Establishing a role for shrimp fortilin in preventing cell death

Aquaculture 255 (2006) 157 – 164 www.elsevier.com/locate/aqua-online

Establishing a role for shrimp fortilin in preventing cell death Potchanapond Graidist a , Kenichi Fujise b , Warapond Wanna a , Kallaya Sritunyalucksana c , Amornrat Phongdara a,⁎ a

b

Department of Biochemistry, Faculty of Science, Prince of Songkla University, Hat-Yai, Songkhla 90112, Thailand Research Center for Cardiovascular Disease, The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Disease and Division of Cardiology, Department of Internal Medicine, The University of Texas-Houston Health Science Center, Houston, Texas 77030, USA c Centex Shrimp, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand Received 6 September 2005; received in revised form 15 December 2005; accepted 15 December 2005

Abstract White spot syndrome virus (WSSV) is a highly virulent and infectious virus to farmed shrimps and represents a serious threat to aquatic industries. Fortilin, also known as translationally controlled tumor protein (TCTP), protects mammalian cells under stress from cell death. A subtraction hybridization screening in our laboratory between healthy and moribund WSSV-infected Penaeus mondon shrimps has identified a fortilin gene whose messages critically decrease during the terminal stage of the WSSV-induced illness. Fortilin/TCTPs are highly conserved throughout the animal and plant kingdoms, and shrimp fortilin has a 64% identity in amino acid composition with human fortilin. In our previous work, the data clearly suggested that fortilin in shrimp protected WSSV-infected shrimps from death. Although human fortilin has a role in apoptosis regulation, it is not known if shrimp fortilin has any role in apoptosis regulation. We report that fortilin is greatly upregulated in shrimp haemolymph during the early phase of WSSV infection and that its expression abruptly decreases as the shrimp becomes moribund. Strikingly, shrimp fortilin, when overexpressed in mammalian cells, protected them from cell death induced by etoposide, staurosporine, cisplatin, hydroxyurea, and 5-fluorouracil (5-FU). These data suggest that shrimp fortilin, like mammalian fortilin, can protect cells under toxic conditions from death. In addition, since shrimp fortilin was capable of protecting cells in a mammalian environment, this indicates that shrimp and human fortilin use a common cellular pathway to achieve this. Shrimp fortilin may play a critical role in their response to WSSVinfection, through regulation of a cell death pathway that is common to shrimp and humans. © 2006 Elsevier B.V. All rights reserved. Keywords: Penaeus monodon (Pm); White spot syndrome virus (WSSV); Anti-apoptotic protein; Fortilin; Translationally controlled tumor protein (TCTP)

1. Introduction In the past decade, white spot syndrome virus (WSSV) has been the most serious viral disease of farmed penaeid shrimp (Zhang et al., 2002b). Once ⁎ Corresponding author. Tel./fax: +66 74 288384. E-mail address: [email protected] (A. Phongdara). 0044-8486/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2005.12.023

introduced, the virus spreads rapidly and infects other species of aquatic organisms including crabs and crayfish (Chen et al., 1997; Flegel, 1997; Lo et al., 1996). The crustacean immune response can be divided into cellular and humoral components. The cellular component is related to haemocytes, that are involved in immediate defensive reactions such as nodulation, encapsulation and phagocytosis. The humoral component

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is characterized by a temporarily enhanced antimicrobial activity in the cell-free haemolymph (Söderhäll and Cerenius, 1992; Niere et al., 1999). Recently, several genes involved in the immune response of Penaeus monodon have been cloned and characterized. These include prophenoloxidase (Sritunyalucksana et al., 1999), the hemolymph clotting protein (Yeh et al., 1999), peroxinectin (Sritunyalucksana et al., 2001) and a β1,3-glucan binding protein (GBP) (Sritunyalucksana et al., 2002). In previous work, we performed subtraction hybridization of mRNAs from healthy and WSSV-infected haemocytes (Bangrak et al., 2002). Several hundred positive clones were obtained. One of the clones, showed a statistically significant similarity with the gene encoding fortilin, also known as translationally controlled tumor protein (TCTP) (Fortilin/TCTP). Intriguingly, as WSSV-infected shrimps began to exhibit signs of serious illness and death, the messages of fortilin/TCTP decreased abruptly (Bangrak et al., 2004). In addition, we found that fortilin/TCTP protein from P. monodon (Pmfortilin/TCTP) bound to Ca2+, the same as its human counterpart. Meanwhile, human fortilin/TCTP was found to bind to MCL1, a protein of the anti-apoptotic BCL-2 family (Li et al., 2001; Zhang et al., 2002a). Human fortilin/TCTP and MCL1 can exert their cytoprotective activities independently of each other (Graidist et al., 2004). On the basis of these observations we have now designated the gene isolated from shrimp as Pm-fortilin/TCTP. Fortilin/TCTP was initially identified as a growthrelated protein in mouse Ehrlich ascites tumor cells and erythroleukemia cells (Yenofsky et al., 1983; Boehm et al., 1989; Chitpatima et al., 1988; Gross et al., 1989). The protein has been known also as P21 (Yenofsky et al., 1983), Q23 (Thomas and Luther, 1981; Thomas, 1986), P23 (Benndorf et al., 1988; Boehm et al., 1989, Bohm et al., 1991), and HRF (MacDonald et al., 1995). It is widely expressed in various organs of both animals and plants (Thiele et al., 1998, 2000). A comparison of various cDNA sequences has revealed a high degree of conservation among all eukaryotes investigated, suggesting that the protein is important in cellular processes. For example shrimp fortilin/TCTPs has a 64% identity in amino acid composition to human fortilin/TCTP. Fortilin/TCTP expression is highly regulated both at the transcriptional and translational level and by a wide range of extracellular signals (Bommer et al., 2002). Fortilin/TCTP has been implicated in important cellular processes such as cell growth, cell cycle progression (Gachet et al., 1999), malignant transformation, protection of cells against various stress conditions (Bommer et

al., 2002; Bonnet et al., 2000; Xu et al., 1999) and apoptosis (Li et al., 2001; Graidist et al., 2004). In order to investigate the function of this gene in the absence of a good shrimp cell culture system, we chose to use a human cell line. The shrimp fortilin/TCTP gene was subcloned into a mammalian expression vector and overexpressed in U2OS, a human osteosarcoma cell line. We then evaluated the property of the shrimp fortilin/ TCTP in these cells. On the basis of these previous observations on shrimp, we decided to use the name fortilin throughout this report. 2. Materials and methods 2.1. WSSV experimental infections Adult P. monodon 20 g in weight were obtained from a shrimp farm in Songkhla province, Thailand. They were kept individually in 60 l aquaria for 2 d for acclimatization before experiments were started. Haemolymph (100–150 μl) was initially withdrawn from individual shrimps and labeled as normal haemolymph. It was stored at − 80 °C until used. Each shrimp was infected by injection with 10 μl of a 1 : 107 dilution of the viral stock solution made in 0.85% NaCl and 100 to 150 μl of haemolymph was subsequently carefully withdrawn at 6, 12, 24, and 72 h postinjection (p.i.). Haemolymph were stored at − 80 °C. The shrimps were raised for another 2 wk in the aquaria and mortality was recorded. 2.2. RNA isolation and cDNA synthesis Total RNA was extracted from normal and WSSVinfected haemolymph using Trizol reagent (Gibco BRL life Technologies) followed by phenol/chloroform extraction and isopropanol precipitation. cDNA synthesis was carried out using oligo (dT)12–18 and SuperScript III reverse transcriptase (Invitrogen) from 300 ng RNA. 2.3. Taqman quantitative PCR Real-time quantitation of Pm-fortilin mRNA was performed on the ABI Prism 7000 Sequence Detection System (Perkin-Elmer Applied Biosystems). Taqman primers and probes were designed using Primer Express 1.0 from Perkin-Elmer Applied Biosystems. Primers and probes were as follows: Forward primer 5′-GAG CCAATCCATCAGCTGA AGA-3′

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Reverse primer 5′-ACATCAACACCAGACTGACT AGTAGT-3′ Probe 5′-CAGTGCCTTCATCTGCC-3′. The probe was labeled with the reporter dye 6-carboxyfluorescein (6-FAM) at the 5′ end and with the quencher dye 6-carboxytetramethylrodamine (TAMRA) at the 3′ end. Quantitative PCR analysis was performed in 10 μl of Taqman PCR master mix (Perkin-Elmer Applied Biosystems), 900 nmol of each primer, and a 200 nmol probe in a final volume of 25 μl. Thermal cycling conditions were as follows: 15 min at 95 °C followed by 40 cycles of 15 s at 95 °C, 15 s at 50 °C and 1 min at 60 °C. A standard curve was constructed by plotting the cross point (Ct) against the known copy number of purified fortilin amplicon (beginning at 10 copies and extending through 107 copies). The Ct is the cycle number at which the fluorescence signal is greater than a defined threshold, one in which all the reactions are in the logarithmic phase of amplification.

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with empty pcDNA4-His-Max vector. Transfected cells were clonally selected after at least 3 weeks using 400 μg/ml Zeocin (Invitrogen, Carlsbad, CA) and characterized by Western blot analysis and detection with antiPm-fortilin antibody, anti-fortilin antibodies raised in our lab and anti-actin (Roche Applied Science) (Li et al., 2001). The resulting lines were named U2OSPm-fortilin, U2OSHuman-fortilin and U2OSempty, respectively. 2.7. Western blot analysis of cell lysates Cells were harvested, washed with cold phosphate buffered saline, counted and centrifuged at 3000 rpm for 7 min. To prevent protein degradation, the cell pellet was immediately transferred into liquid nitrogen. SDS

a 1.E+09 9.E+08 8.E+08

2.4. Cell culture conditions

7.E+08 6.E+08

U2OS cells were maintained at 37 °C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), 1% L-glutamine and 1% antibiotics (penicillin/streptomycin) in the presence of 5% CO2.

5.E+08 4.E+08 3.E+08 2.E+08 1.E+08

2.5. Molecular cloning

0.E+00 Normal

Full-length Pm-fortilin (507 bp) and Human-fortilin cDNA (526 bp) were cloned in frame to the pcDNA4His-MAX mammalian expression vector (Invitrogen, Carlsbad, CA) with Zeocin as selective marker. The cloned constructs namely pcDNA4Human-fortilin and pcDNA4Pm-fortilin, were confirmed by automated dideoxynucleotide sequencing (Lark Technologies, Houston, TX).

6h

12 h

24 h

72 h Moribund

Time after infection with WSSV

b Normal

Infected

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2.6. Generation of stable Pm-fortilin/TCTP and overexpression Pm-fortilin

For the generation of U2OS cells that stably overexpressed Pm-fortilin and Human-fortilin, one million cells of U2OS were transfected, using FuGENE6 reagent (Roche Applied Science). The transfection mixtures contained 160 μl of DMEM without serum, 30 μl of FuGENE6 and 10 μg of cDNA encoding either Pm-fortilin (pcDNA4 Pm-fortilin ) or Human-fortilin (pcDNA4 Human-fortilin). The mixtures were incubated at room temperature for 20 min then dropped wide to U2OS cells. For a control, U2OS cells were transfected

Fig. 1. a) Fortilin mRNA expression in shrimp haemolymph. cDNA samples before and after infection with WSSV were amplified by real time PCR analysis as described in Materials and methods. Results are expressed as the mean of triplicate determinations for each individual sample. b) Proteins were extracted from haemolymph of non-infected and infected shrimps and then 10 μg of proteins were subjected to 12% SDS-PAGE and Western blot analysis, using anti-Pm-fortilin and antiactin antibodies for detection.

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Trypan blue (% Dead)

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U2OSEmpty

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U2OSHuman-fortilin U2OSPm-fortilin

80 60 40 20 0 e

id

s po

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os ur

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et

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a re

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at

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o dr

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ac

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E

Pm10

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H-fortilin-HA H-fortilin

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Fig. 2. a) Effects of apoptosis inducing chemicals on mammalian cell lines that stably express Pm-fortilin (U2OSPm-fortilin) or Human-fortilin (U2OSHuman-fortilin) compared with a control cell line (U2OSEmpty). Chemicals used were 2 mM etoposide and 0.1 μM staurosporine for 5 h, 1 mM cisplatin and 2 mM hydroxyurea for 24 h and 1 mM 5-fluorouracil for 48 h. Cell viability (% of cell death) was determined using the trypan blue exclusion assay. b) Clones were evaluated by Western blot analyses with anti-Human-fortilin, anti-Pm-fortilin and anti-actin antibodies.

loading buffer (5 mM Tris–HCl, pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromophenol blue, and 10% glycerol) was then added to the frozen pellet and the samples were incubated at 45 °C for 1 h. The genomic DNA in the lysate was sheared by passing the lysate through a 27-gauge needle five times. When appropriate, cells were harvested into radioimmune precipitation assay buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate, and protease inhibitors (Complete Protease Inhibitor Mixture Tablets, Roche Applied Science), followed by determination of the protein concentrations using the BCA method (Bio-Rad). The samples were then subjected to SDS-PAGE and Western blot analysis, using anti-Pm-fortilin, anti-Human-fortilin, and antiactin antibodies for detection as described previously (Fujise et al., 2000; Li et al., 2001).

experiments were performed in the absence of Zeocin. In brief, 5 × 104 U2OS cells that stably expressed Pmfortilin and Human-fortilin were seeded into each well of a 24 well plate. Cells were harvested at 24 h after the addition of the following stimuli, 2 mM etoposide or 0.1 μM staurosporine or 1 mM cisplatin (CDDP) or 2 mM hydroxyurea (HU) or 1 mM 5-fluorouracil (5-FU) (Sigma), by a brief trypsinization and centrifuged at 3000 rpm for 7 min. Both floating and attached cells were subjected to the trypan blue staining assay. At least 150 cells were counted per treatment after being stained by trypan blue at a final concentration of 0.2%. Assays were performed in duplicate.

2.8. Trypan blue assay

Real-time PCR analysis of shrimp haemolymph samples indicated that fortilin mRNA was present at a higher level in WSSV-infected shrimp compared with normal shrimp (Fig. 1a). The mean fortilin mRNA levels

Trypan blue assay was performed as previously described (Lissy et al., 2000; Devireddy et al., 2001). All

3. Results 3.1. Expression level of Pm-fortilin in shrimp

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** *

Trypan blue (% Dead)

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50

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40 30 20 10 0 U2OS-Empty U2OS-Pm-fortilin2 U2OS-Pm-fortilin7 U2OS-Pm-fortilin10

b

U2OS

E

Pm2 Pm7 Pm10

Actin Pm-fortilin

Fig. 3. Protection of U2OS cells overexpressing Pm-fortilin from 5-FU-induced cytotoxicity. a) U2OS cells were stably transfected and clonally selected to establish clones harboring empty plasmids (E, U2OSEmpty) or overexpressing fortilin. ⁎⁎p b 0.01, comparing U2OSEmpty and U2OSPm-fortilin7 cells and ⁎⁎⁎p b 0.005 by ANOVA, comparing U2OSEmpty and U2OSPm-fortilin10 cells. (Pm2, U2OSPm-fortilin2; Pm7, U2OSPm-fortilin7; Pm10, U2OSPm-fortilin10). b) Clones were evaluated by Western blot analyses with anti-Pm-fortilin and anti-actin antibodies, HA = Hemagglutinin.

of the WSSV-infected shrimp were 5 fold greater than in normal shrimp whereas shrimps that were dying had lower levels of fortilin mRNA. A higher amount of fortilin protein was also detected by Western blot analysis in infected shrimp compared to those non-infected (Fig. 1b). 3.2. Pm-fortilin and Human-fortilin prevent cell death Ten monoclonal clones that harbored each gene were obtained from the selection process. High expression clones were characterized by Western blot analysis and tested for their susceptibility to various cell death stimuli. Cell viability determinations showed that overexpression of either shrimp or Human-fortilin suppressed cell death induced by etoposide, staurosporine, cisplatin, hydroxyurea and 5-fluorouracil (5-FU). (Fig. 2). Although the reduction of cell death in U2OS cells overexpressing fortilin was rather small, it was statistically significant and fortilin reduced the death rates consistently in cells treated by various apoptosis-inducing reagents.

Fig. 4. Protection from 5-FU induced cytotoxicity in U2OS cells overexpressing fortilin. Cells were challenged with 0–4 mM 5-FU and subjected to trypan blue assay. Overexpression of fortilin was associated with significantly higher survival in U2OSPm-fortilin10 cells comparing U2OSEmpty. ⁎⁎p b 0.01 by ANOVA.

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3.3. The protective activity of Pm-fortilin is dose dependent In testing cells overexpressing different levels of Pm-fortilin for their susceptibility to 1 mM of 5-FU (Fig. 3a), it was found that a high expression level (U2OSPm-fortilin10) made the cells more resistant to the lethal effects of 5-FU treatment than lower expression levels (U2OSPm-fortilin7, U2OSPm-fortilin2) or controls (U2OSEmpty) (P ≤ 0.005). The Pm-fortilin expression level is shown in Fig. 3b. A dose dependence analysis of the effect of exposure to 5-FU for 48 h on the viability of Pm-fortilin expressing cells indicated that U20SPm-fortilin10 cells were significantly more resistant to 5-FU-induced cell death than were U20SEmpty cells (Fig. 4). 4. Discussion Evidence that survivors of WSSV outbreaks survive subsequent challenges with WSSV (Flegel and Pasharawipas, 1998; Venegas et al., 2000) indicates the presence of a protective immune response in shrimp. Thus, many groups have concentrated their work on characterizing the shrimp protective system. Our group (Bangrak et al., 2002, 2004) has done this by using cDNA subtraction technology to differentially screen cDNA libraries from normal and white spot syndrome virus (WSSV) infected shrimp and identified fortilin. A similar result was obtained from WSSV-infected P. japonicus using EST, as reported by Rojtinnakorn et al. (2002). Recently, He et al. (2005) used the technique of suppression subtractive hybridization (SSH) and differential hybridization (DH) to identify genes differentially expressed in the haemocytes of virus-resistant P. japonicus. They found high expression of fortilin, confirming our findings. Results from these three different labs suggest that fortilin may play a critical role in the defense process during viral infection. We have previously shown that a severe systematic illness of the WSSV-infected shrimp correlates with the loss of the fortilin transcript (Bangrak et al., 2004). Here we confirmed those results by quantitative real time PCR and Western blot analysis of infected shrimp compared with non-infected shrimp. Our results strongly indicate that fortilin plays a role in protecting shrimp from viral induced mortality. The marked decline in Pm-fortilin expression in moribund shrimp infected with WSSV supports the hypothesis that apoptosis induced by WSSV is part of the pathophysiology leading to shrimp death (Flegel and Pasharawipas, 1998; Flegel, 2001; Phongdara et al.,

2005). This is also supported by the high Pm-fortilin expression reported for survivors of WSSV (He et al., 2005). Anggraeni and Owens (2000) have used TUNELpositive staining as evidence for apoptosis in lymphoid organ cells of P. monodon infected with WSSV. Sahtout et al. (2001) reported that TdT-mediated dUTP nick-end labeling (TUNEL)-positive cells were present in various tissues of naturally WSSV-infected P. monodon. Shrimp with gross signs of WSSV infection contained up to 40% apoptotic cells and it was suggested that apoptosis might be implicated in shrimp death. Recently, Wongprasert et al. (2003) experimentally infected P. monodon with WSSV and monitored the progression of necrosis and apoptosis by morphological and biochemical methods and strongly suggested that apoptosis occurred following WSSV infection in P. monodon. Pm-fortilin and Human-fortilin cDNA encode 168 and 172 amino acid polypeptides, respectively. Since Pm-fortilin prevents mammalian cells from undergoing damage, in the same way as Human-fortilin and since the amount protection is dose dependent, the results strongly suggests that shrimp fortilin is a protein that uses a conserved mechanism (common to both humans and shrimp) to protect cells from dying. It is important to determine that fortilin is protecting cells from apoptosis, not from necrosis. Our claim that fortilin protects cells against apoptosis is based on our previous observation that fortilin blocks etoposide-induced caspase 3 activation (Li et al., 2001). Caspase 3 activation occurs only in apoptosis, not in necrosis. Furthermore, Tuynder et al. (2002) reported that fortilin blocks the cleavage of PARP. PARP cleavage is a specific event in apoptosis and is not observed in necrosis. Taken together, it is appropriate to speculate that any cell death blocked by fortilin was in fact apoptotic cell death. The fact that Pm-fortilin inhibits mortality in mammalian cells even though Pm-fortilin has only a 64% sequence identity to human-fortilin, suggests that there is a considerable potential for investigating the functions of other shrimp genes using human cell culture systems. It is of interest to examine if fortilin has anything in common with the well known highly conserved family of heat shock proteins (HSPs) that are also known as stress induced proteins or molecular chaperones. Fortilin and HSPs have several similarities, i.e., both fortilin and some HSPs are induced by stressful stimuli and both protect cells against apoptosis. The difference is that while fortilin is shown here to be upregulated by severe viral infection, the same has not been shown for HSPs. In addition, the amino acid sequence of fortilin is entirely different from that of HSPs and lacks both an N-terminus ATPase domain and a C-terminus EEVD motif.

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Furthermore, fortilin is localized predominantly in the nucleus and cytosol while HSPs are located in the mitochondria and cytosol. It is possible that fortilin represents a unique class of molecule that handles stress in a different manner than HSPs. Acknowledgements This work was supported in part by grants from the National Science and Technology Development Agency, Thailand (to A. Phongdara), the Royal Golden Jubilee Graduate Program from the Thailand Research Fund (TRF; to P. Graidist), the American Heart Association (Established Investigator Award, to K. Fujise), and the National Institutes of Health (HL068024, to K. Fujise). We thank Prof. Dr. Brian Hodgson for a checking of the manuscript and valuable comments. References Anggraeni, M.S., Owens, L., 2000. The haemocytic origin of lymphoide organ spheroid cell in the penaeid prawn Penaeus monodon. Dis. Aquat. Org. 40, 85–93. Bangrak, P., Graidist, P., Chotigeat, W., Supamattaya, K., Phongdara, A., 2002. A syntenin-like protein with postsynaptic density protein (PDZ) domains produced by black tiger shrimp Peneaus monodon in response to white spot syndrome virus infection. Dis. Aquat. Org. 49 (1), 19–25. Bangrak, P., Graidist, P., Chotigeat, W., Phongdara, A., 2004. Molecular cloning and expression of a mammalian homologue of a translationally controlled tumor protein (TCTP) gene from Peneaus monodon shrimp. J. Biotechnol. 108, 219–226. Benndorf, R., Nurnberg, P., Bielka, H., 1988. Growth phase-dependent proteins of the Ehrlich ascites tumor analyzed by one- and twodimensional electrophoresis. Exp. Cell Res. 174, 130–138. Boehm, H., Benndorf, R., Gaestel, M., Gross, B., Nurnberg, P., Kraft, R., Otto, A., Bielka, H., 1989. The growth related protein P23 of the Ehrlich ascites tumor: translational control, cloning and primary structure. Biochem. Int. 19, 277–286. Bohm, H., Gross, B., Gaestel, M., Bommer, U.A., Ryffel, G., Bielka, H., 1991. The 5′-untranslated region of p23 mRNA from the Ehrlich ascites tumor is involved in translation control of the growth related protein p23. Biomed. Biochim. Acta 50, 1193–1203. Bommer, U.A., Borovjagin, A.V., Greagg, M.A., Jeffrey, I.W., Russell, P., Laing, K.G., Lee, M., Clemens, M.J., 2002. The mRNA of the translationally controlled tumor protein P23/TCTP is a highly structured RNA, which activates the dsRNA-dependent protein kinase PKR. RNA 8, 478–496. Bonnet, C., Perret, E., Dumont, X., Picard, A., Caput, D., Lenaers, G., 2000. Identification and transcription control of fission yeast genes repressed by an ammonium starvation growth arrest. Yeast 16, 23–33. Chen, X.F., Chen, C., Wu, D.H., Huai, H., Chi, X.C., 1997. A new baculovirus of cultured shrimp. Sci. China Ser. C 40, 630–635. Chitpatima, S.T., Makrides, S., Bandyopadhyay, R., Brawerman, G., 1988. Nucleotide sequence of a major messenger RNA for a 21 kilodalton peptide that is under translational control in mouse tumor cells. Nucleic Acids Res. 16, 2350.

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