Gun-Wook Baeck

Identification and expression analysis of an interferon stimulated gene 15 (ISG15) from black rockfish, Sebastes schlegeli

The interferon stimulated gene 15 (ISG15) is strongly induced in many cell types by IFNs, viral infection and double-stranded RNA (poly I:C). The ISG15 homolog cDNA was isolated from the black rockfish poly I:C stimulated leukocyte cDNA library. The black rockfish ISG15 homolog was found to consist of 1070bp encoding 160 amino acid residues. Compared with other known ISG15 peptide sequences, the most conserved regions of the black rockfish ISG15 peptide were found to be the tandem ubiquitin-like domains and a C-terminal LRLRGG conjugating motif, characteristic of mammalian and non-mammalian ISG15 proteins. A phylogenetic analysis based on the deduced amino acid sequence revealed a homologous relationship between the ISG15 sequence of black rockfish and that of Atlantic salmon, Atlantic cod, crucian carp and rainbow trout. The expression of the black rockfish ISG15 molecule was induced in the peripheral blood leukocytes (PBLs) from 1 to 12h following poly I:C stimulation, with a peak at 6h post-stimulation. The black rockfish gene was predominantly expressed in the PBLs and the spleen.

Molecular cloning and expression of cDNAs for two distinct granulocyte colony stimulating factor genes from black rockfish Sebastes schlegelii.

Granulocyte-colony stimulating factor (G-CSF) is a cytokine that stimulates the proliferation and differentiation of hematopoietic progenitor cells committed to the neutrophil/granulocyte lineage. Here, we report the two distinct granulocyte colony stimulating factor homologues from black rockfish Sebastes schlegelii. The G-CSF homologue cDNAs were isolated from the black rockfish LPS or Con A/PMA stimulated leukocyte cDNA libraries. The cDNA for the Black rockfish G-CSF-1 homologue predicts a peptide of 202 amino acids that is the closest to the Bastard halibut (Paralichthys olivaceus) G-CSF, whereas the cDNA of the Black rockfish G-CSF-2 homologue predicts a peptide of 212 amino acids that is the closest to the Fugu (Takifugu rubripes). In a healthy fish, the mRNAs of both G-CSF homologues were predominantly expressed in leukocytes, spleen, and gill. Expression of the black rockfish G-CSF-1 homologue was induced in peripheral blood leukocytes (PBLs) after stimulation with LPS, Con A/PMA, or Poly I:C, and G-CSF-2 homologue was strongly induced in PBLs after stimulation with Con A/PMA for 24 h only.

First description of programmed cell death10 (PDCD10) in rock bream (Oplegnathus fasciatus): Potential relations to the regulation of apoptosis by several pathogens

In this study, we isolated and characterized programmed cell death10 (PDCD10), which is known to be related to apoptosis, from rock bream (Oplegnathus fasciatus). The full-length rock bream PDCD10 (RbPDCD10) cDNA (1459 bp) contains an open reading frame of 633 bp that encodes 210 amino acids. Furthermore, multiple alignments revealed that the six of the α-helix bundles were well conserved among the other PDCD10 sequences tested. RbPDCD10 was significantly expressed in the liver, RBC (red blood cell), gill, intestine, trunk kidney and spleen. RbPDCD10 gene expression was also examined in several tissues, including the kidney, spleen, liver, and gill, under bacterial and viral challenges. Generally, all of the examined tissues from the fish that were infected with Edwardsiella tarda and the red sea bream iridovirus (RSIV) exhibited significant up-regulations of RbPDCD10 expression compared to the controls. However, RbPDCD10 expression exhibited dramatic down-regulations in all of the examined tissues following injections of Streptococcus iniae, which is major bacterial pathogen that is responsible for mass mortality in rock bream. Our results revealed that rock bream PDCD10 may be involved in the apoptotic regulation of rock bream immune responses.

Expression analysis of lipopolysaccharide-and/or concanavalin A/phorbol myristate acetate-stimulated black rockfish peripheral blood leukocytes using a cDNA microarray to identify up-regulated genes

The responses of peripheral blood leucocytes (PBLs) from the black rockfish Sebastes schlegelii stimulated with lipopolysaccharides (LPS) and/or concanavalin A/phorbol myristate acetate (Con A/PMA) were investigated using a cDNA microarray consisting of 5,088 clones. This analysis showed that 254 unique genes were more than twofold upregulated, and they were selected for sequencing. Among the mitogens, 84 genes were more than twofold upregulated in LPS-stimulated PBLs, and 112 genes were induced in Con A/PMA-stimulated PBLs. Moreover, 58 other genes were more than twofold upregulated in PBLs stimulated with both LPS and Con A/PMA. Other genes were not significantly induced. Overall, these results suggest that certain genes in black rockfish have important roles in physiological and pathological processes. Furthermore, the microarray analysis suggested a promising tool for immune mechanisms in teleost fish.