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Hu Y.-L.,Zhejiang University | Hu Y.-L.,Key Laboratory for Cell and Gene Engineering of Zhejiang Province | Hu Y.-L.,Key Laboratory of Animal Epidemic Etiology and Immunology Prevention | Xiang L.-X.,Zhejiang University | And 5 more authors.
Molecular Immunology

It was generally believed that teleost fish possess only few immunoglobulin isotypes such as IgM and IgD. The newly discovered IgZ/IgZ-like molecules in zebrafish and several other fish species greatly enriched our knowledge of immunoglobulin family in lower vertebrates. In the present study, we report a second IgZ-like isotype in zebrafish, which was designated as IgZ-2, adding a novel member to the immunoglobulin family in teleost fish. The IgZ-2 heavy chain encoding gene exhibited 76.5% nucleotide sequence identity to the previously reported IgZ. The putative IgZ-2 protein consists of four constant regions in its extracellular region, a transmembrane domain, and a short cytoplasmic tail. A number of conserved domains or residues such as the four Ig domains, cysteines required for Ig fold, hydrophobic and hydrophilic residues consistent with the CART domain, and Thr, Ser, Tyr residues known to be essential for association with the B cell co-receptors CD79A/B, were identified. Phylogenetic analysis showed that IgZ-2 grouped with IgZ and other known teleost IgZ-like sequences. The IgZ-2 transcripts were widely expressed in immune-related tissues, and could be significantly up-regulated by in vivo stimulation with LPS in various tissues including head kidney, spleen, intestine, skin, and gill. However, hardly IgZ-2 transcripts were detected during embryonic development until 2 weeks after fertilization. Double immunofluorescence staining showed that IgZ-2 and IgM were co-localized on B cell surfaces. Flow cytometric analysis showed the percentage of IgZ-2-positive cells could be dramatically up-regulated by interleukin-4 (IL-4), a cytokine known to activate the proliferation and differentiation of B cells. These observations indicated that the newly cloned IgZ-2 could be a novel B cell receptor. Our results will add new insights into the immunoglobulin class diversity of teleost fish, and also help us understand better the evolutionary history of adaptive immunity from fish to mammals as a whole. © 2010 Elsevier Ltd. All rights reserved. Source

Dong X.,Shaoxing University | Pan R.,Zhejiang University | Pan R.,Key Laboratory for Cell and Gene Engineering of Zhejiang Province | Zhang H.,Shaoxing University | And 7 more authors.

The multi-potentiality of mesenchymal stem cells makes them excellent options for future tissue engineering and clinical therapy, including liver injury. In this study, we investigated the effects of valproic acid (VPA), a direct inhibitor of histone deacetylase (HDAC), on the hepatic differentiation of human bone marrow mesenchymal stem cells (BMMSCs). The cells were found to differentiate into a more homogeneous hepatocyte-like population when pretreated with 5 mM VPA for 72 h. The expression of liver-specific markers was significantly upregulated in the VPA-treated group at the mRNA and protein levels. VPA treatment also significantly enhanced the hepatic functions of the differentiated cells, including glycogen storage, cytochrome P450 activity, AFP and ALB synthesis, and urea production. Further analysis showed that treatment with 5 mM of VPA for 72 h greatly improved the histones H3 and H4 acetylation. These results demonstrated that VPA could considerably improve the hepatic differentiation of human BMMSCs, probably because the chromatin-acetylated state changes upon VPA treatment through its HDAC inhibitory effect. Thus, this study provides a direct research model for producing human hepatocytes for clinical purposes. © 2013 Dong et al. Source

Shao T.,Zhejiang University | Shao T.,Key Laboratory for Cell and Gene Engineering of Zhejiang Province | Zhu L.-Y.,National University of Defense Technology | Nie L.,Zhejiang University | And 9 more authors.
Developmental and Comparative Immunology

Dendritic cells (DCs) are among the most important professional antigen-presenting cells (APCs) that participate in various biological activities in mammals. However, evidence of the existence of DCs in teleost fish and other lower vertebrates remains limited. In this study, phenotypic and functional characteristics of teleost DCs were described in a zebrafish model. An improved method to efficiently enrich DCs was established. Immunofluorescence staining revealed that the surface phenotypic hallmarks of mammalian DCs, including MHC-II, CD80/86, CD83, and CD209, were distributed on the surfaces of zebrafish DCs (DrDCs). Functional analysis results showed that DrDCs could initiate antigen-specific CD4+ T cell activation, in which MHC-II, CD80/86, CD83, and CD209 are implicated. Hence, teleost DCs exhibit conserved immunophenotypes and functions similar to those of their mammalian counterparts. Our findings contributed to the current understanding of the evolutionary history of DCs and the DC-regulatory mechanisms of adaptive immunity. © 2014 Elsevier Ltd. Source

Xu X.-G.,Zhejiang University | Xu X.-G.,Key Laboratory for Cell and Gene Engineering of Zhejiang Province | Hu J.-F.,Zhejiang University | Hu J.-F.,Key Laboratory for Cell and Gene Engineering of Zhejiang Province | And 11 more authors.
Journal of Immunology

TIM-1 and TIM-4 proteins have become increasingly attractive for their critical functions in immune modulation, particularly in CD4+ Th2 cell activation. Thus, these proteins were hypothesized to regulate adaptive humoral immunity. However, further evidence is needed to validate this hypothesis. This study describes the molecular and functional characteristics of TIM-1 and TIM-4 homologs from a zebrafish (Danio rerio) model (D. rerio TIM [DrTIM]-1 and DrTIM-4). DrTIM-1 and DrTIM-4 were predominantly expressed in CD4+ T cells and MHC class II+ APCs under the induction of Ag stimulation. Blockade or knockdown of both DrTIM-1 and DrTIM-4 significantly decreased Ag-specific CD4+ T cell activation, B cell proliferation, Ab production, and vaccinated immunoprotection against bacterial infection. This result suggests that DrTIM-1 and DrTIM-4 serve as costimulatory molecules required for the full activation of adaptive humoral immunity. DrTIM-1 was detected to be a trafficking protein located in the cytoplasm of CD4+ T cells. It can translocate onto the cell surface under stimulation by TIM-4-expressing APCs, which might be a precise regulatory strategy for CD4+ T cells to avoid self-activation before APCs stimulation. Furthermore, a unique alternatively spliced soluble DrTIM-4 variant was identified to exert a negative regulatory effect on the proliferation of CD4+ T cells. The above findings highlight a novel costimulatory mechanism underlying adaptive immunity. This study enriches the current knowledge on TIM-mediated immunity and provides a cross-species understanding of the evolutionary history of costimulatory systems throughout vertebrate evolution. Copyright © 2016 by The American Association of Immunologists, Inc. Source

Dong W.-R.,Zhejiang University | Dong W.-R.,Key Laboratory for Cell and Gene Engineering of Zhejiang Province | Sun C.-C.,Zhejiang University | Sun C.-C.,Key Laboratory for Cell and Gene Engineering of Zhejiang Province | And 9 more authors.
BMC Microbiology

Background: In an effort to reconstitute the NAD+ synthetic pathway in Escherichia coli (E. coli), we produced a set of gene knockout mutants with deficiencies in previously well-defined NAD+ de novo and salvage pathways. Unexpectedly, the mutant deficient in NAD+ de novo and salvage pathway I could grow in M9/nicotinamide medium, which was contradictory to the proposed classic NAD+ metabolism of E. coli. Such E. coli mutagenesis assay suggested the presence of an undefined machinery to feed nicotinamide into the NAD+ biosynthesis. We wanted to verify whether xanthosine phophorylase (xapA) contributed to a new NAD+ salvage pathway from nicotinamide. Results: Additional knockout of xapA further slowed down the bacterial growth in M9/nicotinamide medium, whereas the complementation of xapA restored the growth phenotype. To further validate the new function of xapA, we cloned and expressed E. coli xapA as a recombinant soluble protein. Biochemical assay confirmed that xapA was capable of using nicotinamide as a substrate for nicotinamide riboside formation. Conclusions: Both the genetic and biochemical evidences indicated that xapA could convert nicotinamide to nicotinamide riboside in E. coli, albeit with relatively weak activity, indicating that xapA may contribute to a second NAD+ salvage pathway from nicotinamide. We speculate that this xapA-mediated NAD + salvage pathway might be significant in some bacteria lacking NAD+ de novo and NAD+ salvage pathway I or II, to not only use nicotinamide riboside, but also nicotinamide as precursors to synthesize NAD+. However, this speculation needs to be experimentally tested. © 2014 Dong et al.; licensee BioMed Central Ltd. Source

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