hnolog

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Cta1lsi(aoskRe(ynsLstisip4bkasi

A

udMC

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Abstracts / Journal of Biotec

ongpim, S., Nontaso, N., Dulgindachabaporn, S., Hintong, W., 2005. Screeningof probiotic bacteria to replace antibiotics normally used in feeding stufffor Tilapia spp. (serial online) available from: ora.kku.ac.th/res kku/Abstract(in Thai).

oi:10.1016/j.jbiotec.2008.07.1289

I3-P-001

unctional characterization of a novel CC chemokine in largeellow croaker (Pseudosciaena crocea)

inhua Chen ∗, Jinzhou Zhang

Key Laboratory of Marine Biogenetic Resources, Third Institute ofceanography, State Oceanic Administration, Xiamen 361005, PRhina

-mail address: Chenxinh@tom.com (X. Chen).

hemokines are small, structurally related and secreted peptideshat play a crucial role in stimulating the recruitment, activation,nd adhesion of cells to sites of infection or injury (Kuang et al.,996; Dixon et al., 1998). A novel CC chemokine gene was iso-ated from large yellow croaker (Pseudosciaena crocea) by expressedequence tag (EST) analysis (LycCC2). Its open reading frame (ORF)s 279 nucleotides (nt) and encodes a polypeptide of 93 amino acidsaa). The deduced LycCC2 contains a 20-aa signal peptide and a 73-a mature polypeptide, which possesses the typical arrangementf four cysteines (C34, C35, C61 and C75) (Zhang and Chen, 2008). Ithares 17.4–38.9% and 10.0–24.5% aa sequence identities to othernown bony fish and mammalian CC chemokines, respectively.ecombinant LycCC2 (rLycCC2) protein produced in Pichia pastorisxhibited marked chemotaxis to the peripheral blood leucocytesPBLs) from large yellow croaker. Tissue expression profile anal-sis showed that LycCC2 gene was constitutively expressed in alline examined, except for liver, although at a different level. Upontimulation with poly(I:C) or inactivated trivalent bacterial vaccine,ycCC2 gene expression was obviously up-regulated in kidney, gills,pleen, liver, intestine, blood and heart at 12 h post-induction. Theime-course of LycCC2 gene expression in spleen and kidney uponnduction was further analyzed using a real-time PCR. The resultshowed that LycCC2 transcripts in these two tissues was quicklyncreased by either poly(I:C) or bacterial vaccine and reached theireak levels at 12 h, followed by a recovery to normal level after8 h. LycCC2 gene expression was more strongly up-regulated byacterial vaccine (37.3- and 17.4-fold mRNA increases in spleen andidney, respectively, at 12 h post-induction) than by poly(I:C) (15.5-nd 8.5-fold increases in spleen and kidney, respectively). Thesetudies suggest that the LycCC2 plays a early acute-phase role innflammation.

cknowledgements

The work was supported by grants from Nation ‘863’ Projectnder contract No. 2006AA100306, National Natural Science Foun-ation of China (40476052), and Fund of Key Laboratory ofarine Biogenetic Resources, State Oceanic Administration of

hina (HY200503).

eferences

ixon, B., Shum, B., Adams, E.J., Magor, K.E., Hedrick, R.P., Muir, D.G., Parham, P., 1998.

CK-1, a putative chemokine of rainbow trout (Oncorhynchus mykiss). Immunol.Rev. 66, 341–348.

uang, Y., Wu, Y., Jiang, H., Wu, D., 1996. Selective G protein coupling by C–Cchemokine receptors. J. Biol. Chem. 271, 3975–3978.

hang, J.Z., Chen, X.H., 2008. Molecular characterization of a novel CC chemokine inlarge yellow croaker (Pseudosciaena crocea) and its involvement in modulation

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of MHC class I antigen processing and presentation pathway. Mol. Immunol. 45,2076–2086.

oi:10.1016/j.jbiotec.2008.07.1290

I3-P-002

olecular and functional characterization of a cystatin ana-ogue in large yellow croaker (Pseudosciaena crocea)

inhua Chen ∗, Shuying Li

Key Laboratory of Marine Biogenetic Resources, Third Institute ofceanography, State Oceanic Administration, PR China

-mail address: Chenxinh@tom.com (X. Chen).

he cDNA of a large yellow croaker cystatin (Lyccys) analogue wassolated from the spleen smart cDNA library of large yellow croaker,n economically important marine fish in China. Its open readingrame (ORF) was 354 nucleotides (nt) encoding a protein of 118mino acids (aa). The deduced Lyccys contained a 21-aa signal pep-ide and a 97-aa mature polypeptide, which contained the threeonsensus motifs known to interact with the active site of cysteineeptidases: QLVAG (residues 69–73), two glycine residues (residues5–26) at the N-terminus and a tryptophan (residue 107) locatedown-stream of the reactive site (Estrada et al., 1998; Turk andode, 1991; Stubbs et al., 1990). The deduced amino acid sequencehowed a high degree of sequence similarity to mammalian cystatin, chicken egg cystatin and other fish cystatins. Genomic analy-is revealed that Lyccys gene, spanning 2297 nt, consisted of threexons and two introns, similar to the genomic structure of humanystatin C, mouse cystatin C and zebrafish cystatin C. Recombinantyccys (rLyccys) protein expressed in E. coli was shown to be ableo inhibit papain, the archetypal activity. Tissue expression profilenalysis showed that Lyccys gene was constitutively expressed inll eight tissues examined, although at different levels. The time-ourse of Lyccys gene expression in spleen, kidney and blood uponnduction with poly(I:C) or inactivated trivalent bacterial vaccineas further analyzed using a real-time PCR. The results showed that

yccys transcripts in spleen and kidney was obviously up-regulated,owever, in blood was down-regulated. An in vivo administration ofLyccys in spleen and kidney could significantly up-regulate mRNAynthesis of tumor necrosis factor �2, a pleiotropic cytokine pro-uced by several cell types (T- and B-cells, mastocytes, naturaliller cells, and macrophages) in response to a large array of stim-li (Beutler and Van Huffel, 1994), and interleukin-10. These resultsuggest that the Lyccys is an inhibitor of cysteine proteinases, anday have an immunomodulatory function in immune response.

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strada, S., Nycander, M., Hill, N., Craven, C.J., Waltho, P.J., Bjork, I., 1998. The role ofgly-4 of human cystatin A (stefin A) in the binding of target proteinase. Char-acterization by kinetic and equilibrium methods of the interaction of cystatinA gly-4 mutants with papain, cathepsinB, and cathepsin, L. Biochemistry 37,7551–7560.

tubbs, M., Laber, B., Bode, W., Huber, R., Jerala, R., Lenarcic, B., Turk, V., 1990. Therefined 2.461505; X-ray crystal structure of recombinant human stefin B in com-plex with the cysteine proteinase papain: a novel type of proteinase inhibitorinteraction. EMBO J. 9, 1939–1947.

oi:10.1016/j.jbiotec.2008.07.1291