliferation inhibition, also cell apoptosis induction and caspase-3/7 activity activation.

Conclusions

TRAF6 may contribute to metastasis and deterioration of the HCC via influencing cell growth and apoptosis. Thus, TRAF6 might become a predictive and therapeutic biomarker for HCC.

     

Genomic organization, single nucleotide polymorphism and functional characterization of natural killer enhancing factor (NKEF-A) in Miichthys miiuy

Peroxiredoxin (Prx) play vital parts in oxidative stress belonging to a cellular antioxidant protein family. Natural killer enhancing factor (NKEF) is a member of the Prx family, which is newly defined. In addition to antioxidant activity, NKEF also can protect DNA from oxidative damage. In order to study immune defense mechanism of NKEF in teleost, NKEF-A gene of miiuy croaker (Miichthys miiuy) was cloned and characterized. The genomic organization containing one non-coding exon, five coding exons and five introns, inclouding one intron located in 5′-terminal untranslated region. The full-length cDNA was 1235 bp, consisting of a 597 bp open reading frame coding for a protein of 198 amino acids. Sequence comparison showed that the deduced amino acid sequence of miiuy croaker NKEF-A had 71.4–90.3 % identity with those of mammal and teleost. Five single nucleotide polymorphisms were detected by direct sequencing of eight samples from three different populations. Phylogenetic analysis revealed that miiuy croaker NKEF-A forms a cluster with other known teleost and mammalian NKEF-As. NKEF-A gene was constitutively expressed in ten examined tissues, and expression level was up-regulated in liver, spleen and kidney after challenge with Vibrio anguillarum. Finally, the NKEF-A was constructed and expressed in Escherichia coli. Then purified recombinant pET-NKEF protein was used to produce the polyclonal antibody and the polyclonal antibody against NKEF-A was tested by Western blot analysis. These results indicate that NKEF may be involved in immune responses as well as homeostatic processes in miiuy croaker.     

Upregulation of tumor necrosis factor receptor-associated factor 6 correlated with synovitis severity in rheumatoid arthritis

Introduction

Rheumatoid arthritis (RA) is a chronic inflammatory disease leading to joint destruction and disability. Focal bone erosion is due to excess bone resorption of osteoclasts. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is one of the critical mediators both in inflammatory signal pathway and differentiation and resorption activity of osteoclasts. Here we aimed to investigate TRAF6 expression in RA synovium and its correlation with histological synovitis severity and radiological joint destruction in RA.

Methods

Synovitis score was determined in needle biopsied synovium from 44 patients with active RA. Synovium from nine patients with osteoarthritis (OA) and seven with orthopedic arthropathies (Orth.A) were enrolled as "less inflamed" disease controls. Serial sections were stained immunohistochemically for TRAF6 as well as CD68 (macrophage), CD3 (T cell), CD20 (B cell), CD38 (plasmocyte), CD79a (B lineage cells from pre-B cell to plasmocyte stage), and CD34 (endothelial cell). Double immunofluorescence staining of TRAF6 and CD68 were tested. Densities of positive staining cells were determined and correlated with histological disease activity (synovitis score) and radiographic joint destruction (Sharp score).

Results

TRAF6 expression was found in the intimal and subintimal area of RA synovium, with intense staining found in the endochylema and nucleus of intimal synoviocytes and subintimal inflammatory cells. Double immunofluorescence staining showed TRAF6 was expressed in most of the intimal cells and obviously expressed in CD68+ cells and some other CD68- cells in subintimal area. Synovial TRAF6 was significantly over-expressed in the RA group compared with the OA and Orth.A group (2.53 ± 0.94 vs. 0.72 ± 0.44 and 0.71 ± 0.49, P < 0.0001). Synovial TRAF6 expression in RA correlated significantly with synovitis score (r = 0.412, P = 0.006), as well as the inflammatory cell infiltration (r = 0.367, P = 0.014). Significant correlation was detected between synovial TRAF6 expression and intimal CD68+ cells, as well as the cell density of subintimal CD68+ cells, CD3+ cells, CD20+ cells, CD38+ cells, and CD79a+ cells (all P < 0.05).

Conclusions

Elevated synovial TRAF6 expression correlated with synovitis severity and CD68+ cell density in RA. It is, therefore, hypothesized that synovial TRAF6 is involved in the pathogenesis of synovial inflammation and osteoclast differentiation in RA.     

Gene structure and spatio-temporal expression of chicken LPIN2

LPIN2 is one of the members of the Lipin family, which acts as a phosphatidate phosphatase enzyme. In this study, we identified the cDNA sequence and exonic variants of chicken LPIN2, and evaluated its spatio-temporal expression patterns. It indicated that chicken LPIN2 cDNA contained a 2,664-bp open reading frame flanked by a 176-bp 5′ untranslated region and a 429-bp 3′ untranslated region, predicted encoding one protein of 886 amino acids. Fourteen variants (three missense mutations) were detected from the coding region of chicken LPIN2. W265L was predicted to affect the gene function (p < 0.01) and eight synonymous mutations were predicted to affect the binding sites of SR proteins, which suggested the important functions of these variants. Real-time quantitative PCR revealed that LPIN2 in two genotypic chickens (LD and HB chickens, with difference in growth rate) presented similar tissue expression patterns, which was liver and ovary enriched with low abundance in skeleton muscles. Chicken LPIN2 exhibited tissue-specific temporal-expression patterns during postnatal development (0–16 weeks). Chicken cutaneous LPIN2 was in steady-state mRNA levels during postnatal development; chicken LPIN2 mRNA in pectoralis major had a prominent level at 0 week-old, then dropped dramatically at 4 week-old and maintained a relatively low level through 4–16 weeks; while chicken hepatic LPIN2 had a relatively high expression at 0 week-old, with a relatively low level through 4–12 weeks and a slight increase at 16 week-old. The studies about the basic gene features of chicken LPIN2 would lay the foundation for further exploring its biological function.