Search

White spot syndrome virus (WSSV) presents a considerable peril to the aquaculture sector, leading to notable financial consequences on a global scale. Previous studies have identified hub proteins, including WSSV051 and WSSV517, as essential binding elements in the protein interaction network of WSSV. This work further investigates the functional structures and potential applications of WSSV hub complexes in managing WSSV infection. Using computational methodologies, we have successfully generated comprehensive three-dimensional (3D) representations of hub proteins along with their three mutual binding counterparts, elucidating crucial interaction locations. The results of our study indicate that the WSSV051 hub protein demonstrates higher binding energy than WSSV517. Moreover, a unique motif, denoted as "S-S-x(5)-S-x(2)-P," was discovered among the binding proteins. This pattern perhaps contributes to the detection of partners by the hub proteins of WSSV. An antiviral strategy targeting WSSV hub proteins was demonstrated through the oral administration of dual hub double-stranded RNAs to the black tiger shrimp, Penaeus monodon, followed by a challenge assay. The findings demonstrate a decrease in shrimp mortality and a cessation of WSSV multiplication. In conclusion, our research unveils the structural features and dynamic interactions of hub complexes, shedding light on their significance in the WSSV protein network. This highlights the potential of hub protein-based interventions to mitigate the impact of WSSV infection in aquaculture.

Abstract The development of non-antibiotic and environmentally friendly agents is a key consideration for health management in shrimp aquaculture. In this study, the probiotic potential in shrimp aquaculture of Pediococcus pentosaceus MR001, isolated from Macrobrachium rosenbergii, was investigated by means of feeding trial and genetic characterization. In the feeding trial, dietary supplementation with P. pentosaceus MR001 significantly increased weight gain and digestive enzyme activity (p

Abstract The 14-3-3 proteins interact with a wide variety of cellular proteins for many diverse functions in biological processes. In this study, a yeast two-hybrid assay revealed that two 14-3-3ε isoforms (14-3-3ES and 14-3-3EL) interacted with Rab11 in the white shrimp Litopenaeus vannamei (LvRab11). The interaction of 14-3-3ε and LvRab11 was confirmed by a GST pull-down assay. The LvRab11 open reading frame was 645 bp long, encoding a protein of 214 amino acids. Possible complexes of 14-3-3ε isoforms and LvRab11 were elucidated by in silico analysis, in which LvRab11 showed a better binding energy score with 14-3-3EL than with 14-3-3ES. In shrimp challenged with the white spot syndrome virus (WSSV), the mRNA expression levels of LvRab11 and 14-3-3ε were significantly upregulated at 48 h after challenge. To determine whether LvRab11 and binding between 14-3-3ε and LvRab11 are active against WSSV infection, an in vivo neutralization assay and RNA interference were performed. The results of in vivo neutralization showed that LvRab11 and complexes of 14-3-3ε/LvRab11 delayed mortality in shrimp challenged with WSSV. Interestingly, in the RNAi experiments, the silencing effect of LvRab11 in WSSV-infected shrimp resulted in decreased ie-1 mRNA expression and WSSV copy number. Whereas suppression of complex 14-3-3ε/LvRab11 increased WSSV replication. This study has suggested two functions of LvRab11 in shrimp innate immunity; (1) at the early stage of WSSV infection, LvRab11 might play an important role in WSSV infection processes and (2) at the late stage of infection, the 14-3-3ε/LvRab11 interaction acquires functions that are involved in immune response against WSSV invasion.

Heading date 3a (Hd3a) is an important flowering regulator of short-day plants, and plays a role in several developmental processes. The oil palm is commercially grown for the edible oil derived from its fruit. Flowering and fruit development are key factors for successful production, contributing to productivity of the oil palm fruit. Because little is known about the gene organization of Hd3a in the oil palm, the present study aimed at isolating the Hd3a and its regulatory region. Furthermore, alternatively spliced transcripts were cloned, and their expression in different tissues was also investigated. The structure of the oil palm Hd3a (EgHd3a-like) gene and its promoter were established based on the isolation of EgHd3a-like cDNA and genomic DNA. The promoter analysis revealed that it contains two key regulatory elements, CCAAT boxes and an ARR1 motif, which are the binding sites of Hd3a inducers. Other cis-elements corresponding to flowering and organ development were also represented. Expression of the transcript variants was investigated by RT-PCR in the anther, pistil, mesocarp and leaf. EgHd3a-like was expressed in all tissues tested, which supports its multifaceted roles in several developmental processes. The variants were found in all tested tissue types but at different levels, showing some level of tissuespecificity by variant. Taken together, these results indicate that EgHd3a-like is regulated under various conditions and that transcript variants might play an important role in gene function and regulation.

Translationally controlled tumor protein (TCTP) is a highly conserved protein found in various organisms and eukaryotes and is broadly expressed in several tissues. TCTP plays a key role in preventing apoptosis and promoting cell survival. The conserved tumor suppressor p53 is a transcription factor which responds to stress signals. p53 controls apoptosis through a series of quantitative and qualitative events which require several binding partners including TCTP. However, the exact coordination of these two molecules in the apoptosis pathway is not clearly understood. In this study, docking simulation successfully identified the binding sites of “human TCTP and human p53”, “Crustacean, Penaeus monodon TCTP and human p53”, and “plant, Elaeis guineensis TCTP and human p53” with binding energies of -843.10 Kcal/mol, -747.80Kcal/mol, and - 786.80 Kcal/mol, respectively. The results implied that non-human TCTPs have less binding activities with human p53 and they are not effective partners compared with human TCTP.

In Thailand, Scoparia dulcis is being used as a traditional medicine. The extract displays the best inhibitory effect against Pseudomonas aeruginosa and its minimum inhibitory concentration (MIC) was 400 µg/mL followed by Escherichia coli and Staphylococcus aureus with MIC 500 µg/mL. The presence of the extract significantly protected Sf9 cells against H2O2- induced cell death. In addition, the extract also showed DNA damage inhibitory effect in a concentration-dependent manner (0.5- 4 mg/mL). However, at higher concentrations (7.5-30 mg/mL) it might induce damage to the DNA. The prevention of DNA damage differs in different parts of the plant. To support secondary metabolite synthesis in different parts of S. dulcis, we investigated the expression of the Calmodulin gene that is involved in secondary metabolite production. The Calmodulin gene showed the highest expression in the fruit. This finding justifies the use of S. dulcis in the treatment of diseases caused by bacteria and free radicals.

Pm-fortilin is a recombinant protein of Penaeus monodon expressed in Escherichia coli. Activities regarding antioxidation and wound healing of the purified Pm-fortilin were investigated in vitro using normal fibroblast cell line L929. Relative viabilities of the cells were determined after exposure to either hydrogen peroxide (0.5 mmol/l for 4 h) or UV radiation (366 nm for 20 min). Significantly decreased viability was observed for the UV exposed cells compared with those incubated with the peroxide. Interestingly, proliferation of the stress-induced cells was improved by Pm-fortilin treatment. The protein’s curing effect was dose dependent. The scratch method was used in the wound healing test on L929 cells. The injured cells distinctively migrated if pre-incubation with Pm-fortilin was carried out. However, such migration ability was compromised if the injured cells were continually in contact with Pm-fortilin. The transcriptions of sydecan-2, dkk1, β-catenin, lrp6, and bmi1 genes in MG63 and HOS cancerous cells were down-regulated following incubation with Pm-fortilin for 5 days compared to those transcribed by L929 cells. The results suggested that Pm-fortilin might be non-oncogenic and is premised for use as a protective or repair agent in topical pharmaceutical products.

In our previous study, we reported that the nucleotide sequence of a 14-3-3ε cDNA clone from Litopenaeus vannamei encodes two transcripts, designated as 14-3-3EL and 14-3-3ES. Based on the retained introns we observed in 14-3-3EL by alternative splicing, we aimed to investigate the genomic structure of this gene. The 14-3-3ε gene, encoded by 2,311 bp of DNA sequence, is interrupted by three introns of 48, 1,124 and 365 bp in size. In addition, using the SWISS-MODEL, the predicted 3D model of 14-3-3EL has twelve α-helices and two β-sheets, whereas 14-3-3ES has only eleven helices in its tertiary structure. Additionally, we generated the 14-3-3EL and 14-3-3ES proteins and measured their thermal stabilities. The results demonstrated that the quantity of the 14-3-3EL protein decreased significantly at all temperatures tested. In contrast, the quantity of the 14-3-3ES protein remained unchanged, suggesting that 143-3-3EL is less stable than 14-3-3ES.

The aim of this study was to identify suitable artificial neural network (ANN) models for the EC-based and flow rate-based prediction of the nitrate nitrogen (NO3-N) concentration in the U-Tapao canal, located in the southern part of Thailand. Two types of four layer ANNs of the feed-forward back propagation (FFBP) and cascade-forward back propagation (CFBP) types were evaluated for this prediction. The selected inputs of the ANNs were EC and flow rate, which were collected daily from December 2014 to March 2015. Overall, the study found that the four layer FFBP with 2 neurons in the input layer, 20 neurons in the first hidden layer, 30 neurons in the second hidden layer, and a single neuron in the output layer with a tan-sigmoid transfer function was the optimal model. The FFBP model produced slightly more accurate results than the CFBP model. Linear regression analysis was used to predict NO3-N, which was compared with the results of the ANNs and the performance of the ANNs was better than that of the linear regression analysis. Therefore, the ANN approach proved to be suitable as an alternative to laboratory-based analysis for the prediction of NO3-N values in the U-Tapao canal.

Gamma-interferon-inducible lysosomal thiol reductase (GILT) contains a CXXC active site motif that possesses thiol reductase activity by catalyzing the disulfide bond reduction of exogenous antigens. Mutating the active site of human GILT to change the cysteine residues to serine residues eliminates this property. Our previous study reported that Penaeus monodon GILT (PmGILT) contained a CXXS active site motif. Therefore, we assessed the enzymatic activity of PmGILT and demonstrated that it displayed identical thiol reductase activity at an acidic pH. In addition, the biological activity of PmGILT against shrimp pathogens, including white spot syndrome virus (WSSV) and Gram-negative bacteria, was investigated. The neutralization of WSSV with PmGILT indicated the inhibition of WSSV invasion into shrimp hemocyte cells. Moreover, the relative percentage survival of shrimp injected with PmGILT-treated virus solution was 75%. Finally, the antimicrobial activity of PmGILT was confirmed by the growth inhibition of Vibrio harveyi. These results establish the role of PmGILT in the inhibition of the virulence of two major shrimp pathogens.

A novel cDNA of a SF21-like protein or NDR-like protein (EgNDL) from Elaeis guineensis Jacq. is 1,044 bp in length and encodes a putative protein with a 347-amino-acid open reading frame. The EgNDL showed 93% identity to the pollenspecific SF21-like protein of Phoenix dactylifera and also showed 79% identity to the NDL protein of Theobroma cacao. Expression analysis of the EgNDL gene in various tissues showed that EgNDL was expressed in the anthers, pistils, mesocarp and leaves. The high expression of EgNDL among three oil palm varieties was significantly expressed in Pisifera (P

Loss of β-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 β-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 β-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-β (IL-1β), 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 β-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 β-cell apoptosis.

Fishmeal quality is of great importance for production of aquatic animals particularly shrimp; however it varies widely depending on raw materials and processing methods. A seven week feeding trial was conducted using five diets to examine effects of fishmeal quality on growth performance, protein digestibility and expression of gene regulating trypsin in Litopenaeus vannamei. Each test diet was fed four times daily to four groups of shrimp with an average initial weight of 2.2 g shrimp-1. The shrimp fed S1-premium grade fish meal (S1-PGFM) diet attained the highest weight gain, specific growth rate with the best feed conversion ratio (FCR) and protein utilization efficiency. Growth of shrimp fed S2-PGFM, grade 1 FM and grade 2 FM containing diets was inferior to those of S1-PGFM fed group. The imported FM diet fed group showed the slowest growth and feed utilization efficiency. FCR of shrimp fed grade 2 FM and imported FM was significantly the poorest (p

Hevea brasiliensis was introduced to Thailand for the production of natural rubber. RRIT251 and RRIM600 are high yield varieties while PB350 produces a high yield of latex and resistance to diseases. The cultivar identity must be ascertained prior to plantation establishment because it is important to relate the genetic background with the production of certain secondary metabolites. In this study, the internal transcribed spacer region was used to evaluate the varieties among these 3 cultivars. Callus induction was initiated with 2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 2 mg/L 6-benzylaminopurine (BAP). Hexadecanoic acid, methyl ester and 9-octadecanoic acid (Z), methyl ester were detected in RRIM600 calli. 2-Furancarboxaldehyde-5-(hydroxymethyl) was dominant in the RRIT251 and PB350 calli. These secondary metabolites are relevant in various high performance commercial sectors.

Oil palm is an economic crop with its oil currently in high demand. In this work, the numbers of EgaccD copies, one of the key genes involved in fatty acid synthesis and oil production, were compared from two populations of oil palm, one a low and the other a high oil content productivity cultivar. Real time PCR was employed and the copy number of the EgaccD from the high oil yielding cultivar was much higher than from the low oil yielding cultivar. The EgaccD was then over expressed in oil palm calli under the control of its promoter to establish if its overexpression could enhance the oil content of transgenic calli. An increase in the lipid accumulation by the modified calli was detected by Sudan black B staining. Based on this finding, genetic manipulation of EgaccD seemed to be one promising method to try to increase the oil content. Also it seemed possible to use the level of this gene as a marker to assist selection of possible high oil yielding cultivars in breeding programs.

The Late-Embryogenesis Abundant group 4 (LEA4) genes is a group of genes that have been reported to be involved in stress and hormone responses. The completed LEA4 cDNA sequence was first obtained from a set of EST sequences of oil palm (Elaeis guineensis Jacq), named as EgLEA4. The open reading frame is 486 bp in length, encoding a deduced amino acid sequence of 161 residues with a molecular weight of 16.5 kDa and a pI value of about 8.0. Five amino acid motif patterns were found in the EgLEA4 (II, I, III, IV and V) and each had a close identity to similar LEA4 patterns of soybean (64%).Comparison of the nucleotide sequences of the cDNA and the genomic DNA demonstrated that the EgLEA4 gene is composed of 2 exons and 1 intron. The 52 untranslated region shows a putative promoter sequence involved in the transcription process, drought stress and hormone responsive elements. RT-PCR analysis showed that the EgLEA4 gene was only expressed in mesocarp, during the late stages of fruit development. It also had a higher expression in induced drought conditions indicating that the EgLEA4 protein may be involved in plant adaptation and stress (drought) responsive pathway.

MicroRNAs (miRNAs) are a distinct class of small non-coding RNAs, ~22 nt long, found in a wide variety of organisms.They play important regulatory roles by silencing gene activities at the post-transcriptional level. In this work, we developeda computational workflow to identify conserved miRNA genes in the 10,536 unique Penaeus monodon expressed sequencetags (ESTs). After removing all simple repeats and coding regions in the ESTs, the workflow uses both the conservationof miRNA sequences and several filters obtained from pre-miRNA secondary structure properties to identify conservedmiRNAs. Finally, we discovered six potential conserved miRNA genes such as mir-4152, mir-466k, miR-32*, lin-4, mir-1346 andmir-4310.

An efficient genetic transformation system using the biolistic method and protocorm-like bodies (PLBs) of Phalaenopsis orchidwas established for introducing the EgTCTP gene obtained from oil palm leaves. A pCAMBIA 1302 vector containing the greenfluorescent protein (mgfp5) as reporter gene and the selectable marker hygromycin phosphotransferase (hpt) gene under the cauliflowermosaic virus (CaMV) 35S promoter were used in this experiment. The transformed PLBs were cultured on MS medium containing 20mg/L hygromycin for 2 months. The surviving PLBs with green fluorescence spots were used to calculate a transformation frequency(93.34%). PLBs containing the transformed EgTCTP gene had the highest percentage of regeneration frequency (95.66%) and numbersof regenerated shoots per explant (3.78±1.89 shoots) compared to the control. The time required for initiation of primordial shoots inthe transformed PLBs (55.22±26.56 days) was much shorter than for the control. Evaluation of the regeneration efficiency, determinedthat the status of the EgTCTP transformants was above average: score=4.04±0.88. The EgTCTP gene was detected in the PLBs overa period of at least 6 months with subculturing every 4 weeks. The stability of the transgenes within the PLBs was confirmed by PCRand this indicated that the transgenes had been integrated into the genome of the transformants. This is the first successful report tointroduce EgTCTP gene into PLBs of Phalaenopsis orchid.

The translationally controlled tumor protein (TCTP) has now been identified in evolutionarily diverse organisms and isthought to play an important role in cell growth and cell division. We have identified an EgTCTP gene from Elaeis guineensisJacq. It is a putative protein of 168 amino acids with a calculated molecular mass of 19.2 kDa. EgTCTP has a high homology(84% - 91% identity) at the amino acid level to other plant TCTPs from Hevea brasiliensis, Arachis hypogaea and Glycinemax. The recombinant EgTCTP protein is a calcium binding protein. Transgenic embryonic calli overexpressing EgTCTP havea faster growth rate than non-transformed and empty vector transformed calli. The results show that the enhancement ofEgTCTP gene expression in oil palm embryogenic calli may result in faster multiplication of the embryogenic calli. EgTCTPacts as another Ca2+-modulated protein that is involved in the cell cycle progression.

Latex from Hevea brasiliensis contains 30-50% (w/w) of natural rubber (cis-1,4-polyisoprene), the important rawmaterial for many rubber industries. We have constructed a cDNA library from the latex of H. brasiliensis to investigate theexpressed genes and molecular events in the latex. We analyzed 412 expressed sequence tags (ESTs). More than 90% of theEST clones showed homology to previously described sequences in public databases. Functional classification of the ESTsshowed that the largest category were proteins of unknown function (30.1%), 11.4% of ESTs encoded for rubber synthesisrelatedproteins (RS) and 8.5% for defense or stress related proteins (DS). Those with no significant homology to knownsequences (NSH) accounted for 8.7%, primary metabolism (PM) and gene expression and RNA metabolism were 7.8% and6.6%, respectively. Other categories included, protein synthesis-related proteins (6.6%), chromatin and DNA metabolism(CDM 3.9%), energy metabolism (EM 3.4%), cellular transport (CT 3.2%), cell structure (CS 3.2%), signal transduction (ST2.2%), secondary metabolism (SM 1.7%), protein fate (PF 2.2%), and reproductive proteins (RP 0.7%).

Zygotic embryos of oil palm (Elaeis guineensis Jacq. var. tenera) were excised and cultured on MS medium containing 3 mg/l 2, 4-D either with or without 0.05% activated charcoal (AC). Improved growth of embryos was obtained on MS medium supplemented with 0.05% AC. Callus cultures were initiated from embryos, young leaves and roots on MS medium containing 2, 4-D, NAA and 0.05% AC. On these media, two morphologically distinct types of white and yellow compact calluses were produced. Green shoots regenerated after several transfers of the yellow compact calluses from zygotic embryos to MS medium supplemented with 15 mg/l chitosan either with or without 5 mg/l 2, 4-D. Histological sectioning revealed that regenerated shoots originated from a clump of meristematic cells that had dense cytoplasm. Regenerated shoots rooted when transferred to MS medium in the presence of 0.05% AC. Transfer of plantlets to soil was achieved. Callus from young seedling leaves and roots did not regenerate shoots or roots in medium containing 2, 4-D or TDZ, with or without chitosan. This finding shows that chitosan can initiate organogenesis in oil palm callus.

A crustin gene (Amk1) was identified from a haemocyte library of the black tiger shrimp, Penaeus monodon. The full-length cDNA consists of 411 bp encoding a deduced precursor of 136 amino acids with a signal peptide of 17 aminoacids. Amk1 contains a hydrophobic and a Gly-rich region at the N-terminus and a 12 conserved cysteine domain (6-DSC) at the C-terminus. Transcripts of Amk1 are mainly detected in haemocytes and gills by RT-PCR analysis. A recombinant Amk1was overexpressed and purified from Escherichia coli. This has a molecular mass of 43.66 kDa with a predicted pI of 8.23. Antibacterial assays demonstrated that recombinant Amk1 exhibited antibacterial activity against Gram-positive and Gramnegativebacteria with strong inhibition against Staphylococcus aureus and Vibrio harveyi.

Babylonia areolata is an aquatic mollusk in the genus of the gastropod family Buccinidae. Genetic variations were observed in the 542 base pairs of the sequences from its cytochrome oxidase subunit I (COI) gene of mtDNA. COI alignments showed that there were two monophyletic clades on the phylogenetic tree, referred as B. areolata. These results point towards the possibility of B. areolata being a single species with strong phylogeographic subdivisions. About 520 base pairs of the internal transcribed spacer 1 (ITS1) sequence were able to distinguish between the shell colors (brown, cream and white) of B. areolata.

An efficient genetic transformation system for oil palm using particle bombardment was established. The transformation was performed using the pCAMBIA 1302 DNA which contains the green fluorescent protein (mgfp5) reporter gene and the selectable marker hygromycin phosphotransferase (hph) gene. Oil palm explants were bombarded under the following conditions: rupture disk to macrocarrier distance, 11 mm; macrocarrier to target tissue, 90 mm and using 1 μm gold particles as microcarrier. Four different pressures of helium were tested with three types of target tissues (mature embryo, embryogenic callus and young seedlings). From the transformation efficiency, calli were much more efficiently transformed in the biolistic process compared with mature embryos and seedlings. A 100% transformation efficiency for DNA delivery into callus oil palm explants was obtained at 850 psi helium pressures, for embryos a maximum 81.8% efficiency required 850 psi and for seedlings a maximum 75.9% efficiency required 1,550 psi. Using a confocal laser scanning microscope, and appropriate filters to block out the red fluorescence of chlorophyll, expression of the GFP gene was observed in all three bombarded explant types by a bright-green fluorescence. The mgfp5 gene was still present more than 8 months after bombardment, hence it indicated the stability of transgene in those transformants.

Sinus gland from Penaeus merguiensis De man, 1888 was extracted and proteins were fractionated on a HPLC column (μBondapack-phenyl column, 9 mm i.d., 10 μm particle size, Waters). Three major peaks (peaks 3, 5 and 6) were collected and investigated for crustacean hyperglycemic hormone (CHH) and gonad inhibiting hormone activity (GIH). The CHH activity of the peak 5 fraction was significantly higher than the control while peak 6 did not have significant CHH activity. No CHH activity was found in the peak 3 fraction. GIH activity was determined by inhibition of total protein and vitellin synthesis in ovarian tissue. An antivitellin antibody was prepared and used for immunoprecipitation in the GIH activity assay. Hence GIH activity was detected in peaks 5 and 6 while peak 3 had gonad stimulating hormone like activity (GSH-like activity).

Fucoidan, a sulfated polysaccharide with several biological activities, is usually isolated from marine seaweeds or from echinoderms. Here, we report on the anti-tumour activity of fucoidan isolated from the aquatic plant Utricularia aurea Lour (Lentibulariaceae). A crude extract (CE) prepared by incubating U.aurea with hot water at 95ºC for 12 hr was partially purified by Sephadex G-50, eluting with a 50mM sodium acetate buffer, at pH 5.0, containing 0.5M NaCl. Partially purified fucoidan (PPF) had a 3- fold increase in fucose content when compared with the CE and a molecular weigÄt of 11.6 kDa as determined by Sephadex G-200. Chemical analysis showed that CE consisted of 62.5% glucuronic acid, 5.0% fucose, 1.7% sulfate and 12.0% proteins while PPF consisted of 65.0% glucuronic acid, 15.3% fucose, 2.1 % sulfate and 8.3% proteins.The anti-tumour activity of the CE and PPF was determined by the MTT test. The CE at 125 μg/mL fucoidan and PPF at 250 μg/mL inhibited the growth of KB cells (a nasopharynx tumour cell line), but did not inhibit that of normal fibroblast cells. The inhibition was postulated to occur via apoptosis as significantlymore apoptotic cells were found after treatment than in the untreated KB cells (P

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.

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.

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.

BackgroundLitopenaeus Vannamei, well known as pacific white shrimp, is the most popular shrimp in the world shrimp market. Identification and characterization of shrimp muscle regulatory genes are not only important for shrimp genetic improvement, but also facilitate comparative genomic tools for understanding of muscle development and regeneration.Methodology/principal findingsA novel mRNA encoding for a putative DNA-binding protein LvDBP23 was identified from Litopenaeus vannamei abdominal muscle cDNA library. The LvDBP23 cDNA contains 639 nucleotides of protein-coding sequence with deduced 212 amino acids of predicted molecular mass 23.32 kDa with glycine-rich domain at amino acid position 94-130. The mRNA sequence is successfully used for producing LvDBP23 recombinant protein in sf9 insect cell expression system. The expression of LvDBP23 mRNA is presented in abdominal muscle and swimming leg muscle, as well as other tissues including intestine, lymphoid and gill. The mRNA expression has the highest level in abdominal muscle in all tested tissues. LVDBP23 transcript during the molt cycle is highly expressed in the intermolt stage. In vitro nucleic acid-binding assays reveal that LvDBP23 protein can bind to both ssDNA and dsDNA, indicating its possible role of regulation of gene transcription.Conclusions/significanceWe are the first to report a DNA-binding protein identified from the abdominal muscle tissue of marine shrimp L. Vannamei. Its high-level specific expression during the intermot stage suggests its role in the regulation of muscle buildup during the growth phase of shrimp molt cycle.