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Ghrelin plays important roles, such as regulating growth hormone release and energy metabolism, but little is known about its developmental changes in the proventriculi of chicken embryos. This study was designed to elucidate the distributions and developmental changes of ghrelin and ghrelin-O-acyltransferase (GOAT) expression in broiler embryos using qRT-PCR and immunohistochemistry. Our results demonstrated the following: (1) on E18, ghrelin and GOAT are ubiquitously expressed in every tissue examined. The expression level of ghrelin mRNA was the highest in the proventriculus, reaching a level that was 50-fold higher than that in the hypothalamus, while GOAT mRNA expression was low in the proventriculus and it was only 67.6% as high as that of hypothalamus; (2) ghrelin and GOAT mRNA expression were detected in the proventriculus on E9, but only at 1.9% and 1.7% of the level expressed on E18, respectively, and their expression levels increased rapidly from E18 to E21. There was similar developmental pattern in the ghrelin and GOAT mRNA expression; and (3) ghrelin-immunopositive cells were first detected in the proventriculus on E15, were located only in the compound tubular glands of the proventriculus, and were of the closed-cell type. The density of ghrelin-immunopositive cells increased significantly from E15 to E21. These results suggest that ghrelin may be an important regulating factor that plays a vital role during the development of chicken embryos. [ABSTRACT FROM AUTHOR]

Abstract: As a crucial component in TLR/IL-1R signaling pathways, IRAK-4 plays a central role in innate and adaptive immunity. In the present study, the cDNA of IRAK-4 was cloned for the first time from half-smooth tongue sole (Cynoglossus semilaevis). The full-length cDNA of csIRAK-4 was 2149bp and contained a 168bp 5′ UTR, a 580bp 3′ UTR and a 1401bp CDS. The predicted protein sequence of csIRAK-4 had two typical domains, a death domain (DD) at the N terminus and a serine/threonine/tyrosine protein kinase domain (STYKc) at the C terminus. RT-PCR showed that csIRAK-4 mRNA was detected in all tested tissues, especially in immune-related organs, gonads and brain. After injected with inactivated Vibrio anguillarum, the expressions of csIRAK-4 were up-regulated significantly (P <0.05) in spleen and head kidney. During development, csIRAK-4 was expressed at all selected stages and low-level expression was detected at metamorphosis. Taken together, the present study indicated that csIRAK-4 played a crucial role in immune responses and might be involved in the process of development. [Copyright &y& Elsevier]

Abstract: Toll-like receptors (TLRs) are considered as key sensors to trigger the host''s innate immune system and adaptive immune responses by recognizing various PAMPs and initiating signal transduction. TLR9, as a member of TLR family, mediates the recognition of unmethylated CpG dinucleotide motifs commonly found in both bacterial and viral genomes. In the current study, the TLR9 gene was isolated from one of flatfish species, half-smooth tongue sole (Cynoglossus semilaevis). In the 4588 bp genomic sequence, three exons, two introns, and 5′ UTR of 23 bp and 3′ UTR of 342 bp were identified. Putative amino acid sequence was 1062 residues long, including a typical conserved cytosolic Toll/interleukin-1 receptor (TIR) domain, 14 leucine-rich repeat (LRR) motifs, with greater than 60% identity to gilthead sea bream Sparus aurata and Japanese flounder Paralichthys olivaceus orthologs. Quantitative RT-PCR analysis indicated a broad expression of csTLR9, especially in spleen and gonads. No statistically significant changes were observed for csTLR9 mRNA levels in spleen and head kidney after inactive Vibrio anguillarum immunisation. In C. semilaevis ontogeny, the expression of csTLR9 appeared to be developmentally regulated. The presence of maternal TLR9 mRNA and the dramatic decrease of TLR9 expression at metamorphic stage indicated TLR9 might be involved in C. semilaevis development. Comparing sequence and expression profile of csTLR9 with mammalian and other piscine TLR9s suggested that the main function of TLR9 might be conserved across vertebrates, although species-specific features were present. [Copyright &y& Elsevier]

Abstract: To investigate the biological effect of mdm2 in human colorectal adenocarcinoma LoVo cells, three mdm2siRNA constructions were recombinated and transient transfected into human colorectal adenocarcinoma LoVo cells with low differentiation character in vitro. The results showed that mdm2siRNA3 reduced mRNA level of mdm2 and protein level of mdm2, leading to proliferation inhibition on LoVo cells, and reduced tumor growth in nude mice. It was found that depletion of MDM2 in this pattern promoted apoptosis of LoVo cells and Cisplatin (DDP) treated in the mdm2siRNA3 transfected cell population would result in a substantial decrease by MTT colorimetry. Decreasing the MDM2 protein level in LoVo cells by RNAi could significantly inhibit tumor growth both in vitro and in vivo, which indicated that mdm2 gene played a definite role in the development and aggressiveness of human colon carcinoma. It also could be a therapeutic target in colorectal carcinoma. The synergistic activation of RNAi and cell toxicity agents indicated that the combination of chemotherapy and gene therapy will be a promising approach in the future. [Copyright &y& Elsevier]

Layer chickens were artificially challenged with infectious bursal disease virus (IBDV), and the kinetics of IFN-λ and antiviral genes in the bursa were explored using quantitative real-time PCR. Data showed that after the chickens were infected with IBDV, the virus load in the bursa of the Fabricius peaked at 96 h and gradually decreased. The relative mRNA expression levels of IFN-λ and antiviral genes (zinc-finger antiviral protein ZAP , interferon alpha-inducible protein 6 IFI6 , laboratory of genetics and physiology 2 LGP2 , virus inhibitory protein Viperin , and Mx) of the infected group dramatically increased at 24–168 h compared with those of the negative-infected group. Furthermore, the ZAP mRNA expression peaked at 24 h (3.97-fold). The Viperin mRNA transcript level was highest at 48 h (384.60-fold). The mRNA expression levels of IFI6 (96.31-fold), LGP2 (18.29-fold), and Mx (88.85-fold) peaked at 72 h, and that of IFN-λ was most remarkable at 96 h (2978.81-fold). Furthermore, the ZAP change rule was significantly positively correlated with the change rule of the IBDV load. The mRNA expression levels of IFN-λ and antiviral genes (ZAP , IFI6 , LGP2 , Viperin , and Mx) increased as the virus expression increased and then decreased. These results further corroborated that the IBDV infection seriously interfered with the chicken's innate immune response. • The expression levels of IFN-λ , ZAP , IFI6 , LGP2 , Viperin , and Mx were remarkably increased along with different IBDV loads. • Expression of IFN-λ , ZAP , IFI6 , LGP2 , Viperin , and Mx mRNA peaked at different time points, some early on, some later. • The kinetics of ZAP relative expression level had a significant positive correlation with the change in IBDV load. [ABSTRACT FROM AUTHOR]

Abstract: Monokine induced by IFN-γ (Mig) is a member of CXC-chemokines and recruits T-lymphocytes to activate the immune response. In recent years, it has raised much interest in the areas of autoimmune disease and allograft rejection, as the production of recombinant human Mig (rHuMig) would be of considerable significance for both research and potential clinical use. Here we report the expression, preparation and characterization of non-tagged recombinant human Mig (rHuMig) using a prokaryotic expression system. Following expression in Escherichia coli (E. coli) BL21, the 103 amino acid residue of rHuMig was purified from bacteria inclusion bodies with a one-step S-Sepharose cation exchange chromatography. The product was immunologically characterized via Western blot and its purity was determined via SDS–PAGE and silver staining to be above 99%, with an endotoxin level <0.5EU/μg via a chemotaxis assay, rHuMig demonstrated chemotactic activity on mouse spleen lymphocytes with an ED50 of 15ng/mL. Additionally, using a proliferation assay, rHuMig significantly inhibited proliferation of the human bladder cell line T24. In vivo experiments revealed that rHuMig could inhibit mouse bone marrow mononuclear cells cycling into the S-phase and reduced intestinal cell proliferation. Our results demonstrate that rHuMig is fully functional in the mouse model. [Copyright &y& Elsevier]

Abstract: Background: The study was performed to observe the systemic release and kinetics of high mobility group box-1 protein (HMGB1) in burned patients. Methods: 106 patients were included, and they were divided into three groups with different burn sizes: group I, group II and group III. Healthy volunteers served as normal controls (n =25). The peripheral blood samples were collected on postburn days (PBD) 1, 3, 7, 14, and 21. The blood samples were used to detect levels of HMGB1 in plasma by ELISA kits for human. Gene expression of HMGB1 in peripheral blood mononuclear cells was assessed by real-time quantitative PCR taking GAPDH as the internal standard. Results: The levels of HMGB1 were significantly elevated on PBD 1–21 in patients with various burn sizes compared with normal controls, and there were obvious differences between group I and group III. The HMGB1 levels were significantly higher in septic patients than those without sepsis on PBD 7–21. Among septic patients, the HMGB1 levels in the survival group were markedly lower than those with fatal outcome on PBD 3–21. Conclusions: Extensive burn injury could result in significantly increased HMGB1 levels, which appears to be associated with the development of sepsis and fatal outcome of major burns. [Copyright &y& Elsevier]

Abstract: Regenerating gene (Reg) IV is a newly discovered member of the regenerating gene family belonging to the calcium (C-type) dependent lectin superfamily. Reg IV is highly expressed in the gastrointestinal tract and markedly up-regulated in colon adenocarcinoma, pancreatic cancer, gastric adenocarcinoma, and inflammatory bowel disease. However, the physiological and pathological functions of Reg IV are largely unknown, partly due to the limited access of the bioactive protein. We report here the first expression and purification of Reg IV proteins using a prokaryotic system. Human Reg IV was expressed in Escherichia coli as an insoluble protein which was identified in the fraction of inclusion body after ultrasonication of the bacteria. After the protein aggregate was solubilized by guanidine–HCl, it was refolded by sucrose and arginine-assisted procedures and purified using cation-exchange chromatography. The protein identity and purity of the final preparation were confirmed by analysis of the protein mass and immune specificity in SDS–PAGE, Western blotting, and HPLC assay. The biological activity of the protein was determined by the HCT116 and HT29 cell proliferation assays. The highly purified bioactive human Reg IV should aid in further characterization of its physiological and pathological functions. [Copyright &y& Elsevier]

Abstract: Chemerin is a novel chemokine that binds to the G protein-coupled receptor (GPCR) ChemR23, also known as chemokine-like receptor 1 (CMKLR1). It is secreted as a precursor and executes pro-inflammatory functions when the last six amino acids are removed from its C-terminus by serine proteases. After maturation, Chemerin attracts dendritic cells and macrophages through binding to ChemR23. We report a new method for expression and purification of mature recombinant human Chemerin (rhChemerin) using a prokaryotic system. After being expressed in bacteria, rhChemerin in inclusion bodies was denatured using 6M guanidine chloride. Soluble rhChemerin was prepared by the protein-specific renaturation solution under defined conditions. It was subsequently purified using ion-exchange columns to more than 95% purity with endotoxin level <1.0 EU/μg. We further demonstrated its biological activities for attracting migration of human dendritic cells and murine macrophages in vitro using established chemotaxis assays. [Copyright &y& Elsevier]