Abalone NF-κB transcription factor requires a nuclear localization signal for nuclear import

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Abstracts / Journal of Biotechnology 136S (2008) S548–S557

Wang, L.L., Zhang, H., Song, L.S., Guo, X.M., 2007. Loss of allele diversity in introduced populations of the hermaphroditic bay scallop Argopecten irradians. Aquaculture 271, 252–259.

doi:10.1016/j.jbiotec.2008.07.1306 VI3-P-022 Abalone NF-␬B transcription factor requires a nuclear localization signal for nuclear import Yusheng Zhang 1

Jiang 2,∗ , Jinyan

Shang 1 , Xinzhong

Wu 1,2 , Chuanxi

1

Zhejiang University, Hangzhou 310029, PR China College of Life Sciences and Technology, Dalian Fisheries University, Dalian116023, PR China 2

E-mail address: [email protected] (Y. Jiang). Abalone, Haliotis diversicolor supertexta is economically important marine gastropod cultured in the south coast of China. However, this industry has often been devastated by disease outbreak. Up to date, few studies on the molecular mechanism of immunology were reported on this ancient invertebrate. A homologue of Rel\NF␬B transcription factor, named Ab-Rel, was identified from this species, and proved to play a possible role in the immune response (Jiang and Wu, 2007; Jiang et al., 2007). Since the DNA-binding capacity of NF-␬B in hemocytes can be specifically up-regulated by immune challenge, it is presumed that proper nuclear transport of Ab-Rel was occurred within this process (Birbach et al., 2002). To investigate the mechanisms of nuclear import, we analyzed the effect of truncated mutations on subcellular distribution of Ab-RelEGFP using the modified bac to bac insect cell expression system. It demonstrated that a consensus nuclear localization signal (NLS) is involved in the translocation event. Our data provide further evidence that Ab-Rel is functionally conserved as one of Rel\NF-␬B members, allowing us to study this important transcription factor in an evolutionary context. References Birbach, A., Gold, P., Binder, B.R., Hofer, E., Martin, R.D., Schmid, J.A., 2002. Signaling molecules of the NF-␬B pathway shuttle constitutively between cytoplasm and nucleu. J. Biol. Chem. 277, 10842–10851. Jiang, Y.S., Wu, X.Z., 2007. Characterization of a Rel\NF-␬B homologue in a gastropod abalone, Haliotis diversicolor supertexta. Dev. Comp. Immunol. 31 (2), 121–131. Jiang, Y.S., Wu, X.Z., Zhang, Y., 2007. Magnetocapture of abalone transcription factor NF-␬B: a new strategy for isolation and detection of NF-␬B both in vitro and in vivo. J. Biotechnol. 127 (3), 385–391.

doi:10.1016/j.jbiotec.2008.07.1307 VI3-P-023 Abalone NF-␬B transcription factor is composed of two different subunits Yusheng Jiang 2,∗ , Junfeng Li 2 , Xinzhong Wu 1,2 , Yue Ma 1,2 , Gang Liu 2 1

College of Animal Sciences, Zhejiang University, Hangzhou 310029, PR China 2 College of Life Sciences and Technology, Dalian Fisheries University, Dalian 116023, PR China E-mail address: [email protected] (Y. Jiang). Rel\NF-␬B signal transduction pathway is evolutionarily conserved and involved in numerous biological processes (Hayden and Ghosh, 2004). Previously, we cloned and functionally characterized a

homologue of Rel\NF-␬B transcription factor in a gastropod Haliotis diversicolor supertexta, named Ab-Rel (Jiang and Wu, 2007). Using an antibody raised against the Ab-Rel RHD (Rel homology domain) which is the signature motif of the Rel families, detected two specific immunoreactive bands on the Western blot, leading us to extrapolate that abalone NF-␬B is consisted of two subunits. To confirm this hypothesis, a plasmid containing a specific fragment corresponding to transactivation domain (TAD) of Ab-Rel was constructed to express recombinant protein in E. coli. The expressed products were purified to immunize rabbits, and the antibody was obtained with the titer determined by an indirect ELISA. Western blot analysis of total extracts of abalone haemocytes using the AbRel specific antibody detected one specific immunoreactive band. These results are consistent with northern blot analysis using the nucleotide probe to Ab-Rel RHD or TAD. Taken together, all results indicated that Ab-Rel is one of the two subunits of abalone NF␬B (Huxford et al., 1998). This is the first report on the molecular structure of NF-␬B protein in shellfish. References Hayden, M.S., Ghosh, S., 2004. Signaling to NF-␬B. Genes Dev. 18, 2195–2224. Huxford, T., Huang, De-B., Malek, S., Ghosh, G., 1998. The crystal structure of the I␬Ba/NF-␬B complex reveals mechanisms of NF-␬B inactivation. Cell 95, 759–770. Jiang, Y.S., Wu, X.Z., 2007. Characterization of a Rel\NF-␬B homologue in a gastropod abalone, Haliotis diversicolor supertexta. Dev. Comp. Immunol. 31 (2), 121–131.

doi:10.1016/j.jbiotec.2008.07.1308 VI3-P-024 Effect of l-ascorbic acid as immunity enhancer for juvenile sea cucumber, Apostichopus japonicus Kailai Wang ∗ , Ziru Liu, Yongping Xu, Gang Liu, Tongju Ren, Liji Jin School of Environmental and Biological Science and Technology, Dalian University of Technology, Dalian, 116024, People’s Republic of China E-mail address: [email protected] (K. Wang). A trail was conducted to determine the efficiency of vitamin C (ascorbic acid, AsA) as an immunity enhancer to stimulate the non-specific immune response and to protect juvenile sea cucumbers from infectious pathogen. Five diets of different AsA levels were fed to five groups of juvenile sea cucumbers (G1—0, G2—487, G3—1372, G4—4836, G5—13,857 mg/kg diet), with an average initial weight 1.19 ± 0.46 g. During the 40 days feeding trial, higher but not significantly survival rates were observed in groups G3, G4, G5 than the AsA free group G1. The bactericidal activity of skin mucus and coelom fluid (CF) of sea cucumbers from group G3, G4 were found significantly (P < 0.05) enhanced. Moreover, a significantly higher lysozyme activity of CF was showed in G3. The coelom cytocrit demonstrated an increased trend along with the increased diet AsA level, while the coelom cells of G4, G5 showed significantly higher phagocytic activity and respiratory burst activity. A greater CF lectin titer was described significantly in G4, and higher mucus lectin titer activity was observed in animals from G3, G4 and G5. In G1, both alkaline phosphatase (ALP) and acid phosphatase (ACP) were found to possess a significantly lower activity than AsA containing groups. However, there was no significant difference observed in phenoloxidase activity in all the five groups. Bacterial challenged study showed that typical infection signs were first observed among the five groups, respectively, on day 4, 6, 8,

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