Treatment with Pluchea indica (L.) Less. leaf ethanol extract alleviates liver injury in multiple low-dose streptozotocin-induced diabetic BALB/c mice.

Hyperglycemia-induced oxidative stress and inflammation are hallmarks of liver damage in diabetes mellitus. Accumulating evidence has demonstrated that Pluchea indica leaf ethanol extract (PILE) possesses strong antioxidant and anti-inflammatory properties. However, studies of its effects on liver damage in streptozotocin (STZ)-induced diabetic animals remain insufficient. To the best of our knowledge, the present study was the first to illustrate that PILE mitigated liver injury in STZ animals. Mice were first pretreated with PILE at either 50 mg/kg (PILE 50) or 100 mg/kg (PILE 100) 2 weeks prior to the induction of hyperglycemia by multiple low doses of STZ. The mice were then fed with PILE 50 or PILE 100 for 4 or 8 weeks, following which liver weight, pathological changes, oxidative stress parameters, inflammation-related markers and caspase-mediated apoptosis were measured at each time point. Untreated STZ mice exhibited abnormal increases in liver weight and severe pathological changes. However, PILE 100 reduced the severity of the STZ-induced diabetic phenotype at both time points. A significant decrease in the levels of superoxide dismutase and catalase, in addition to an increase in malondialdehyde, were observed in the livers of untreated STZ mice, all of which were significantly reversed by treatment with PILE 100 for 8 weeks. Western blot analysis revealed reduced levels of liver inflammatory markers, including interleukin-6, tumor necrosis factor-α, NF-κB p65, transforming growth factor-ß1 and protein kinase C following PILE 100 treatment. Additionally, changes in the levels of apoptotic markers indicated that PILE 100 significantly attenuated caspase-9 and -3 expression, whilst preserving that of the Bcl-2 protein. In conclusion, the present study revealed that PILE alleviates hyperglycemia-induced liver injury by normalizing the various mediators of oxidative stress, inflammation and apoptosis.

Copper and zinc differentially affect root glutathione accumulation and phytochelatin synthase gene expression of Rhizophora mucronata seedlings: Implications for mechanisms underlying trace metal tolerance.

Mangroves are susceptible to contamination due to their proximity to shores and human activities. Exposure to excessive trace metals can disturb their physiological functions and may eventually lead to death. Rhizophora mucronata is a common species growing in the mangrove forests of Thailand. Previous studies have shown that seedlings of R. mucronata are tolerant of trace metal and that they accumulate a large metal content in their root tissue. However, knowledge of their tolerance mechanisms is still lacking. To elicit the role of metal detoxification and sequestration by phytochelatins (PC) in the roots of R. mucronata seedlings, the impacts of Cu and Zn exposure were assessed on 1) physiological characteristics 2) the concentration of glutathione (GSH), a precursor of PC and 3) the level of the transcripts encoding phytochelatin synthase (PCS), the key enzyme for PC biosynthesis. Seedlings of R. mucronata were exposed to Cu and Zn in a hydroponic experiment (200 mg Cu or Zn/L in 1/4× Hoagland solution containing 8‰ NaCl, single addition). We found that both trace metals were largely accumulated in the roots. Only Cu-treated seedlings showed a decrease in the photosynthetic efficiency, in line with observed toxicity symptoms (i.e. bent stems and slight wilting of leaves). Metal accumulation, however, did not induce oxidative stress in the roots as indicated by similar level of total reactive species and lipid peroxidation across treatments. The GSH content in the roots exposed to Cu was significantly reduced while no change was observed in Zn-exposed roots. Coordinated semi-quantitative PCR and RT-qPCR revealed pcs down-regulation in Cu-treated roots, whereas Zn-treated roots showed a down-regulation on day 1 and a subsequent recovery on day 5. Failure of detoxification and sequestration of excess Cu due to GSH limitation and down-regulation of pcs may lead to the phytotoxic effects observed in Cu-treated plants. Our results suggest that both GSH and PC play an important role in trace metal tolerance in R. mucronata seedlings.

Penaeus monodon GILT enzyme restricts WSSV infectivity by reducing disulfide bonds in WSSV proteins.

Gamma-interferon-inducible lysosomal thiol reductase (GILT) is involved in the adaptive immune response via its effects on major histocompatibility complex (MHC)-restricted antigen presentation. In addition to antigen presentation, GILT exerts its antiviral activity by reducing disulfide bonds in proteins involved in viral infection and assembly, thereby inhibiting viral envelope-mediated infection and viral progeny production. In black tiger shrimp, Penaeus monodon GILT (PmGILT) was cloned and characterized, and found to be involved in the shrimp innate immune response and to exert neutralizing activity against white spot syndrome virus (WSSV) infection. However, the anti-WSSV mechanism of PmGILT in the shrimp innate immune response has not been defined. To explore the anti-WSSV activity of PmGILT, a yeast 2-hybrid (Y2H) assay was performed to identify WSSV proteins targeted by PmGILT. The assay revealed 4 potential PmGILT-interacting WSSV proteins: WSSV002, WSSV164, WSSV189, and WSSV471. Three of these 4 WSSV proteins (WSSV002, WSSV164 and WSSV189) were successfully produced and confirmed to interact with PmGILT in in vitro pull-down assays. WSSV189 and WSSV471 were previously identified as structural proteins, whereas WSSV164 is an immediate-early protein which has anti-melanization activity, and WSSV002 is an unknown. Because of the thiol reductase activity of PmGILT, WSSV164 and WSSV189, both of which are cysteine-containing WSSV proteins, were chosen for disulfide bond reduction assays. PmGILT reduced intrachain disulfide bonds in both WSSV proteins, suggesting that PmGILT exerts its anti-WSSV activity via its thiol reductase activity to disrupt the WSSV protein complex and restore the melanization activity of PmproPO1 and PmproPO2.

Phosphoproteome Profiling of Isogenic Cancer Cell-Derived Exosome Reveals HSP90 as a Potential Marker for Human Cholangiocarcinoma.

The northeastern region of Thailand is well known to have a high incidence and mortality of cholangiocarcinoma (CCA). Protein phosphorylation status has been reported to reflect a key determinant of cellular physiology, but identification of phosphoproteins can be a problem due to the presence of phosphatase. Exosomes are stable toward circulating proteases and other enzymes in human blood and can be recognized before the onset of cancer progression. Here an in vitro metastatic model of isogenic CCA cells is used to provide insight into the phosphorylation levels of exosomal proteins derived from highly invasive cells. Gel-based and gel-free proteomics approaches are used to reveal the proteins differentially phosphorylated in relation to tumor cell phenotypes. Forty-three phosphoproteins are identified with a significant change in phosphorylation level. Phos-tag western blotting and immunohistochemistry staining are then employed to validate the candidate phosphoproteins. Heat shock protein 90 is successfully confirmed as being differentially phosphorylated in relation to tumor malignancy. Importantly, the aberrant phosphorylation of exosomal proteins might serve as a promising tool for the development of a biomarker for metastatic CCA.

Ethanolic extracts of Pluchea indica (L.) leaf pretreatment attenuates cytokine-induced ß-cell apoptosis in multiple low-dose streptozotocin-induced diabetic mice.

Loss of ß-cell mass and function is a fundamental feature of pathogenesis for type 1 and type 2 diabetes. Increasing evidence indicates that apoptosis is one of the main mechanisms of ß-cell death in both types. Ethanolic extracts of Pluchea indica leaf (PILE) have been reported to possess blood glucose lowering actions in vivo. Nevertheless, further study is required to determine the underlying mechanisms. In this report, we have investigated the preventive effects of PILE on multiple low doses of streptozotocin (MLDS)-induced ß-cell apoptosis. Mice were pre-treated with PILE at 50 mg/kg (PILE 50) or 100 mg/kg (PILE 100) for 2 weeks before streptozotocin (STZ) stimulation, and the treatment continued for 4 or 8 weeks. Results revealed that PILE 100 mice exhibited improved blood biochemistry, maintained a higher body weight, had decreased hyperglycemia, and restored islet architectures compared to non-treated STZ mice. Significantly, PILE 100 decreased levels of inflammatory response markers interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and interlukin1-ß (IL-1ß), concomitant with the inhibition of caspase-3, caspase-8, capsepase-9, phosphorylation of signal transducer and activator of transcription 1 (pSTAT1), nuclear factor-κBp65 (NF-κBp65), and inducible nitric oxide synthase (iNOS). Additionally, survival and proliferative ability of ß-cells was mediated by up-regulated Bcl-2 and Ki67, respectively. These results provide strong evidence that pretreatment with PILE 100 effectively attenuated STZ-induced diabetes-related symptoms and these effects could be associated with the inhibition of cytokine-induced ß-cell apoptosis.

Elevation of serum fortilin levels is specific for apoptosis and signifies cell death in vivo.

BACKGROUND: Billions of cells undergo apoptosis each day in the average normal adult. The ability to readily assess the degree of apoptosis in human diseases is hampered by the lack of sensitive and specific serum biomarkers of apoptosis. Fortilin is a novel prosurvival molecule that protects cells against various noxious stimuli. While fortilin is secreted into the extracellular space under certain conditions, the relationship between the serum concentration of fortilin and the presence and extent of apoptosis in vivo remains unknown. METHODS & RESULTS: Using a newly developed fortilin ELISA system, we show here that fortilin exists in the normal human and mouse circulation. We further demonstrate that fortilin serum levels are significantly elevated in patients with solid cancer, in response to anti-cancer chemo- or radiation therapy. The elevation of fortilin serum levels is more robust and sensitive than that of such previously-reported serum biomarkers of apoptosis as fragmented cytokeratin-18, cytochrome c, and nucleosomal DNA. In addition, targeted apoptotic liver damage induced by Jo2 anti-Fas (CD95) antibody consistently and significantly increased serum fortilin levels in C57BL/6J mice. Finally, when challenged by anti-human-Fas IgM antibody, Jurkat leukemic T cells apoptosed and released fortilin into the medium before plasma membrane integrity was compromised. CONCLUSIONS: Taken together, these data suggest that serum fortilin levels reflect the degree and extent of apoptosis occurring in vivo. GENERAL SIGNIFICANCE: Fortilin is a viable serum biomarker of in vivo apoptosis and can be utilized to noninvasively assess the status of in vivo apoptosis in humans.

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.

ß-thymosins and hemocyte homeostasis in a crustacean.

Thymosin proteins are well known for their actin-binding activity. Thymosin beta 4 (Tß4) 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 ß-subunit of ATP synthase. Here, we identified five different ß-thymosins from Pacifastacus leniusculus, designated Pl-ß-thymosin1-5. The two dominant isoforms in brain, HPT and hemocytes, Pl-ß-thymosin1 and 2, were chosen for functional studies. Both isoforms could bind to the ß-subunit of ATP-synthase, and Pl-ß-thymosin1, but not Pl-ß-thymosin2, significantly increased extracellular ATP formation. Moreover, Pl-ß-thymosin1 stimulated HPT cell migration in vitro and Ast1 blocked this effect. Pl-ß-thymosin2 increased the circulating hemocyte number at an early stage after injection. Additionally, in vivo injection of Pl-ß-thymosin1 resulted in significant reduction of reactive oxygen species (ROS) production in crayfish HPT whereas Pl-ß-thymosin2 had a similar but transient effect. Both Pl-ß-thymosins induced the expression of Ast1 and superoxide dismutase (SOD) transcripts, while silencing of endogenous Pl-ß-thymosin 1 and 2 by RNAi resulted in significant reduction of the Ast1 and SOD transcripts. The diverse effects exhibited by Pl-ß-thymosin1 and Pl-ß-thymosin2 indicates that these proteins are involved in a complex interaction that regulates the hematopoietic stem cell proliferation and differentiation.

Characterization of a novel binding protein for Fortilin/TCTP--component of a defense mechanism against viral infection in Penaeus monodon.

The Fortilin (also known as TCTP) in Penaeus monodon (PmFortilin) and Fortilin Binding Protein 1 (FBP1) have recently been shown to interact and to offer protection against the widespread White Spot Syndrome Virus infection. However, the mechanism is yet unknown. We investigated this interaction in detail by a number of in silico and in vitro analyses, including prediction of a binding site between PmFortilin/FBP1 and docking simulations. The basis of the modeling analyses was well-conserved PmFortilin orthologs, containing a Ca(2+)-binding domain at residues 76-110 representing a section of the helical domain, the translationally controlled tumor protein signature 1 and 2 (TCTP_1, TCTP_2) at residues 45-55 and 123-145, respectively. We found the pairs Cys59 and Cys76 formed a disulfide bond in the C-terminus of FBP1, which is a common structural feature in many exported proteins and the "x-G-K-K" pattern of the amidation site at the end of the C-terminus. This coincided with our previous work, where we found the "x-P-P-x" patterns of an antiviral peptide also to be located in the C-terminus of FBP1. The combined bioinformatics and in vitro results indicate that FBP1 is a transmembrane protein and FBP1 interact with N-terminal region of PmFortilin.

Association of genetic polymorphisms in the RET-protooncogene and NRG1 with Hirschsprung disease in Thai patients.

Hirschsprung disease (HSCR) is a congenital developmental defect of the enteric nervous system known to be associated with the RET-protooncogene and other candidates. Recently, a genome-wide association study has added NRG1, a regulator of the development of the enteric ganglia precursors, as a new candidate gene. The aim of this study is to validate the association of the RET-protooncogene and the NRG1 in HSCR in Thai patients. The study used TaqMan single-nucleotide polymorphism (SNP) genotyping and PCR-restriction fragment length polymorphism for genotyping of 10 SNPs within the RET-protooncogene and four SNPs within the NRG1, in 68 Thai sporadic HSCR cases and 120 ethnic-matched controls. On univariate disease association analysis, 9 of 10 RET-protooncogene SNPs and all four NRG1 SNPs showed an association with HSCR. The rs2435357 (RET-protooncogene) and rs2439305 (NRG1) showed the strongest associations with the disease at P-values of 8.17E-09 (odds ratio (OR)=6.43, 95% confidence intervals (CI)=3.33-12.40) and 6.94E-03 (OR=3.28, 95% CI=1.28-8.38), respectively. The RET-protooncogene rs2435357 (TT genotype) in combination with the NRG1 rs2439305 (GG genotype) was strongly associated with an increased risk of HSCR with a P-value of 1.99E-04 (OR=20.34, 95% CI; 2.54-162.78) when compared with a single SNP of the RET-protooncogene or NRG1. Genetic variation of the RET-protooncogene and NRG1 is involved in the risk of HSCR development in the Thai population. Moreover, the study also detected a combined effect of SNPs by SNP-SNP interaction, which may help in predicting HSCR risk.

Isolation of the detoxification enzyme EgP450 from an oil palm EST library.

CONTEXT: Sequencing of cDNA clones from plant tissue to generate expressed sequence tags (ESTs) is an effective tool for gene discovery. Together with powerful bioinformatics tools, EST sequences allow the prediction of functions of putative bioactive compounds that can later be confirmed. OBJECTIVE: To isolate a detoxification enzyme from an EST library from the oil palm (Elaeis guineensis Jacq. Arecaceae). METHODS: In total, 750 clones from an oil palm cDNA library were randomly sequenced and analyzed. A clone homologous to cytochrome P450 monooxygenases (P450) was selected from the list of highly expressed genes. The full-length cDNA of P450 from E. guineensis (EgP450) was generated and transformed into a bacterial host to produce recombinant protein. A 3D model of EgP450 was generated and used in a molecular docking analysis to screen for target herbicide substrates. Finally, the detoxification activity of EgP450 was confirmed by an herbicide tolerance test with rice seedlings. RESULTS AND DISCUSSION: The full-length EgP450 has an open reading frame (ORF) of 1515 bp that encodes a protein of 505 amino acids. Docking analysis showed that EgP450 bound to phenylurea-like herbicides such as isoproturon, chlortoluron and fluometuron. The herbicide tolerance test demonstrated that the presence of EgP450 protected the rice seedlings from the killing action of the phytotoxic agent isoproturon. CONCLUSIONS: The gene EgP450 was detected in the roots and stems of oil palm tissues, and its recombinant product was shown to protect rice seedlings from exogenous herbicides of the phenylurea family.

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.

Molecular cloning and expression analysis of the interferon-γ-inducible lysosomal thiol reductase gene from the shrimp Penaeus monodon.

The interferon-γ-inducible lysosomal thiol reductase enzymes (GILT) have been shown to play an important role in the processing of exogenous antigens by catalyzing disulfide bond reduction, that facilitates unfolding of the native protein antigen to simplify further cleavage by cellular proteases. In this study a Penaeus monodon GILT (PmGILT) gene was isolated from an EST library of white spot syndrome virus (WSSV)-infected P. monodon. The full-length cDNA of the PmGILT gene was 780 bp and contained an open reading frame of 657 bp that encoded 218 amino acid residues with a predicted protein molecular weight of 24 kDa. The deduced amino acid sequence of PmGILT contains an active site CXXS motif, a GILT signature sequence (CQHGX(2)ECX(2)NX(4)C) and 10 conserved cysteines together with other signature characteristics of GILT proteins. RT-PCR analysis showed that the PmGILT mRNA expression level was clearly up-regulated in the lymphoid organ of both the LPS-induced and WSSV-infected shrimp, compared to normal shrimp. In response to WSSV infection, the penaeid shrimp JAK/STAT pathway is reported to play an important role in the lymphoid organ. We hypothesize that this activated STAT may stimulate GILT expression so that it can be involved in the shrimp immune response system.

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 fortilin's 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.

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.

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.

Antiapoptotic protein partners fortilin and MCL1 independently protect cells from 5-fluorouracil-induced cytotoxicity.

Fortilin, a potent 172-amino acid antiapoptotic polypeptide (Li, F., Zhang, D., and Fujise, K. (2001) J. Biol. Chem. 276, 47542-47549), binds MCL1, a protein of the antiapoptotic Bcl-2 family. The fortilin-MCL1 interaction stabilizes and increases the half-life of fortilin but not necessarily of MCL1 (Zhang, D., Li, F., Weidner, D., Mnjoyan, Z. H., and Fujise, K. (2002) J. Biol. Chem. 277, 37430-37438). It is not known to what extent each protein depends on the other for its apoptotic activity. Here, we present evidence that fortilin and MCL1 are capable of functioning as antiapoptotic proteins independently of each other. Using a robust small interfering RNA (siRNA)-mediated gene silencing system developed in our laboratory, we analyzed the cytoprotective effects of fortilin and MCL1 together and apart in U2OS cell lines exposed to 5-fluorouracil (5-FU) in both monoclonal and polyclonal cell populations. When MCL1 was silenced by MCL1-targeted siRNA, fortilin was still able to protect cells from 5-FU-induced cytotoxicity in a dose-dependent manner. Conversely, when fortilin was silenced by fortilin-targeted siRNA, MCL1 was also able to protect cells from 5-FU-induced cytotoxicity in a dose-dependent manner. Together, these data clearly suggest that fortilin and MCL1 can exert their cytoprotective activities independently of each other. The silencing of fortilin and MCL1 did not qualitatively change the subcellular localization of MCL1 and fortilin, respectively. The biological significance of fortilin-MCL1 interaction may be that it increases cellular resistance to apoptosis by allowing MCL1, an independently antiapoptotic protein, to stabilize another independently antiapoptotic protein, fortilin.

Molecular cloning and expression of a mammalian homologue of a translationally controlled tumor protein (TCTP) gene from Penaeus monodon shrimp.

White spot syndrome, caused by white spot syndrome virus (WSSV), is a deadly disease of shrimps, causing a catastrophic loss in shrimp industries worldwide. In order to investigate molecular response of shrimp haemocyte to WSSV infection, we performed subtraction hybridization of mRNAs from healthy and WSSV-infected haemocyte. One of the genes that were severely down-regulated in moribund WSSV-infected-haemocyte was translationally controlled tumor protein (TCTP) (or fortilin). Strikingly, while there was a slight difference in the amount of TCTP message between normal and early WSSV-infected shrimps, shrimps that exhibited severe symptoms uniformly had very little TCTP in their haemocyte. Taken together with the fact that TCTP functions as an anti-apoptotic protein in mammals, our data suggest that TCTP in shrimp protects WSSV-infected shrimps from death.

A syntenin-like protein with postsynaptic density protein (PDZ) domains produced by black tiger shrimp Penaeus monodon in response to white spot syndrome virus infection.

We report the isolation and characterization of products from a subtractive cDNA library from the haemolymph of Penaeus monodon experimentally infected with white spot syndrome virus (WSSV). One cDNA derived from up-regulated mRNA was identified. A homology search indicated similarity to the putative protein syntenin (TE8). The nearly complete nucleotide sequence of TE8 was obtained by rapid amplification of cDNA (RACE). Its putative protein product contained a tandem repeat of PDZ domains (postsynaptic density protein or PSD-95, DlgA and ZO-1). We propose that TE8 may function as an adapter that couples PDZ-binding protein(s) in a signaling pathway involved in the shrimp response to WSSV.