Cinobufagin-induced DNA damage response activates G2/M checkpoint and apoptosis to cause selective cytotoxicity in cancer cells.

BACKGROUND: Processed extracts from toad skin and parotoid gland have long been used to treat various illnesses including cancer in many Asian countries. Recent studies have uncovered a family of bufadienolides as the responsible pharmacological compounds, and the two major molecules, cinobufagin and bufalin, have been shown to possess robust antitumor activity; however, the underlying mechanisms remain poorly understood. METHODS: Intracellular reactive oxygen species (ROS) were measured by DCFH-DA staining and flow cytometry, and DNA damage was analyzed by immunofluorescent staining and the alkaline comet assay. Cytotoxicity was measured by MTT as well as colony formation assays, and cell cycle and apoptosis were analyzed by flow cytometry. In addition, apoptosis was further characterized by TUNEL and mitochondrial membrane potential assays. RESULTS: Here we showed that sublethal doses of cinobufagin suppressed the viability of many cancer but not noncancerous cell lines. This tumor-selective cytotoxicity was preceded by a rapid, cancer-specific increase in cellular ROS and was significantly reduced by the ROS inhibitor N-acetyl cysteine (NAC), indicating oxidative stress as the primary source of cinobufagin-induced cancer cell toxicity. Sublethal cinobufagin-induced ROS overload resulted in oxidative DNA damage and intense replication stress in cancer cells, leading to strong DNA damage response (DDR) signaling. Subsequent phosphorylation of CDC25C and stabilization of p53 downstream of DDR resulted in activation of the G2/M checkpoint followed by induction of apoptosis. These data indicate that cinobufagin suppresses cancer cell viability via DDR-mediated G2 arrest and apoptosis. CONCLUSION: As elevated oxidative pressure is shared by most cancer cells that renders them sensitive to further oxidative insult, these studies suggest that nontoxic doses of cinobufagin can be used to exploit a cancer vulnerability for induction of cancer-specific cytotoxicity.

Identification and comparative analysis of the ovary and testis microRNAome of mud crab Scylla paramamosain.

The role of microRNA (miRNA) in reproductive regulation is attracting increasingly more attention. In this study, we obtained 9,643,114 and 15,498,999 raw reads from the ovary and testis library of important farmed mud crab Scylla paramamosain, respectively. After data mining, a total of 4,096,464 and 11,737,973 mappable small RNA sequences remained for analysis. By mapping to the reference genome and expressed sequence tag (EST) of Daphnia pulex and other crabs, a total of 1,417 miRNAs were identified. On the basis of 1,417 miRNAs, 514 (36.3%) unique miRNAs coexpressed in the gonad of female and male libraries, and 336 (23.7%) and 567 (40%) expressed preferentially in female and male libraries, respectively. Analysis of library sequencing data resulted in the identification of 108 miRNAs (out of 1,417; 7.6%) that showed significant differential expression between the two samples. Of these, 13 miRNAs were expressed only in the testis, two miRNAs were expressed only in the ovary, and 93 miRNAs were coexpressed: 57 (61.3%) were upregulated (ovary/testis) and 36 (38.7%) were downregulated (ovary/testis). To confirm the expression patterns of the predicted miRNAs, we randomly selected 14 candidate miRNAs from 108 differentially expressed miRNAs and performed stem-loop real time quantitative PCR (RT-qPCR) assays in five ovary developing stages. Five miRNAs showed similar expression patterns in almost every stage as those revealed by identification of differentially expressed genes (IDEG6) analysis. The above five miRNAs were predicted to match the 3'-untranslated region of the published S. paramamosain gene. Four out of five miRNA had a regulation effect on many genes, especially the genes related to gonadal development.

VIH from the mud crab is specifically expressed in the eyestalk and potentially regulated by transactivator of Sox9/Oct4/Oct1.

Vitellogenesis-inhibiting hormone (VIH) is known to regulate ovarian maturation by suppressing the synthesis of vitellogenin (Vtg) in crustaceans, which belongs to a member of crustacean hyperglycemic hormone (CHH) family synthesized and secreted from the X-organ/sinus gland complex of eyestalks. In this study, the cDNA, genomic DNA (gDNA) and the 5'-upstream regulatory (promoter region) sequences of VIH gene were obtained by conventional PCR, genome walker and tail-PCR techniques according to our transcriptomic database of Scylla paramamosain. The full-length cDNA of SpVIH is 634bp including 105bp 5'UTR, 151bp 3'UTR and 378bp ORF that encodes a peptide of 125 amino acids. The full length gDNA of SpVIH is 790bp containing two exons and one intron. The 5'-flanking promoter regions of SpVIH we isolated are 3070bp from the translation initiation (ATG) and 2398bp from the predicted transcription initiation (A), which consists of putative core promoter region and multiple potential transcription factor binding sites. SpVIH was only expressed in eyestalk. The expression level of SpVIH in eyestalk of female crab decreased gradually along with the development of ovary. As there is not cell line of crabs available, we chose the mature transfection system HEK293FT cell lines to explore the mechanism of transcription regulation of SpVIH in crabs. Sequential deletion assays using luciferase reporter gene in HEK293FT cells revealed that the possible promoter activity regions (including positive and negative transcription factors binding sites simultaneously) presented between pSpVIH-4 and pSpVIH-6. In order to further identify the crucial transcription factors binding site in this region, the site-directed mutagenesis of Sox9/Oct4/Oct1 binding site of pSpVIH-4 was created. The results demonstrated that the transcriptional activity of pSpVIH-4△ decreased significantly (p<0.05). Thus, it is reasonable to deduce that the Sox9/Oct4/Oct1 may be the essential positive transcription factors which regulate the expression of SpVIH.

Functional analysis of the promoter of the molt-inhibiting hormone (mih) gene in mud crab Scylla paramamosain.

In this study, the 5'-flanking region of molt-inhibiting hormone (MIH) gene was cloned by Tail-PCR. It is 2024 bp starting from the translation initiation site, and 1818 bp starting from the predicted transcription start site. Forecast analysis results by the bioinformatics software showed that the transcription start site is located at 207 bp upstream of the start codon ATG, and TATA box is located at 240 bp upstream of the start codon ATG. Potential transcription factor binding sites include Sp1, NF-1, Oct-1, Sox-2, RAP1, and so on. There are two CpG islands, located at -25- +183 bp and -1451- -1316 bp respectively. The transfection results of luciferase reporter constructs showed that the core promoter region was located in the fragment -308 bp to -26 bp. NF-kappaB and RAP1 were essential for mih basal transcriptional activity. There are three kinds of polymorphism CA in the 5'-flanking sequence, and they can influence mih promoter activity. These findings provide a genetic foundation of the further research of mih transcription regulation.

Molecular Characterization and Expression Profiles of Sp-uchl3 and Sp-uchl5 during Gonad Development of Scylla paramamosain.

Ubiquitin C-terminal hydrolases (UCHLs) are a subset of deubiquitinating enzymes, and are involved in numerous physiological processes. However, the role of UCHLs during gonad development has not been studied in crustaceans. In this study, we have first cloned and analyzed expression profiling of Sp-uchl3 and Sp-uchl5 genes from mud crab Scylla paramamosain. The full-length cDNA of Sp-uchl3 is of 1804 bp. Its expression level in the ovary was significantly higher than in other tissues (p < 0.01), and during gonadal development, its expression in both O1 and O5 stages was significantly higher than in the other three stages of ovaries (p < 0.05), while in T3 it was higher than in the former two stages of testes (p < 0.05). Meanwhile, the full-length cDNA of Sp-UCHL5 is 1217 bp. The expression level in the ovary was significantly higher than in other tissues (p < 0.01). Its expression in ovaries was higher than in testes during gonadal development (p < 0.05). The expression level in the O5 stage was the highest, followed by the O3 stage in ovarian development, and with no significant difference in the testis development (p > 0.05). These results provide basic data showing the role of Sp-UCHL3 and Sp-UCHL5 in the gonad development of the crab.

Induction of ROS Overload by Alantolactone Prompts Oxidative DNA Damage and Apoptosis in Colorectal Cancer Cells.

Cancer cells typically display higher than normal levels of reactive oxygen species (ROS), which may promote cancer development and progression but may also render the cancer cells more vulnerable to further ROS insult. Indeed, many of the current anticancer therapeutics kill cancer cells via induction of oxidative stress, though they target both cancer and normal cells. Recently, alantolactone (ATL), a natural sesquiterpene lactone, has been shown to induce apoptosis by increasing ROS levels specifically in cancer cells; however, the molecular mechanisms linking ROS overproduction to apoptosis remain unclear. Here we show that the ATL-induced ROS overload in human SW480 and SW1116 colorectal cancer cells was followed by a prominent accumulation of cellular oxidized guanine (8-oxoG) and immediate increase in the number of DNA strand breaks, indicating that increased ROS resulted in extensive oxidative DNA damage. Consequently, the G1/S-CDK suppresser CDKN1B (p21) and pro-apoptotic proteins Bax and activated caspase-3 were upregulated, while anti-apoptotic Bcl-2 was downregulated, which were followed by cell cycle arrest at G1 and marked apoptosis in ATL-treated cancer but not non-cancer cells. These results suggest that the ATL-induced ROS overload triggers cell death through induction of massive oxidative DNA damage and subsequent activation of the intrinsic apoptosis pathway.

Differential expression of three estrogen receptors mRNAs in tissues, growth development, embryogenesis and gametogenesis from large yellow croaker, Larimichthys crocea.

The biological activity of estrogens in target organs is mainly mediated by estrogen receptors (ERs). Herein, we addressed the isolation and expression analysis of three nuclear estrogen receptors, namely LcERα, LcERß1, and LcERß2 from Larimichthys crocea by means of SMART-RACE, qRT-PCR, and in situ hybridization. Results in different tissues showed that both LcERα and LcERß2 had the highest expression levels in female liver, followed by testis, but LcERß1 expression level was significantly higher in testis and ovary than in other tissues. Expression of LcERα and LcERß2 was significantly higher than LcERß1 in female liver, and LcERß2 was significantly higher than LcERα and LcERß1 in male liver. Moreover, we analyzed the expression of LcERs in gonad and liver at three different growth stages during the same breeding season. Significant up-regulated expression of LcERα and LcERß2 were found in female liver at 1000dph compared with at 270dph. The expression of LcERß2 was prominently higher in male liver than LcERα, LcERß1 and LcAR, while LcERß1 was lower than other receptors in male and female liver at all the three stages. In ovary, LcERα at 270dph was lower than at 635dph and 1000dph, but had no significant change in testis. The two LcERß subtypes and LcAR highly expressed in the early testis, and gradually decrease with the development of testis. In embryogenesis, a significant increase in the expression of LcERα and LcERß2 were observed after appearance of optic vesicles phase (11.8hpf). LcERß1 gradually decrease with the embryogenesis but increased dramatically at 1dph. Results of in situ hybridization showed that signals of LcERα and LcERß1 mRNA were mainly detected in Stage I-Stage IV oocytes, as well as in follicle cells around the Stage II-Stage IV and degenerated oocytes. Signals of LcERß2 were detected in the cytoplasm of Stage I and Stage II oocytes but not in the follicle cells of all oocytes stages. In parallel, LcERα and LcERß1 were detected in all cell types of spermatogenesis, but in terms of LcERß2, little or no signals were detected during spermatogenesis. Based on these results, we deduced that both LcERα and LcERß2 play a major role in mediating the physiological effects of estrogen in female liver, and LcERß2 maybe also play an important role in regulation of vitellogenesis in male liver. Differential expression of LcERs and LcAR imply their physiological functions during development and differentiation of gonad. The signals for LcERα and LcERß1 in follicle cells suggested that the follicle cell maybe an important site of estrogen action, by which estrogens exert influences on the maturation oocytes and ovulation. Furthermore, the steroid hormones produced by follicle cells may be related to the differential distributions among ER subtypes. Besides, we deduced that LcERα and LcERß1 rather than LcERß2 may play a major role in spermatogenesis of croaker. However, the differential expression of LcERß2 during gametogenesis also implicates its certain functions in mediating physiological process of estrogen action.

Echinacoside Induces Apoptosis in Human SW480 Colorectal Cancer Cells by Induction of Oxidative DNA Damages.

Echinacoside is a natural compound with potent reactive oxygen species (ROS)-scavenging and anti-oxidative bioactivities, which protect cells from oxidative damages. As cancer cells are often under intense oxidative stress, we therefore tested if Echinacoside treatment would promote cancer development. Surprisingly, we found that Echinacoside significantly inhibited the growth and proliferation of a panel of cancer cell lines. Treatment of the human SW480 cancer cells with Echinacoside resulted in marked apoptosis and cell cycle arrest, together with a significant increase in active caspase 3 and cleaved PARP, and upregulation of the G1/S-CDK blocker CDKN1B (p21). Interestingly, immunocytochemistry examination of drug-treated cancer cells revealed that Echinacoside caused a significant increase of intracellular oxidized guanine, 8-oxoG, and dramatic upregulation of the double-strand DNA break (DSB)-binding protein 53BP1, suggesting that Echinacoside induced cell cycle arrest and apoptosis in SW480 cancer cells via induction of oxidative DNA damages. These results establish Echinacoside as a novel chemical scaffold for development of anticancer drugs.

Transcriptome analysis of the differences in gene expression between testis and ovary in green mud crab (Scylla paramamosain).

BACKGROUND: The green mud crab (Scylla paramamosain) is the most prevalent crustacean on the southeast coast of China. The molecular regulatory mechanism of sex determination and gonadal differentiation in this species has received considerable attention in recent years because of the huge differences--both biological and economic--between male and female crabs. In this study, next-generation sequencing technology was used to develop deep-coverage transcriptomic sequencing data for the testis and ovary of S. paramamosain. RESULTS: A total of 365,116 reads (testis 171,962, ovary 193,154) with an average sequence length of 285 bp were produced from testis and ovary cDNA libraries. After filtering out contaminating reads, the clean reads were assembled, producing a total of 21,791 isotigs and leaving 22,814 reads as singlets. Using the BLASTX program, 3,471 unique sequences (2,275 isotigs and 1,196 singletons) were annotated with known protein sequences from the NCBI non-redundant (Nr) protein sequence database. The Gene Ontology and KEGG (Kyoto Encyclopedia of Genes and Genomes) analyses allowed the 224 unique sequences that were annotated with enzyme code (EC) numbers to be mapped into 174 KEGG pathways. After comparing the ovary and testis libraries, 4,021 gonad-differentially, 10,522 ovary-specifically, and 19,013 testis-specifically expressed genes were identified. Moreover, 33 ovary-specific, 14 testis-specific, and 34 gonad-differential transcripts were confirmed by semi-quantitative PCR and quantitative real-time PCR. In addition, 8,610 putative simple sequence repeats (SSRs) and 23,879 potential single nucleotide polymorphisms (SNPs) were identified. CONCLUSION: This is the first large-scale RNA sequencing of S. paramamosain to be reported. We have identified many important functional genes and made a preliminary attempt to construct the regulatory network involved in the gonadal development of crustaceans. The annotated transcriptome data will provide fundamental support for future research into the reproduction biology of S. paramamosain. A large number of candidate SSRs and SNPs were detected, which could be used as genetic markers for population genetics and functional genomics in this species.

Molecular cloning, characterization and expression analysis of three heat shock responsive genes from Haliotis diversicolor.

In this study, molecular characterization and expression of three heat shock responsive genes were analyzed as indicators to understand the mechanism of heat shock response of small abalone Haliotis diversicolor under stresses. The full length cDNA of heat shock transcriptional factor 1 (HdHSF1), heat shock factor binding protein 1(HSBP1), and heat shock protein 90 (HdHSP90) are 1548 bp, 809 bp, and 2592 bp respectively, encoding a protein of 515 aa, 75 aa, and 728 aa respectively. Real time quantitative PCR analysis revealed that these three genes are constitutively expressed in 7 selected tissues. The expression level of HdHSF1 in gills was higher than that in other tissues (p < 0.05). The highest expression level of HdHSBP1 was detected in hemocytes. The highest expression level of HdHSP90 was in the digestive tract and colleterial gland. The HdHSF1 expression level in the gills was up-regulated significantly (p < 0.05) after thermal stress and hypoxia exposure respectively. On the contrary, HdHSBP1 was down-regulated both in gills and hemocytes after thermal stress and the same as in gills after hypoxia stress. HdHSP90 expression level was also up-regulated in gills and hemocytes after both thermal and hypoxia stresses. These results indicated that these three heat shock responsive genes play important roles in response to thermal and hypoxia stress.

Identification and expression analysis of immune-related genes linked to Rel/NF-κB signaling pathway under stresses and bacterial challenge from the small abalone Haliotis diversicolor.

Inhibitor of NF-κB (IκB), nuclear factor-κB (NF-κB), and Akirin2 are all important members of Rel/NF-κB signaling pathway, which plays a pivotal role in regulating the innate immune response of vertebrates and invertebrates. In this study, the IκB (SaIκB) and Akirin2 (SaAkirin2) cDNAs of small abalone Haliotis diversicolor were cloned and characterized. The full length cDNA of SaIκB and SaAkirin2 were 1748 bp and 1452 bp respectively, encoding a protein of 401 aa and 187 aa respectively. A conserved degradation motif (DS56GIYS60) and six ankyrin repeats were identified in the SaIκB by SMART analysis. Meanwhile, a typical nuclear localization signal (NLS) was found at the N-terminal region of the SaAkirin2 protein. Also, the mRNA expression level of SaIκB, SaAkirin2, and AbNF-κB were detected by quantitative real-time PCR. The results revealed that all these three genes were ubiquitously expressed in 7 selected tissues. The expression level of SaIκB in gills was higher than that in other tissues (P < 0.05) while the expression level of AbNF-κB was significantly higher in hepatopancreas and haemocytes. The highest expression level of SaAkirin2 was detected in hepatopancreas, followed by mantle. The mRNA expression levels in either gills or haemocytes of SaIκB, SaAkirin2, and AbNF-κB were significantly up-regulated (P < 0.05) post thermal stress, hypoxia exposure, thermal plus hypoxia stress and the injection of Vibrio parahaemolyticus. These results indicated that these three NF-κB signaling pathway-related genes are involved in response to bacterial infection and play essential roles in response to thermal and hypoxia stress.

Identification and function analysis of steroid hormone synthesis pathway-related gene-Hsd3b in Scylla paramamosain.

Mud crab (Scylla paramamosain) has become an important mariculture crab along the southeast coast of China due to its strong adaptability, delicious taste, and rich nutrition. Several vertebrate steroid hormones and their synthesis-related genes and receptors have been found in crustaceans, but there are few reports on their synthesis process and mechanism. 3-beta-hydroxysteroid dehydrogenase (HSD3B) is a member of the Short-chain Dehydrogenase/Reductase (SDR) family, and an indispensable protein in vertebrates' steroid hormone synthesis pathway. In this study, the SpHsd3b gene sequence was obtained from the transcriptome data of S. paramamosain, and its full-length open reading frame (ORF) was cloned. The spatial and temporal expression pattern of SpHsd3b was performed by quantitative real-time PCR (qRT-PCR). SpHsd3b dsRNA interference (RNAi) and HSD3B inhibitor (trilostane) were used to analyze the function of SpHSD3B. The results showed that the SpHsd3b gene has an 1113 bp ORF encoding 370 amino acids with a 3ß-HSD domain. SpHSD3B has lower homology with HSD3B of vertebrates and higher homology with HSD3B of crustaceans. SpHsd3b was expressed in all examined tissues in mature crabs, and its expression was significantly higher in the testes than in the ovaries. SpHsd3b expression level was highest in the middle stage of testicular development, while its expression was higher in the early and middle stages of ovarian development. RNAi experiment and trilostane injection results showed that SpHSD3B had regulatory effects on several genes related to gonadal development and steroid hormone synthesis. 15-day trilostane suppression could also inhibit ovarian development and progesterone level of hemolymph. According to the above results, crustaceans may have steroid hormone synthesis pathways like vertebrates, and the Hsd3b gene may be involved in the gonadal development of crabs. This study provides further insight into the function of genes involved in steroid hormone synthesis in crustaceans.

Molecular cloning, characterization, and gene expression of the androgen receptor in the large yellow croaker, Larimichthys crocea.

Androgens mediate a wide range of physiological responses and developmental processes in vertebrates, involving both reproductive and nonreproductive systems. The activity of androgens is mediated by the androgen receptor (AR), a member of the nuclear receptor superfamily. In this study, an AR gene was cloned from the large yellow croaker (Larimichthys crocea) for the first time. qRT-PCR revealed ubiquitous expression of AR in all adult tissues examined, with higher expression in the gonad and liver of both sexes and highest expression in the blastula stage of embryonic development. Using in situ hybridization, we detected positive signals of AR in the spermatogonium, spermatocyte, spermatid, and spermatozoon during spermatogenesis, in the cytoplasm of all oocytes during oogenesis and in the follicle cells of stage IV oocytes. Our findings support the important role that AR plays in gametogenesis, gonadal development, and the early stages of embryonic development.

Synergistic lethality between auranofin-induced oxidative DNA damage and ATR inhibition in cancer cells.

AIMS: Studies in the past have shown that inhibition of the ataxia telangiectasia and Rad3-related (ATR) kinase sensitizes cancer cells to genotoxic anticancer treatments, however, clinical use of ATR inhibitors in combination with DNA damaging chemotherapy is limited due to toxicity in healthy tissues. In this study, we investigated the synergistic anticancer effect between ATR inhibition and oxidative DNA damage induced by the thioredoxin reductase inhibitor auranofin. MAIN METHODS: Cytotoxicity was evaluated by cell viability assays. Western blot, comet assay, immunostaining and flow cytometry were performed to dissect the underlying mechanisms. In vivo efficacy was examined against tumor xenografts. KEY FINDINGS: Nontoxic doses of auranofin alone increased the levels of reactive oxygen species (ROS) in cancer but not noncancerous cells, resulting in oxidative DNA damage and activation of the ATR DNA damage response pathway selectively in cancer cells. Inhibition of ATR in auranofin-treated cancer cells resulted in unscheduled firing of dormant DNA replication origins, abrogation of the S phase cell cycle checkpoint and extensive DNA breakage, leading to replication catastrophe and potent synergistic lethality. Both the antioxidant NAC and the DNA polymerase inhibitor aphidicolin reduced replication stress and synergistic cytotoxicity, implicating replication stress-driven catastrophic cell death resulted from collision between oxidative DNA damage and dysregulated DNA replication. In vivo, auranofin and VE822 coadministration enabled marked regressions of tumor xenografts, while each drug alone had no effect. SIGNIFICANCE: As increased generation of ROS is a universal feature of tumors, our findings may open new routes to broaden the therapeutic potential of ATR inhibitors.

Tissue-specific promoters regulate the transcription of cyp19a1 in the brain-pituitary-gonad axis of Japanese eel (Anguilla japonica).

Aromatase is a key enzyme that catalyzes the biosynthesis of estrogens. Previous study indicated that putative tissue-specific promoters of the one aromatase gene (cyp19a1) may drive the differential regulatory mechanisms of cyp19a1 expression in Anguilla japonica. In the present study, for elucidating the transcription characteristics and the function of putative tissue-specific promoters of cyp19a1 in the brain-pituitary-gonad (BPG) axis during vitellogenesis, we investigated the transcriptional regulation of cyp19a1 by 17ß-estrogen (E2), testosterone (T), or human chorionic gonadotropin (HCG) in A. japonica. The expression of estrogen receptor (esra), androgen receptor (ara), or luteinizing hormone receptor (lhr) was up-regulated as cyp19a1 in response to E2, T, or HCG, respectively in the telencephalon, diencephalon, and pituitary. The expression of cyp19a1 was also upregulated in the ovary by HCG or T in a dose-dependent manner. Unlike in the brain and pituitary, the expression of esra and lhr, rather than ara, was upregulated by T in the ovary. Subsequently, four primary subtypes of 5'-untranslated terminal regions of cyp19a1 transcripts and the corresponding two 5' flanking regions (promoter P.I and P.II) were identified. The P.II existed in all BPG axis tissues, whereas the P.I with strong transcriptional activity was brain- and pituitary-specific. Furthermore, the transcriptional activity of promoters, the core promoter region, and the three putative hormone receptor response elements were validated. The transcriptional activity did not change when the HEK291T cells co-transfected with P.II and ar vector were exposed to T. These results suggested that the expression of cyp19a1 was upregulated indirectly through esra and lhr rather than ara by T in the ovary, whereas the expression of cyp19a1 was upregulated directly through androgen receptor and the downstream androgen response element of tissue-specific P.I in the brain and pituitary. The results of the study reveal the regulatory mechanisms of estrogen biosynthesis and provide a reference for optimizing the technology of artificially induced maturation in eels.

Insulin-like growth factor binding protein 7, a member of insulin-like growth factor signal pathway, involved in immune response of small abalone Haliotis diversicolor.

Insulin-like growth factor binding protein 7 (IGFBP7), the only member of the IGFBP superfamily that binds strongly to insulin, may have different functions from other IGFBPs. Unlike other IGFBPs, there is no knowledge available on aquatic invertebrate IGFBP7. In this study, a molluscan IGFBP7 gene, saIGFBP7, was cloned for the first time from the small abalone Haliotis diversicolor. Its full-length cDNA sequence is 1812 bp, with a 720 bp open reading frame encoding a protein of 239 aa. The molecular mass of the deduced protein is approximately 25.37 kDa with an estimated pI of 5.00, and it shares highest 41% identity to IGFBP7 of Amblyomma americanum. Analysis of conserved domains revealed the presence of an IGFBP N-terminal domain (IB), a kazal-type serine proteinase inhibitor domain (KI), and an immunoglobulin-like C2 domain (IgC2) in saIGFBP7. Furthermore, the 12 cysteine residues and the signature amino acid motif 'xCGCCxxC' which are characterized by the amino terminus region of the IGFBP superfamily are all presented in saIGFBP7. Quantitative real-time PCR and western blot were employed to investigate the tissue distribution of saIGFBP7, and its expression under bacterial challenge. The saIGFBP7 mRNA and protein could be detected in all examined tissues, with the highest expression level in hemocytes, higher expression level in gills, and was up-regulated in hemocytes and gills after bacterial injection. In addition, saIGFBP7 mRNA transcripts were observed in a subset of the branchial epithelium and the nucleus of hemocytes using the in situ hybridization method. Interestingly, saIGFBP7 was detected mainly in the goblet-like cell of the branchial epithelium by immunohistochemistry. These results suggested that saIGFBP7 was likely to be involved in a function associated with pathogenic infection and may play an important role in the adult abalone immune system.

Selenium-dependent glutathione peroxidase gene expression during gonad development and its response to LPS and H2O2 challenge in Scylla paramamosain.

A selenium-dependent glutathione peroxidase cDNA was obtained from green mud crab Scylla paramamosain (SpGPx) by homology PCR technique and rapid amplification of cDNA ends (RACE) methods. The 1135 bp full-length cDNA contains a 9 bp 5'-untranslated region (UTR), an open reading frame (ORF) of 564 bp encoded a deduced protein of 187 amino acids (aa), and a 562 bp 3'-UTR with a 100 bp conserved eukaryotic selenocysteine insertion sequence (SECIS). It involves a putative selenocysteine (Sec4°, or U4°) residue which is encoded by an opal codon, ¹²7TGA¹²9, and forms an active site with residues Q74 and W¹4². Sequence characterization revealed that SpGPx contain a characteristic GPx signature motif 2 (64LAFPCNQF7¹), an active site motif (¹5²WNFEKF¹57), a potential N-glycosylation site (76NTT78), and two residues (R9° and R¹68) which contribute to the electrostatic architecture by directing the glutathione donor substrate. Multiple sequence alignment and phylogenetic analysis showed that SpGPx share a high level of identities and closer relationship with other selected invertebrate GPxs and vertebrate GPx1 and GPx2. Molecular modelling analysis results also supported these observations. Real time quantitative PCR analysis revealed that SpGPx was constitutively expressed in 10 selected tissues, and its expression level in gill and testis was higher than that in the other tissues (p < 0.05). The SpGPx expression increased and then declined during ovarian and testicular development implying thatnscrpits yowed that SpGPx might play an important role in gonad development by protecting them from oxidative stress. The expression of SpGPx mRNA was induced by lipopolysaccharide (LPS) and hydrogen peroxide (H2O2) in hepatopancreas and haemocytes. The results suggested that SpGPx was implicated in the immune response induced by LPS and H2O2.

A vasa gene from green mud crab Scylla paramamosain and its expression during gonadal development and gametogenesis.

VASA is one of the important regulatory factors that determine the development of the reproductive system. However, no information on vasa gene from Pleocyemata Brachyura is available. By using Race, we obtained a full-length cDNA of Sp-vasa of the green mud crab Scylla paramamosain. The full-length (2,851 bp) cDNA of vasa encodes a peptide of 631 amino acids. Real-time PCR results indicated that the expression level of Sp-vasa in the growth stage of ovary was higher than in the maturation stage, and in stage I and II of testis, the expression level of Sp-vasa were higher than in stage III. By using in situ hybridization, Sp-vasa RNAs were detected in the large part of oocyte plasm in stage I, nucleus zone in stage III and perinuclear zone in stage V. As the size of oocytes increases during oogenesis, the signals change from strong to weak. In addition, in stage I and II of testis, the expression levels of Sp-vasa were higher than in stage III, and the hybridization intensity of Sp-vasa gene gradually increased during spermatogenesis from spermatogonia to spermatids. However, no hybridization signal was detected in spermatozoon. Real-time PCR and in situ hybridization were consistent. These findings suggest that Sp-vasa is likely to serve as a useful and specific marker for germ cell development of S. paramamosain.

Gene expression profiling in respond to TBT exposure in small abalone Haliotis diversicolor.

In this study, we investigated the gene expression profiling of small abalone, Haliotis diversicolor by tributyltin (TBT) exposure using a cDNA microarray containing 2473 unique transcripts. Totally, 107 up-regulated genes and 41 down-regulated genes were found. For further investigation of candidate genes from microarray data and EST analysis, quantitative real-time PCR was performed at 6 h, 24 h, 48 h, 96 h and 192 h TBT exposure. 26 genes were found to be significantly differentially expressed in different time course, 3 of them were unknown. Some gene homologues like cellulose, endo-beta-1,4-glucanase, ferritin subunit 1 and thiolester containing protein II CG7052-PB might be the good biomarker candidate for TBT monitor. The identification of stress response genes and their expression profiles will permit detailed investigation of the defense responses of small abalone genes.

Molecular cloning and expression of interleukin-1 receptor-associated kinase 4, an important mediator of Toll-like receptor signal pathway, from small abalone Haliotis diversicolor.

Mammal interleukin-1 receptor-associated kinases (IRAKs) have been demonstrated to play important functions in TLRs (Toll-like receptor) signal pathway and T cell proliferation, but there is less knowledge available on mollusc IRAKs. In this study, a molluscan IRAK-4 gene, saIRAK-4, was cloned for the first time from the small abalone (Haliotis diversicolor). Its full-length cDNA sequence was 2062 bp, with a 1548 bp open reading frame encoding a protein of 516 aa. The molecular mass of the deduced protein was approximately 57.8 kDa with an estimated pI of 5.23, and showed highest identity (47%) to acorn worm Saccoglossus kowalevskii. Amino acid sequence analysis revealed saIRAK-4 shares conserved signature motifs with other IRAK-4 proteins, including the death domain (DD), serine/threonine/tyrosine protein kinase domain (STYKc), protein kinases ATP-binding region signature, serine/threonine protein kinases active-site signature and prokaryotic membrane lipoprotein lipid attachment site. Quantitative real-time PCR was employed to investigate the tissue distribution of saIRAK-4 mRNA, and its expression in abalone under bacteria challenge and larvae at different developmental stages. The saIRAK-4 mRNA could be detected in all examined tissues, with the highest expression level in gills, and was up-regulated in hemocytes and gills after bacteria injection. Additionally, saIRAK-4 was constitutively expressed at all examined developmental stages. These results indicate that saIRAK-4 could respond to pathogenic infection and may play an important role in the adult abalone immune system and early innate immunity in the process of abalone larval development.