Amornrat Phongdara

Shrimp Pm-fortilin inhibits the expression of early and late genes of white spot syndrome virus (WSSV) in an insect cell model

Fortilin plays an important role in anti-apoptotic mechanisms and cell proliferation in many eukaryotic organisms. This work confirmed previous reports that Sf9 can support the replication of white spot syndrome virus (WSSV) genomic material by using immunohistochemistry with a specific antibody to detect the immediate early gene 1 (ie1) and by amplification of WSSV DNA and mRNA products. Using this insect-cell model system, we show that overexpression of Pm-fortilin in Sf9 cells inhibited the expression of WSSV early genes and late genes (WSSV-DNA polymerase, VP15 and VP28) but not an immediate early gene ie1. This is the first time that an insect cell line has been used to demonstrate interaction between a shrimp gene and genes of a shrimp virus.

Fortilin binds Ca2+ and blocks Ca2+-dependent apoptosis in vivo

Fortilin, a 172-amino-acid polypeptide present both in the cytosol and nucleus, possesses potent anti-apoptotic activity. Although fortilin is known to bind Ca2+, the biochemistry and biological significance of such an interaction remains unknown. In the present study we report that fortilin must bind Ca2+ in order to protect cells against Ca2+-dependent apoptosis. Using a standard Ca2+-overlay assay, we first validated that full-length fortilin binds Ca2+ and showed that the N-terminus (amino acids 1-72) is required for its Ca2+-binding. We then used flow dialysis and CD spectropolarimetry assays to demonstrate that fortilin binds Ca2+ with a dissociation constant (Kd) of approx. 10 mM and that the binding of fortilin to Ca2+ induces a significant change in the secondary structure of fortilin. In order to evaluate the impact of the binding of fortilin to Ca2+ in vivo, we measured intracellular Ca2+ levels upon thapsigargin challenge and found that the lack of fortilin in the cell results in the exaggerated elevation of intracellular Ca2+ in the cell. We then tested various point mutants of fortilin for their Ca2+ binding and identified fortilin(E58A/E60A) to be a double-point mutant of fortilin lacking the ability of Ca2+-binding. We then found that wild-type fortilin, but not fortilin(E58A/E60A), protected cells against thapsigargin-induced apoptosis, suggesting that the binding of fortilin to Ca2+ is required for fortilin to protect cells against Ca2+-dependent apoptosis. Together, these results suggest that fortilin is an intracellular Ca2+ scavenger, protecting cells against Ca2+-dependent apoptosis by binding and sequestering Ca2+ from the downstream Ca2+-dependent apoptotic pathways.

The role of Pm-fortilin in protecting shrimp from white spot syndrome virus (WSSV) infection.

Crustacean fortilin or the product of the translationally controlled tumor protein (TCTP) gene isolated from Penaeus monodon, is well conserved and has a Ca(++) binding domain. Pm-fortilin has anti-apoptotic properties and is present at high levels during the onset of viral infections in P. monodon. The possibility of using rFortilin to protect against white spot syndrome virus (WSSV) infection was tested. Injection of shrimp with rFortilin, after infection with WSSV, resulted in 80-100% survival and detection of very low levels of WSSV by PCR, whereas in moribund samples WSSV levels were very high. This result implies that injection of recombinant rFortilin decreases viral infection by an unknown mechanism, but probably by inhibiting viral replication. Using a yeast two-hybrid screen for cellular protein partners to rFortilin we identified an unknown protein that bound to fortilin. This is a novel polypeptide of 93 amino acids with a number of XPPX signature sequences that are often reported to have a function in antiviral peptides.

Shrimp laminin receptor binds with capsid proteins of two additional shrimp RNA viruses YHV and IMNV.

Laminin receptor (Lamr) in shrimp was previously proposed to be a potential receptor protein for Taura syndrome virus (TSV) based on yeast two-hybrid assays. Since shrimp Lamr bound to the VP1 capsid protein of TSV, we were interested to know whether capsid/envelope proteins from other shrimp viruses would also bind to Lamr. Thus, capsid/envelope encoding genes from 5 additional shrimp viruses were examined. These were Penaeus stylirostris densovirus (PstDNV), white spot syndrome virus (WSSV), infectious myonecrosis virus (IMNV), Macrobrachium rosenbergii nodavirus (MrNV), and yellow head virus (YHV). Protein interaction analysis using yeast two-hybrid assay revealed that Lamr specifically interacted with capsid/envelope proteins of RNA viruses IMNV and YHV but not MrNV and not with the capsid/envelope proteins of DNA viruses PstDNV and WSSV. In vitro pull-down assay also confirmed the interaction between Lamr and YHV gp116 envelope protein, and injection of recombinant Lamr (rLamr) protein produced in yeast cells protected shrimp against YHV in laboratory challenge tests.

Embryonic lethality of fortilin-null mutant mice by BMP-pathway overactivation

BACKGROUND Fortilin negatively regulates apoptosis and is overexpressed in cancer. However, the role of fortilin in mammalian development is not clear. METHODS AND RESULTS In order to evaluate the physiological role of fortilin in vivo, we performed a targeted disruption of the fortilin gene in mice. Fortilin(+/-) mice have the ability to survive and exhibit normal growth, while fortilin(-/-) mice are embryonically lethal around the 3.5 days post-coital (dpc). Cultured blastocysts from fortilin(+/-) embryos undergo normal outgrowth to produce inner cell mass (ICM) and trophoblasts (TB), while ICM of fortilin(-/-) embryos either fails to outgrow or prematurely disintegrates. Mouse embryonic fibroblasts (MEF) derived from fortilin(+/-) embryos are more susceptible to noxious stimuli than are wild type embryos. It has been consistently shown in Xenopus embryos that the depletion of fortilins message severely compromises the formation of neural tissue, even in the brain, while overexpression of fortilin induces the partial double body axis in embryos and is capable of blocking BMP4-induced transcription of Vent1, Vent2, and Msx1 genes. This suggests that fortilin is an inhibitor of the BMP pathway. Strikingly, when fortilin levels are reduced by siRNA, BMP4 causes MEF to undergo extensive DNA-fragmentation, while DNA fragmentation is minimal in the presence of fortilin. In addition, BMP4 induces more Msx2 in the absence of fortilin than in its presence. Furthermore, Msx2 overexpression causes MEF to undergo apoptotic cell death. CONCLUSION We conclude that in early phase of development, fortilin functions as an inhibitor of the BMP pathway. The presence of fortilin in the very early stages of development is required for the survival of embryos. GENERAL SIGNIFICANCE Abnormalities in the fortilin gene may be associated with early pregnancy loss.

Molecular phylogenetic analysis of white prawns species and the existence of two clades in Penaeus merguiensis

Closely related species, Penaeus merguiensis and Penaeus silasi from Thai waters, were genetically examined using variation observed in 558 base pairs (bp) of sequence from cytochrome oxidase subunit I (COI) gene of mtDNA. The sequence divergences of COI between P. merguiensis and other Penaeus species were 5.76–6.15% (P. silasi), 13.17–13.97% (Penaeus indicus), 16.43% (Penaeus vannamei), 16.63% (Penaeus monodon), and 18.37% (Penaeus japonicus). From the alignment reported that there were four clades on phylogenetic tree, the distinction of the two monophyletic clades was referred as P. merguiensis, one monophyletic clade within P. silasi and P. indicus. These results point toward the possibility of P. merguiensis being a complex of two cryptic species or a single species with strong phylogeographic subdivision.

WSSV: VP26 binding protein and its biological activity.

White spot syndrome virus (WSSV) is one of the major causes of disease in the shrimp culture industry causing enormous economic losses. In this study, we displayed peptides from a cDNA library obtained from the hemolymph of shrimp infected with WSSV, on the surface of phage and screened for the peptides that interacted with the WSSV. One WSSV binding protein (WBP) gene was found to consist of 171 bp that had no matches in the NCBI database. This WBP was shown to bind to the VP26 protein of the WSSV by Western blotting. In addition, WBP reduced the binding of WSSV to shrimp haemocytes from 2.0 × 10(7)copies in the control to 6.0 × 10(2) after treatment with 80 μg of WBP. The survival rate of shrimp after WSSV were mixed with WBP at 80 μg, was 89% and the binding of WBP remained unchanged for at least 24h. Therefore, the results indicate that the WBP can bind to VP26 and inhibit the invasion of WSSV into host cells. This finding may introduce another future way to try to fight this disease in shrimp culture.

Morelloflavone blocks injury-induced neointimal formation by inhibiting vascular smooth muscle cell migration

BACKGROUND In-stent restenosis, or renarrowing within a coronary stent, is the most ominous complication of percutaneous coronary intervention, caused by vascular smooth muscle cell (VSMC) migration into and proliferation in the intima. Although drug-eluting stents reduce restenosis, they delay the tissue healing of the injured arteries. No promising alternative anti-restenosis treatments are currently on the horizon. METHODS In endothelium-denudated mouse carotid arteries, oral morelloflavone-an active ingredient of the Thai medicinal plant Garcinia dulcis-significantly decreased the degree of neointimal hyperplasia, without affecting neointimal cell cycle progression or apoptosis as evaluated by Ki-67 and TUNEL staining, respectively. At the cellular level, morelloflavone robustly inhibited VSMC migration as shown by both scratch wound and invasion assays. In addition, morelloflavone prevented VSMCs from forming lamellipodia, a VSMC migration apparatus. Mechanistically, the inhibition by morelloflavone of VSMC migration was through its negative regulatory effects on several migration-related kinases, including FAK, Src, ERK, and RhoA. Consistently with the animal data, morelloflavone did not affect VSMC cell cycle progression or induce apoptosis. RESULTS These data suggest that morelloflavone blocks injury-induced neointimal hyperplasia via the inhibition of VSMC migration, without inducing apoptosis or cell cycle arrest. GENERAL SIGNIFICANCE We propose morelloflavone to be a viable oral agent for the prevention of restenosis, without compromising effects on the integrity and healing of the injured arteries.

β-thymosins and hemocyte homeostasis in a crustacean.

Thymosin proteins are well known for their actin-binding activity. Thymosin beta 4 (Tβ4) has been associated with biological activities in tissue repair and cell migration via interaction with ATP-synthase in vertebrates, while the information of similar thymosin functions in invertebrates is limited. We have shown previously that ATP-synthase is present on the surface of crayfish hematopoietic tissue (HPT) cells, and that astakine 1 (Ast1, an invertebrate cytokine) was found to interact with this β-subunit of ATP synthase. Here, we identified five different β-thymosins from Pacifastacus leniusculus, designated Pl-β-thymosin1-5. The two dominant isoforms in brain, HPT and hemocytes, Pl-β-thymosin1 and 2, were chosen for functional studies. Both isoforms could bind to the β-subunit of ATP-synthase, and Pl-β-thymosin1, but not Pl-β-thymosin2, significantly increased extracellular ATP formation. Moreover, Pl-β-thymosin1 stimulated HPT cell migration in vitro and Ast1 blocked this effect. Pl-β-thymosin2 increased the circulating hemocyte number at an early stage after injection. Additionally, in vivo injection of Pl-β-thymosin1 resulted in significant reduction of reactive oxygen species (ROS) production in crayfish HPT whereas Pl-β-thymosin2 had a similar but transient effect. Both Pl-β-thymosins induced the expression of Ast1 and superoxide dismutase (SOD) transcripts, while silencing of endogenous Pl-β-thymosin 1 and 2 by RNAi resulted in significant reduction of the Ast1 and SOD transcripts. The diverse effects exhibited by Pl-β-thymosin1 and Pl-β-thymosin2 indicates that these proteins are involved in a complex interaction that regulates the hematopoietic stem cell proliferation and differentiation.

Astakine 2—the Dark Knight Linking Melatonin to Circadian Regulation in Crustaceans

Daily, circadian rhythms influence essentially all living organisms and affect many physiological processes from sleep and nutrition to immunity. This ability to respond to environmental daily rhythms has been conserved along evolution, and it is found among species from bacteria to mammals. The hematopoietic process of the crayfish Pacifastacus leniusculus is under circadian control and is tightly regulated by astakines, a new family of cytokines sharing a prokineticin (PROK) domain. The expression of AST1 and AST2 are light-dependent, and this suggests an evolutionarily conserved function for PROK domain proteins in mediating circadian rhythms. Vertebrate PROKs are transmitters of circadian rhythms of the suprachiasmatic nucleus (SCN) in the brain of mammals, but the mechanism by which they function is unknown. Here we demonstrate that high AST2 expression is induced by melatonin in the brain. We identify RACK1 as a binding protein of AST2 and further provide evidence that a complex between AST2 and RACK1 functions as a negative-feedback regulator of the circadian clock. By DNA mobility shift assay, we showed that the AST2-RACK1 complex will interfere with the binding between BMAL1 and CLK and inhibit the E-box binding activity of the complex BMAL1-CLK. Finally, we demonstrate by gene knockdown that AST2 is necessary for melatonin-induced inhibition of the complex formation between BMAL1 and CLK during the dark period. In summary, we provide evidence that melatonin regulates AST2 expression and thereby affects the core clock of the crustacean brain. This process may be very important in all animals that have AST2 molecules, i.e. spiders, ticks, crustaceans, scorpions, several insect groups such as Hymenoptera, Hemiptera, and Blattodea, but not Diptera and Coleoptera. Our findings further reveal an ancient evolutionary role for the prokineticin superfamily protein that links melatonin to direct regulation of the core clock gene feedback loops.