魚病研究 Fish Pathology,39(4),189-196,2004.

12 2004 The Japanese Society of Fish Pathology

Characterization of Expressed Genes in Kidney Cells of

Japanese Flounder Paralichthys olivaceus Following
Treatment with ConA/PMA and LPS

Nur Rahmawaty Arma, Ikuo Hirono and Takashi Aoki*

Graduate School of Marine Science and Technology, Tokyo University of Marine Science and
Technology, Tokyo 108-8477, Japan

(Received June 21, 2004)

ABSTRACT-Two cDNA libraries were prepared from Japanese flounder Paralichthys olivaceus
kidney cells that had been treated with LPS or a combination of ConA and PMA. From these two
libraries, we sequenced 373 and 379 clones, respectively. Of these 752 clones, 109 clones were
characterized as immune-related. Among the immune-related cDNAs, the following have not been

previously reported in fish: NK-lysin, perforin, complement C1 q, CD9 antigen, CD63 antigen, CD82
antigen, ISGF-3, IP-30, G-CSF, equistatin precursor, TLS-CHOP, fas ligand and CARD4. This
study highlights the success of using ConA/PMA and LPS as mitogens to increase the number of
immune-related genes expressed in Japanese flounder and demonstrates that different stimuli
induce the expression of different immune-related genes.

Key words: expressed sequence tag, Paralichthys olivaceus, Japanese flounder, biodefense
gene, cDNA sequence, kidney

Although most of the genomes of tiger puffer Fugu immunostimulant to induce the expression of immune
rubripes (Aparicio et al., 2002) and zebrafish Danio rerio and biodefense-related genes. Other mitogens might
(Woods et al., 2000) have been sequenced, only a lim induce a greater number of immune-related genes in a
ited number of immune-related genes has been identi given cDNA library and mitogenic activation can gener
fied from fish. As a result, fish immunology is far behind ate more lymphocyte clones. We previously showed
that of higher vertebrates. A better understanding of that EST analysis is a good technique for investigating
the fish immune system is needed to address disease biodefense molecules encoded in the cDNAs and genes
problems, which are of great concern to the aquaculture of fish (Inoue et al., 1997; Aoki et al., 1999; Nam et al.,
industry. Knowing which genes in fish are immune 2000, 2003).
related can be useful because these genes have poten Concanavalin-A (ConA) induces proliferation of T
tial for use as genetic markers, drug targets and vaccine lymphocytes (Miyamoto et al., 2000), phorbol myristate
adjuvants. acetate (PMA) induces proliferation of both T and B lym
Kono and Sakai (2001) examined the genes phocytes (McMahan et al., 1999), and lipopolysaccha
expressed in kidney that were stimulated by injecting ride (LPS) induces proliferation of B lymphocytes (Abbas
Japanese flounder Paralichthys olivaceus with pepti et al., 1994). Thus, we used these mitogens in an
doglycan, which stimulates macrophages and lympho attempt to stimulate expression of unidentified immune
cytes. The authors putatively identified 92 cDNA and biodefense-related genes in Japanese flounder kid
clones and found that only 9 (9.8%) were related to ney cells. cDNA libraries were prepared from the stimu
immune and defense functions. However, their ESTs lated cells and a total of 752 clones were sequenced.
didn't include genes known to be expressed following

stimulation with interleukin-lƒÀ (Suzuki et al., 1990) and

Materials and Methods
macrophage-activating factor (Siwicki et al., 1996).

This may reflect the low efficiency of peptidoglycan as an Construction of cDNA library
A portion of the whole kidney was taken from an
* Corresponding author apparently healthy Japanese flounder (body weight:
E-mail: aoki@s.kaiyodai.ac.jp approx. 900 g). The cells were suspended in cold
190 N. R. Arma, I. Hirono and T. Aoki

RPMIl640 medium (Nipro, Japan) and divided into six known genes, as described by Adams et al. (1995).

aliquots. Three of the aliquots were stimulated with a

combination of 50 ƒÊg/mL of ConA and 0.35 ,ug/mL of

Results and Discussion
PMA, and the other three were stimulated with 500 ƒÊg/

mL of LPS. The three aliquots of each type were incu Gene expression of kidney cells stimulated with mito

bated for 1, 3 and 6 h, respectively, at 25•Ž. The kid gens

ney cells were collected by centrifugation at 1,500 •~ g for cDNA libraries of 1.6 •~ 106 cfu/mL and 5.9 •~ 105 cfu/

10 min at 20•Ž and washed twice with lx PBS (137 mm mL clones were constructed from poly-adenylated

NaCI, 2.7 mm KCI, 10 mm Na2HPO4, 2 mm KH2PO4). mRNA of Japanese flounder kidney cells stimulated with

The mRNAs from the three different time periods were ConA/PMA (ConA/PMA cDNA library) and LPS (LPS

isolated using a micro mRNA purification kit (Amersham cDNA library), respectively. The numbers of genes

Biotech, USA). These were subsequently pooled to sequenced and the numbers in different functional

ensure complete coverage of expressed genes in the al groups are shown in Table 1. All the sequences were
lotted time frames (1, 3, and 6 h) and used to construct a registered in GenBank under accession numbers

cDNA library. The cDNA library was prepared with a AU260402- AU261275. Each EST was assigned to

cDNA synthesis kit (Gibco, USA) with a NotI/SalI the functional group of its most commonly known

adapter, and constructed in the pSPORT 1 vector, function. The two cDNA libraries had a total of 109

according to the instructions of the manufacturers. clones related to immune function. Of these, 63 were

unique genes. Among these clones, 14 single genes

Plasmid preparation and DNA sequencing were isolated from both libraries (marked with asterisks
The recombinant plasmid DNA was isolated by in Table 2). The frequency of each identified clone in
the alkaline lysis method (Sambrook and Russell, volved in defense mechanisms in the ConA/PMA and
2001). The cDNA clones were sequenced using LPS cDNA libraries is shown in Table 2.
ThermoSequenase with M13 forward or M13 reverse Clones involved in gene/protein expression account

primers or both and an automated DNA sequencer for the highest percentage of identified ESTs in both
4200L (Li-Cor, USA). libraries (Table 1). This result was in agreement with

previously reported Japanese flounder ESTs (Inoue et

Data analysis al., 1997; Aoki et al., 1999; Nam et al., 2000, 2003; Kono
Each DNA sequence was compared with and Sakai, 2001; Hirono et al., 2004). The clones with
sequences in the GenBank databases using the BLAST the highest redundancy in the ConA/PMA cDNA library
algorithm (Altschul et al., 1990). Assignment of putative were those for a dnaK-type molecular chaperone (12
identities for ESTs required a minimum P value of 10-4. clones) and HSP90 a (10 clones) (Table 3). On the
ESTs that putatively matched known genes were cata other hand, the dominant clones found in the LPS cDNA
logued into seven general categories (cell division, cell library were 40S ribosomal protein S4 (6 clones), a
signaling/cell communication, cell structure/motility, cell/ dnaK-type molecular chaperone (6 clones), and a
organism defense, gene/protein expression, metabolism, ubiquitin like-protein (6 clones) (Table 3).
and unclassified) based on the putative functions of the

Table 1. Summary of seauences and clones renresented

 Table 2. List of putatively defense and immune related genes from cDNA libraries of Japanese flounder Paralichthys olivaceus mitogen-stimulated kidney cells

191
ESTs of mitogen-stimulated kidney
 Table 2. Continue

N.R.Arma, l. Hirono and T.Aoki

192
 ESTs of mitogen-stimulated kidney cells 193

Table 3. Redundancy of expressed genes in this study perforin, were found in the ConA/PMA cDNA

library. Clone HKC040 has similarities to pig NK-lysin

precursor. Each of the cysteine residues within the

clone HKC040 protein sequence aligns with the six-con

served cysteine residues found in the saposin-like pro

tein family, to which NK-lysin belongs, strongly suggest

ing a common structural organization between these

proteins (Andra and Leippe, 1999). The antibacterial

activity of NK-lysin is dependent on its pore-forming

potential, which is mainly attributable to its single highly

chargeƒ¿-helical segment (Miteva et al., 1999).

Perforin is a cytolytic mediator produced by NK cells

and CD8+ T lymphocytes (Liu et al., 1995). Perforin

has been purified from mice (Liu et al., 1995), rats

(Lowrey et al., 1989), and humans (Li et al., 2001). The

only teleost perforin sequence data available are

partial sequences for zebrafish (EST accession

numbers BI842922 and AW018698) and fugu

Defense-and immunere-lated genes from ConA/PMA (FRUP00000132628). The nucleotide sequences of

and LPS-stimulated kidney cells these clones showed 41.8% and 68.9% identity with that

Different types of cytokine genes were isolated from of clone HKC346, respectively. Krensky (2000) reported

the two libraries. A clone homologous to CC that NK-lysin assists the cytotoxic effect of perforin,

chemokine-1 was isolated from the LPS cDNA library. granzymes and other membrane-destabilizing polypep

CC chemokines are well-characterized in mammals, tides to facilitate cytolysis.

while in fish they have been identified only in carp Although no IFN ƒ¿, ƒÀor ƒÁgenes were obtained, IFN

Cyprinus carpio (Fujiki et al., 1999), rainbow trout inducible protein genes, such as Mx, ISGF-3 and IP-30,

Oncorhynchus mykiss (Dixon et al., 1998) and Japanese were isolated only from LPS-stimulated cell. Mx have

flounder (Kono and Sakai, 2001; Khattiya et al., been reported in Japanese flounder (Lee et al., 2000;

2003). Granulocyte colony-stimulating factor (G-CSF) Caipang et al., 2003), while ISGF-3 and IP-30 have not

precursor and FasL were also isolated from the ConA/ been previously reported. Schindler et al. (1992)

PMA cDNA library. However, the identities of amino reported that cytoplasmic activation of ISGF-3 is a very

acid sequences between these cytokines and their mam prompt event since it interacts directly with its specific

malian counterparts were low (approximately 30%), but ligand. The function of IP-30 is not yet fully understood,

functionally important amino acid residues were but it may play a role in IFNƒÁ-mediated immune reac

conserved. Now, we are studying the function of these tions (Schuelke et al., 1998).

molecules using recombinant proteins. Clone HKC053 had 55% homology with an

The mitogens lead to processes in which molecular equistatin precursor, a protease inhibitor, that was iso

oxygen is converted to reactive oxygen species (ROS), lated from sea anemone Actinia equina (Galesa et al.,

which are responsible for many of the microbicidal, 2000). Equistatin is a cysteine protease inhibitor that

tumoricidal or inflammatory activities of cells (Maeng et belongs to the thyropins family (Strukelj et al., 2000).

al., 2004). In response to the generation of ROS, the This group currently includes equistatin, MHC class II

host cells produce antioxidants that act to detoxify associated p41 invariant chain fragment, and the chum

ROS. However, when the balance of pro-oxidants and salmon Oncorhynchus keta egg cysteine proteinase

anti-oxidants is disturbed, the host cells undergo auto inhibitor (Lenarcic et al., 1997; Rogelj et al., 2000).

oxidative stress, which causes DNA damage (Hsieh et The MHC class IIassociated invariant chain was

al., 2004). Thus, the appearance of the anti-oxidant isolated in both cDNA libraries (clone numbers, HKC322

encoding cDNAs in the present ESTs may have been to and HKL276). This gene has not previously been

reported in Japanese flounder, although proteins that

protect against oxidative damage triggered by the
mitogens. HSPs, which act as molecular chaperones, share domain structure with the mammalian MHC class

II invariant chain have been reported in rainbow trout

protect leucocytes from oxidative damage associated
with reactive oxygen molecules (Teshima et al., 1996). (Dijkstra et al., 2003). In mammals, these proteins

It is significant that 5 types of HSPs appeared in both have multiple functions, which include the binding of

libraries. This suggests that the function of the HSP class II molecules in the endoplasmic reticulum and

inhibiting the activity of specific proteases (Yoder et al.,

genes is to repair or remove oxidative damage in the
Japanese flounder. 1999). The nucleotide sequences of HKC322 and

Two cytolytic mediators, NK-lysin precursor and HKL27 have some identities to the nucleotide sequences
194 N. R. Arma, I. Hirono and T. Aoki

of the corresponding genes in rainbow trout. Another et al., 2002). These genes are known to associate with
clone, HKC053, which is identified as a putative the up-regulation of cytokines and with the function of
equistatin precursor, has 96.7% homology to clone macrophages and granulocytes (poli, 1998). The find
HKC322. This suggests that clone HKC322 also has a ing of the above three types of C/EBP raises the possi
specialized role as a protease inhibitor. Clone HKL276, bility that all C/EBP members exist in fish. Another
which has only 49.2% homology to clone HKC053 prob clone homologous to TLS-CHOP, which plays a role in
ably represents a different type of MHC class II invariant tumor formation (Rapp et al., 2002), was also
chain. It is of considerable interest to determine isolated. Therefore, this reflects the biological signif-i
whether this clone is involved in Japanese flounder class cance of C/EBPs as regulators of immune-related
II antigen presentation or whether it functions as a protease genes.
inhibitor. Clone HKL222 showed 74% identity with a human
Two types of Fc receptors were identified. The Fc gene that is associated with the differentiation of mono
receptor a chain was identified from the LPS cDNA cytes to macrophages. LPS and PMA have been
library and the Fc receptor ƒÁchain was identified from reported to inhibit the differentiation of primary mono
the ConA/PMA cDNA library. Fc receptors, like the cytes to macrophages (Basia et al., 2001).
T-cell receptor, form a multisubunit complex, in which A total of 3,609 ESTs of Japanese flounder have
only the a chain is responsible for ligand recognition, been now stored in GenBank (dbEST release January,
while the ƒÁchain, which is closely related to the T-cell 30, 2004). An additional 752 ESTs from this study have
receptorƒÌchain, mediates signal transduction by most been added to the GenBank database and 55% (344
Fc receptors (Janeway et al., 1999). clones) of the latter ESTs represent new findings in
A clone homologous to the caspase recruitment fish. This study determined 468,147 base pairs of
domain (CARD) 4 was isolated from ConA/PMA cDNA nucleotides of the Japanese flounder genome. This
library. CARD4 plays critical roles in stress-activated work should contribute to a better understanding of the
signaling pathways and has been suggested to be network system of immune and biodefense-expressed
involved in the innate immune response (Bertin et al., genes in fish through comprehensive expression analy
1999). CARD4 belongs to the Nod family, which con sis of the genes so far identified.
tains intracellular activators of the caspase and NF-ƒÈB
signaling pathways (Geddes et al., 2001).
Acknowledgement
A clone homologous to G protein-coupled receptor

(GPCR) 42 was identified in the ConA/PMA cDNA This study was supported in part by the Grants-in

library, while a clone homologous to G protein pathway Aid for Scientific Research (S) from the Ministry of Edu

suppressor (GPS) 1 was identified in the LPS cDNA cation, Culture, Sports, Science and Technology of

library. GPCRs mediate numerous intracellular signal- Japan.

ling pathways on binding extracellular agonists, e.g. hor

mones, neurotransmitters, and odorants (Giorelli et al.,

References
2002). In human, GPS suppresses the G protein and

mitogen-activated protein kinase-mediated signal trans Abbas, A. K., A. H. Lichtman and J.S. Pober (1994): Cellular

and molecular immunology. USA, 457 p.

duction (Jin et al., 1997).
Adams, M. D, A. R. Kerlavage, R. D. Fleischmann, R. A.
Six clones of the ConA/PMA cDNA library and 10
Fuldner, C. J. Bult, N. H. Lee, E. F. Kirkness, K. G.
clones of the LPS cDNA library were found to have Weinstock and J. D. Gocayne, O. White, G. Sutton, J. A.
homologies to genes involved in the regulation of Blake, R. C. Brandon, C. Man-Wai, R. A. Clayton, T. R.

apoptosis pathways. Although cytolytic mediator genes Cline, M. D. Cotton, J. Earle-Hughes, L. D. Fine, L. M.
Fitzgerald, W. M. Fitzhugh, J. L. Fritchman, N. S.
were not isolated from the LPS cDNA library, genes
Geoghagen, A. Glodek, C. L. Gnehm, M. C. Hanna, E.
associated with the proteolytic pathway such as genes
Hedblom, P. S. Hinkle Jr., J. M. Kelley, J. C. Kelley, L.-I.
for proteasomes and ubiquitins were isolated from both Li‚•, S. M. Marmaros, J. M. Merrick, R. F. Moren

libraries. Palanques, L. A. McDonald, D. T. Nguyen, S. M. Pelligrino,

‚•-

Five clones were homologous to genes encoding C. A. Phillips, S. E. Ryder, J. L. Scott, D. M. Saudek, R.

Shirley, K. V. Small, T. A. Spriggs, T. R. Utterback, J. F.

transcription factors for immune-related genes. C/
Weidman, Y. Li, D. P. Bednarik, L. Cao, M. A. Cepeda, T.
EBPs are members of a family of leucine zipper tran
A. Coleman, E. J. Collins, D. Dimke, D. -F. Feng, A. Ferrie,
scription factors comprising six members ( C. Fischer, G. A. Hastings, W. W. He, J.S. Hu, J. M.
andĀ) that are critically involved in the regulation
ƒ¿,ƒÀ,ƒÂ,ƒÃ,ƒÁ
of nor Greene, J. Gruber, P. Hudson, A. K. Kim, D. L. Kozak, C.

mal cellular differentiation and function in multiple tis Kunsch, J. Hungjun, H. Li, P. S. Meissner, H. Olsen, L.

Raymond, Y. F. Wei, J. Wing, C. Xu, G. L. Yu, S. M.

sues (Lekstrom-Himes and Xanthopoulos, 1998).
Ruben, P. J. Dillion, M. R. Fannon, C. A. Rosen, W. A.
Clones homologous to C/EBP ƒ¿and ƒÀ3 were isolated in
Haseltine, C. Fields, C. M. Fraser and J. C. Venter (1995):
this study. C/EBP ƒÀ3 and ƒÃwere previously reported in Initial assessment of human gene diversity and expression

kidney, heart and spleen of Japanese flounder (Tucker patterns based upon 83 million nucleotides of cDNA
 ESTs of mitogen-stimulated
sequence. Nature, 377, 3-174.
Altschul, S. F., W. Gish, W. Miller, E. W. Myers and E. W.
Lipman (1990): Basic local alignment search tool. J. Mol.
Biol., 215, 403-410.
Andra, J. and M. Leippe (1999): Candidacidal activity of short
ened synthetic analogs of amoebapores and NK-lysin.
Med. Microbiol. Immnunol., 188, 117-124.
Aoki, T., B. H. Nam, I. Hirono and E. Yamamoto (1999):
Sequences of 596 cDNA clones (565,977 bp) of Japanese
flounder (Paralichthys olivaceus) leukocytes infected with
Hirame rhabdovirus. Mar. Biotechnol., 1, 477-488.
Aparicio, S., J. Chapman, E. Stupka, N. Putnam, J. M. Chia, P.
Dehal, A. Christoffels, S. Rash, S. Hoon, A. Smit, M. D.
Gelpke, J. Roach, T. Oh, I. Y. Ho, M. Wong, C. Detter, F.
Verhoef, P. Predki, A. Tay, S. Lucas, P. Richardson, S. F.
Smith, M. S. Clark, Y. J. Edwards, N. Doggett, A. Zharkikh,
S. V. Tavtigian, D. Pruss, M. Barnstead, C. Evans, H.
Baden, J. Powell, G. Glusman, L. Rowen, L. Hood, Y. H.
Tan, G. Elgar, T. Hawkins, B. Venkatesh, D. Rokhsar and
S. Brenner (2002): Whole-Genome Shotgun Assembly and
Analysis of the Genome of Fugu rubripes. Science, 297,
1301-1310.
Basta, S., S. Knoetig, A. Summerfield and K. C. McCullough
(2001): Lipopolysaccharide and phorbol l2-myristate 13
acetate both impair monocyte differentiation, relating cellu-
lar function to virus susceptibility. Immunol., 103, 488
497. - of cytolytic T lymphocytes
Bertin, J., W-J. Nir, C. M. Fischer, O. V. Tayber, P. R. Errada, J.
R. Grant, J. J. Keilty, M. L. Gosselin, K. E. Robison, G. H.
W. Wong, M. A. Glucksmann and P. S. DiStefano. (1999):
Human CARD4 Protein is a novel CED-4/Apaf-1cell death
family member that activates NF-kB. J. Biol. Chem., 274,
12955-12958.
Caipang, C. M. A, I. Hirono and T. Aoki (2003): In vitro inhibition
of fish rhabdoviruses by Japanese flounder, Paralichthys
olivaceus Mx. Virol., 317, 373-382.
Dijkstra, J. M., I. Kiryu, B. Kollner, Y. Yoshiura and M. Ototake
(2003): MHC class II invariant chain homologues in rain
bow trout (Oncorhynchus mykiss). Fish Shellfish
Immunol., 15, 91-105.
Dixon, B., B. Shum, E. J. Adams, K. E. Magor, R. P. Hedrick, D.
G. Muir and P. Parham (1998): CK-1, a putative chemokine
of rainbow trout. Immunol. Rev., 166, 341-348.
Fujiki, K., D. H. Shin, M. Nakao and T. Yano (1999): Molecular
cloning of carp (Cyprinus carpio) CC-chemokine, CXC
chemokine receptors, allograft inflammatory factor-1, and
natural killer cell enhancing factor by use of suppression
subtractive hybridization. Immunogenetics, 49, 909-914.
Galesa, K., B. Strukelj, S. Bavec, V. Turk and B. Lenarcic
(2000): Cloning and expression of functional equistatin.
Biol. Chem., 381, 85-88.
Geddes, B. J., L. Wang, W-J. Huang, M. Lavellee, G. A. Manji,
M. Brown, M. Jurman, J. Cao, J. Morgenstern, S. Merriam,
M. A. Glucksmann, P. S. Distefano, and J. Bertin (2001):
Human CARD12 Is a Novel CED4/Apaf-1 Family Member
That Induces Apoptosis. Biochem. Biophys. Res. Comm.,
284, 77-82.
Giorelli, M., P. Livrea, G. Defazio, L. lacovelli, L. Capobianco, A.
Picascia, M. Sallese, D. Martino, M. S. Aniello, M. Trojano
and A. De Blasi (2002): Interferon beta-1a counteracts
effects of activation on the expression of G-protein-coupled
receptor kinases 2 and 3, b-arrestin-1, and regulators of G
protein signalling 2 and 16 in human mononuclear
leukocytes. Cell. Signal., 14, 673-678.
Hirono, I., R. Yazawa, T. Aoki (2004): Expressed sequence tag
of Japanese flounder Paralichthys olivaceus skin cells.
Fish. Sci., 70, 195-197.
kidney cells 195
Hsieh, T. J., T. Z. Liu, Y. C. Chia, C. L. Chern, F. J. Lu, M. C.
Chuang, S. Y. Mau, S. H. Chen, Y. H. Syu, C. H. Chen
(2004): Protective effect of methyl gallate from Toona
sinensis (Meliaceae) against hydrogen peroxide-induced
oxidative stress and DNA damage in MDCK cells. Food
Chem. Toxicol. 42, 843-850.
Inoue, S., B. H. Nam, I. Hirono and T. Aoki (1997): A survey of
expressed genes in Japanese flounder (Paralichthys
olivaceus) liver and spleen. Mol. Mar. Biol. Biotechnol., 6,
376-380.
Janeway, C. A, P Travers, M. Walport and J. D. Capra (1999):
Immunobiology: The immune system in health and
disease. Current Biology Publications, London, 635p.
Jin, D. Y., H. Teramoto, C. Z. Giam, R. F. Chun, J. S. Gutkind
and K. T. Jeang (1997): A human suppressor of c-Jun N
terminal kinase 1 activation by tumor necrosis factor alpha.
J. Biol. Chem., 272, 25816-25823.
Khattiya, R., I. Hirono and T. Aoki (2003): Molecular cloning,
gene structure and expression of two CC chemokines from
Japanese flounder Paralichthys olivaceus. Fish. Sci., 69,
1065-1074.
Kono, T. and M. Sakai (2001): The analysis of expressed genes
in the kidney of Japanese flounder, Paralichthys olivaceus,
injected with the immunostimulant peptidoglycan. Fish
Shellfish Immunol., 11, 357-366.
Krensky, A. M. (2000): Granulysin. A novel antimicrobial peptide
and natural killer cells.
Biochem. Pharmacol., 59, 317-320.
Lee, J.Y., I. Hirono and T. Aoki (2000): Cloning and analysis of
expression of Mx cDNA in Japanese flounder, Paralichthys
olivaceus. Dev. Comp. Immunol., 24, 407-415.
Lekstrom-Himes, J. and K.G. Xanthopoulos (1998): Biological
role of the CCAAT/enhancer-binding protein family of tran
scription factors. J. Biol. Chem., 273, 28545-28548.
Lenarcic, B., A. Ritonja, B. Strukelj, B. Turk and V. Turk (1997):
Equistatin a new inhibitor of cysteine proteinases from
Actinia equina, is structurally related to thyroglobulin type-1
domain. J. Biol. Chem., 272, 13899-13903.
Li, F., X. Zhou, W. Qin and J. Wu (2001): Full-length cloning and 3
'terminal portion expression of human perforin cDNA.
Clin. Chim. Acta, 313, 25-131.
Liu, C. C., C. M. Walsh, and J. D. Young (1995): Perforin: struct
ure and function. Immunol. Today, 16, 194-201.
Lowrey, D. M., T. Aebischer, K. Olsen, M. Lichtenheld, F. Rupp,
H. Hengartner and E. R. Podack (1989): Cloning, analysis,
and expression of murine perforin 1 cDNA, a component of
cytolytic T-cell granules with homology to complement
component C9, Proc. Natl. Acad. Sci., USA, 86, 247-251.
Maeng, O., Y. C. Kim, H. J. Shin, J. O. Lee, T. L. Huh, K. I.
Kang, Y. S. Kim, S. G. Paik H. Lee (2004): Cytosolic
NADP(+)-dependent isocitrate dehydrogenase protects
macrophages from LPS-induced nitric oxide and reactive
oxygen species. Biochem. Biophys. Res. Commun., 317,
558-564.
McMahan, R., R. O. Johnson, L. N. Ruben and R. H. Clothier
(1999): Apoptosis and the cell cycle in Xenopus laevis:
PHA and PMA exposure of splenocytes. Immunol. Lett.,
70, 179-183.
Miteva, M., M. Andersson, A. Karshikoff and G. Otting (1999):
Molecular electroporation: a unifying concept for the de
scription of membrane pore formation by antibacterial pep
tides, exemplified with NK-lysin. FEBS Lett., 462, 155
158. -
Miyamoto, S., S. R. Kimball and B. Saver (2000): Signal trans
duction pathways that contribute to increased protein syn
thesis during T-cell activation. Biochim. et Biophys. Acta,
1494, 28-42.
196 N. R. Arma, I. Hirono
Nam, B. H, E. Yamamoto, I. Hirono and T. Aoki (2000): A sur
vey of genes in the leukocytes of Japanese flounder,
Paralichthys olivaceus, infected with Hirame rhabdovirus.
Dev. Comp. Immunol., 24, 13-24.
Nam, B. H, I. Hirono and T. Aoki (2003): Bulk isolation of
immune response-related genes by expressed sequenced
tags of Japanese flounder Paralichthys olivaceus
leucocytes stimulated with Con A/PMA. Fish Shellfish
Immunol., 14, 467-476.
oli, V. (1998): The P role of C/EBP isoforms in the control of
inflammatory and native immunity functions. J. Biol.
Chem., 273, 29279-29282.
Rapp, T. B., L. Yang, E. U. Conrad III, N. Mandahl, and H. A.
Chansky. (2002): RNA splicing mediated by YB-1 is inhib
ited by TLS/CHOP in human myxoid liposarcoma cells, J.
Orthopaedic Res., 20, 723-729.
Rogelj, B., B. Strukelj, D. Bosch and M. A. Jongsma (2000):
Expression, purification, and characterization of equistatin
in Pichia pastoris. Protein Expr. Purif. 19, 329-334.
Sambrook, J., and D. W. Russel (2001): Molecular cloning: A
Laboratory Manual. Cold Spring Harbor Laboratory, New
York.
Schindler, C., X. Y. Fu, T. Improta, R. Aebersold, J. E. Darnell Jr
(1992): Proteins of transcription factor ISGF-3: one gene
encodes the 91-and 84-kDa ISGF-3 proteins that are acti
vated by interferon alpha. Proc. Natl. Acad. Sci., USA, 89,
7836-7839.
Schuelke, M., J. Loeffen, E. Mariman, J. Smeitink and L. van
den Heuvel (1998): Cloning of the human mitochondrial 51
kDa subunit (NDUFV1) reveals a 100% antisense homol
ogy of its 3'UTR with the 5'UTR of the gamma-interferon
inducible protein (IP-30) precursor: is this a link between
and T. Aoki
mitochondrial myopathy and inflammation. Biochem.
Biophys. Res. Comm., 245, 599-606.
Siwicki, A. K., T. Miyazaki, I. Komatsu and T. Matsuzato (1996):
In vitro influence of heat extract from firefly squid
Watasenia scintillans on the phagocyte and lymphocyte
activities in rainbow trout Oncorhynchus mykiss. Fish
Pathol., 31, 1-7.
Strukelj B, B. Lenarcic, K. Gruden, J. Pungercar, B. Rogelj, V.
Turk, D. Bosch and M.A. Jongsma (2000): Equistatin, a
protease inhibitor from the sea anemone Actinia equina, is
composed of three structural and functional domains.
Biochem. Biophysic. Res. Comm., 269, 732-736.
Suzuki, I., H. Tanaka, A. Kinoshita, S. Oikawa, M. Osawa and
T. Yadome (1990): Effect of orally administered beta
glucan on macrophage function in mice. Int. J.
Immunopharmacol., 12, 675-684.
Teshima, S., K. Rokutan, M. Takahashi, T. Nikawa and K. Kishi
(1996): Induction of heat shock proteins and their possible
roles in macrophages during activation by macrophage
colony-stimulating factor. Biochem. J., 315, 497-504.
Tucker, C. S., I. Hirono and T. Aoki (2002): Molecular cloning
and expression of CCAAT/enhancer binding proteins in
Japanese flounder Paralichthys olivaceus. Dev. Comp.
Immunol., 26, 271-282.
Woods, I. G., P. D. Kelly, F. Chu, P. Ngo-Hazelett, Y. L. Yan, H.
Huang, J. H. Postlethwait, W. S. Talbot (2000): A comparat
ive map of the zebrafish genome. Genome Res., 10,
1903-1914.
Yoder, J. A., R. N. Haire and G. W. Litman (1999): Cloning of
two zebrafish cDNAs that share domains with MHC class
II-associated in variant chain. Immunogenetics, 50, 84--88.