Development of expressed sequence tags from the bay scallop, Argopecten irradians irradians

The bay scallop, Argopecten irradians irradians, introduced from North America, has become one of the most important aquaculture species in China. Inan effort to identify scallop genes involved in host defense, a high-quality cDNA library was constructed from whole body tissues of the bay scallop. A total of 5828 successful sequencing reactions yielded 4995 expressed sequence tags (ESTs) longer than 100 bp. Cluster and assembly analyses of the ESTs identified 637 contigs (consisting of 2853 sequences) and 2142 singletons, totaling 2779 unique sequences. Basic Local Alignment Search Tool (BLAST) analysis showed that the majority (73%) of the unique sequences had no significant homology (E-value >= 0.005) to sequences in GenBank. Among the 748 sequences with significant GenBank matches, 160 (21.4%) were for genes related to metabolism, 131 (17.5%) for cell/organism defense, 124 (16.6%) for gene/protein expression, 83 (11.1%) for cell structure/motility, 70 (9.4%) for cell signaling/communication, 17 (2.3%) for cell division, and 163 (21.8%) matched to genes of unknown functions. The list of host-defense genes included many genes with known and important roles in innate defense such as lectins, defensins, proteases, protease inhibitors, heat shock proteins, antioxidants, and Toll-like receptors. The study provides a significant number of ESTs for gene discovery and candidate genes for studying host defense in scallops and other molluscs.

Discovery of the genes in response to white spot syndrome virus (WSSV) infection in Fenneropenaeus chinensis through cDNA microarray

We used microarray technology to study differentially expressed genes in white spot syndrome virus (WSSV)-infected shrimp. A total of 3136 cDNA targets, including 1578 unique genes from a cephalothorax cDNA library and 1536 cDNA clones from reverse and forward suppression subtractive hybridization (SSH) libraries of Fenneropenaeus chinensis, plus 14 negative and 8 blank control clones, were spotted onto a 18 x 18 mm area of NH2-modified glass slides. Gene expression patterns in the cephalothorax of shrimp at 6 h after WSSV injection and moribund shrimp naturally infected by WSSV were analyzed. A total of 105 elements on the arrays showed a similar regulation pattern in artificially infected shrimp and naturally infected moribund shrimp; parts of the results were confirmed by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The up-regulated expression of immune-related genes, including heat shock proteins (HSP70 and HSP90), trehalose-phosphate synthase (TPS), ubiquitin C, and so forth, were observed when shrimp were challenged with WSSV. Genes including myosin LC2, ATP synthase A chain, and arginine kinase were found to be down-regulated after WSSV infection. The expression of housekeeping genes such as actin, elongation factor, and tubulin is not stable, and so these genes are not suitable as internal standards for semiquantitative RT-PCR when shrimp are challenged by WSSV. As a substitute, we found that triosephosphate isomerase (TPI) was an ideal candidate of interstandards in this situation.

Evaluation of induced triploid shrimp Penaeus (Fenneropenaeus) chinensis cultured under laboratory conditions

Triploid Penaeus (Fenneropenaeus) chinensis was successfully produced by heat shock. Their metamorphosis, the relationship between body weight and length and difference in appearance between triploids and their diploid siblings under laboratory culture were studied. Hematological studies showed a smaller number of haemocytes, but larger cell volume, in triploids than in diploids. Triploid shrimp did not show higher growth during the immature stage, but exhibited superior growth during the maturation stage. Characteristics of reproductive organs indicated that triploid shrimp may be sterile and sex ratio can be changed through triploidization of shrimp. This paper summarizes the progress made in triploid shrimp research which would be helpful in understanding more about triploids of crustaceans. (c) 2006 Elsevier B.V. All rights reserved.

Screening of genes related to ovary development in Chinese shrimp Fenneropenaeus chinensis by suppression subtractive hybridization

The ovary of triploid shrimp Fenneropenaeus chinensis was apparently impaired compared to that of the diploid shrimp at the same age. Therefore triploid shrimp ovary is possible to be taken as a model to understand the mechanism of ovary development of shrimp compared to that of the ovary of diploid shrimp at the same age. In the present study, a suppression subtractive hybridization (SSH) technique was applied to identify differentially expressed genes in the ovary between diploid and triploid shrimp. For the forward library (RNA from the ovary of triploid shrimp as the tester), 54 genes were identified. For the reverse library (RNA from the ovary of diploid shrimp as the tester), 16 genes were identified. The identified genes encoded proteins with multiple functions, including extracellular matrix components, cytoskeleton, cell growth and death, metabolism, genetic information processing, signal transduction/transport or immunity related proteins. Eleven differentially expressed genes were selected to be confirmed in the ovaries of triploid and diploid shrimp by semi-quantitative RT-PCR. Genes encoding spermatogonial stem-cell renewal factor, cytochrome c oxidase subunits I and II, clottable protein, antimicrobial peptide and transposase showed up-regulated expressions in the ovary of triploid shrimp. Genes encoding tubulin, cellular apoptosis susceptibility protein, farnesoic acid O-methyltransferase, thrombospondin and heat shock protein 90 genes showed higher expressions in the ovary of diploid shrimp. The differential expressions of the above genes are suggested to be related to the ovary development of shrimp. It will provide a new clue to uncover the molecular mechanisms underlying the ovarian development in penaeid shrimp. (C) 2010 Elsevier Inc. All rights reserved.

Cloning and expression analysis of a HSP70 gene from Pacific abalone (Haliotis discus hannai)

Heat shock protein 70 (HSP70), the primary member of HSPs that are responsive of thermal stress, is found in all multicellular organisms and functions mostly as molecular chaperon. The inducible HSP70 cDNA cloned from Pacific abalone (Haliotis discus hannai) using rapid amplification of cDNA ends (RACE), was highly homologous to other HSP70 genes. The full-length cDNA of the Pacific abalone HSP70 was 2631 bp, consisting of a 5'-terminal untranslated region (UTR) of 90 bp, a 3'-terminal UTR of 573 by with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame of 1968 bp. The HSP70 cDNA encoded a polypeptide of 655 amino acids with an ATPase domain of 382 amino acids, the substrate peptide binding domain of 161 amino acids and a C-terminus domain of 112 amino acids. The temporal expression of HSP70 was measured by semi-quantitative RT-PCR after heat shock and bacterial challenge. Challenge of Pacific abalone with heat shock or the pathogenic bacteria Vibrio anguillarum resulted in a dramatic increase in the expression of HSP70 mRNA level in muscle, followed by a recovery to normal level after 96 h. Unlike the muscle, the levels of HSP70 expression in gills reached the top at 12 h and maintained a relatively high level compared with the control after thermal and bacterial challenge. The upregulated mRNA expression of HSP70 in the abalone following heat shock and infection response indicates that the HSP70 gene is inducible and involved in immune response. (c) 2006 Elsevier Ltd. All rights reserved.

Proteomic analysis of the larval stage of the parasite Echinococcus granulosus: causative agent of cystic hydatid disease

Gustavo Chemale, Arjan J. van Rossum, James R. Jefferies, John Barrett, Peter M. Brophy, Henrique B. Ferreira, Arnaldo Zaha (2003). Proteomic analysis of the larval stage of the parasite Echinococcus granulosus: causative agent of cystic hydatid disease. Proteomics, 3(8), 1633-1636. Sponsorship: CNPq / PADCT/CNPq / FAPERGS (Brazil)/ BBSRC (UK) RAE2008

Salicylic acid dependent signaling promotes basal thermotolerance but is not essential for acquired thermotolerance in Arabidopsis thaliana

Clarke, S. M., Mur, L. A. J., Wood, J. E., & Scott, I. M. (2004). Salicylic acid dependent signaling promotes basal thermotolerance but is not essential for acquired thermotolerance in Arabidopsis thaliana. The Plant Journal, 38(3), 432-447. Sponsorship: BBSRC RAE2008

Proteomic approach to oxidative stress in Daphnia magna

The keystone aquatic organism Daphnia magna is extensively used to assess the toxicity of chemicals. This has recently lead to an increase in the omics literature focusing on daphnids, an increase fuelled by the sequencing of the Daphnia pulex genome. Yet, no omics study has looked directly at oxidative stress (OS) in daphnids, even though OS is of primary importance in the response of aquatic organisms to their changing environment and is often induced by anthropogenic xenobiotics. This thesis thus focuses on the application of redox-proteomics, the study of the oxidative modification of proteins, to D. magna Specifically, daphnids were exposed to copper or paraquat, two well studied prooxidants, and protein carbonyls were labelled with fluorescein-5-thiosemicarbazide prior to twodimensional electrophoresis (2DE). This showed clearly that both compounds affect a different portion of the proteome. The identified proteins indicated that energy metabolism was affected by paraquat, while copper induced a reduction of the heat shock response (heat shock proteins, proteases and chaperones) a counterintuitive result which may be adaptative to metal toxicity in arthropods. The same approach was then applied to the study of the toxicity mechanism of silver nanoparticles (AgNP), an increasingly utilised form of silver with expected environmental toxicity, and its comparison to silver nitrate. The results demonstrate that, although less toxic than silver ions, AgNP toxicity functions through a different mechanism. AgNP toxicity is thus not a product of silver dissolution and increased protein carbonylation indicates that AgNP cause OS. Interestingly three of the four tested compounds altered vitellogenin levels and oxidation. Vitellogenins could thus represent an interesting subproteome for the detection of stress in daphnids. Finally, an experiment with oxidised BSA demonstrates the applicability of solid phase hydrazide in the enrichment of undigested carbonylated proteins.

A binding site for the cyclic adenosine 3',5'-monophosphate-response element-binding protein as a regulatory element in the grp78 promoter.

The 78-kDa glucose-regulated protein (GRP78) is ubiquitously expressed in many cell types. Its promoter contains multiple protein-binding sites and functional elements. In this study we examined a high affinity protein-binding site spanning bp -198 to -180 of the rat grp78 promoter, using nuclear extracts from both B-lymphoid and HeLa cells. This region contains a sequence TGACGTGA which, with the exception of one base, is identical to the cAMP-response element (CRE). Site-directed mutagenesis reveals that this sequence functions as a major basal level regulatory element in hamster fibroblast cells and is also necessary to maintain high promoter activity under stress-induced conditions. By gel mobility shift analysis, we detect two specific protein complexes. The major specific complex I, while immunologically distinct from the 42-kDa CRE-binding protein (CREB), binds most strongly to the grp site, but also exhibits affinity for the CRE consensus sequence. As such, complex I may consist of other members of the CREB/activating transcription factor protein family. The minor specific complex II consists of CREB or a protein antigenically related to it. A nonspecific complex III consists of the Ku autoantigen, an abundant 70- to 80-kDa protein complex in HeLa nuclear extracts. By cotransfection experiments, we demonstrate that in F9 teratocarcinoma cells, the grp78 promoter can be transactivated by the phosphorylated CREB or when the CREB-transfected cells are treated with the calcium ionophore A23187. The differential regulation of the grp78 gene by cAMP in specific cell types and tissues is discussed.

Ligation of cell surface GRP78 with antibody directed against the COOH-terminal domain of GRP78 suppresses Ras/MAPK and PI 3-kinase/AKT signaling while promoting caspase activation in human prostate cancer cells.

We have previously shown that treatment of prostate cancer and melanoma cells expressing GRP78 on their cell surface with antibody directed against the COOH-terminal domain of GRP78 upregulates and activates p53 causing decreased cell proliferation and upregulated apoptosis. In this report, we demonstrate that treatment of 1-LN prostate cancer cells with this antibody decreases cell surface expression of GRP78, Akt(Thr308) and Akt(Ser473) kinase activities and reduces phosphorylation of FOXO, and GSK3beta. This treatment also suppresses activation of ERK1/2, p38 MAPK and MKK3/6; however, it upregulates MKK4 activity. JNK, as determined by its phosphorylation state, is subsequently activated, triggering apoptosis. Incubation of cells with antibody reduced levels of anti-apoptotic Bcl-2, while elevating pro-apoptotic BAD, BAX and BAK expression as well as cleaved caspases-3, -7, -8 and -9. Silencing GRP78 or p53 gene expression by RNAi prior to antibody treatment abrogated these effects. We conclude that antibody directed against the COOH-terminal domain of GRP78 may prove useful as a pan suppressor of proliferative/survival signaling in cancer cells expressing GRP78 on their cell surface.

Thermodynamic analysis of a molecular chaperone binding to unfolded protein substrates.

Molecular chaperones are a highly diverse group of proteins that recognize and bind unfolded proteins to facilitate protein folding and prevent nonspecific protein aggregation. The mechanisms by which chaperones bind their protein substrates have been studied for decades. However, there are few reports about the affinity of molecular chaperones for their unfolded protein substrates. Thus, little is known about the relative binding affinities of different chaperones and about the relative binding affinities of chaperones for different unfolded protein substrates. Here we describe the application of SUPREX (stability of unpurified proteins from rates of H-D exchange), an H-D exchange and MALDI-based technique, in studying the binding interaction between the molecular chaperone Hsp33 and four different unfolded protein substrates, including citrate synthase, lactate dehydrogenase, malate dehydrogenase, and aldolase. The results of our studies suggest that the cooperativity of the Hsp33 folding-unfolding reaction increases upon binding with denatured protein substrates. This is consistent with the burial of significant hydrophobic surface area in Hsp33 when it interacts with its substrate proteins. The SUPREX-derived K(d) values for Hsp33 complexes with four different substrates were all found to be within the range of 3-300 nM.

The prevalence and regulation of antisense transcripts in Schizosaccharomyces pombe.

A strand-specific transcriptome sequencing strategy, directional ligation sequencing or DeLi-seq, was employed to profile antisense transcriptome of Schizosaccharomyces pombe. Under both normal and heat shock conditions, we found that polyadenylated antisense transcripts are broadly expressed while distinct expression patterns were observed for protein-coding and non-coding loci. Dominant antisense expression is enriched in protein-coding genes involved in meiosis or stress response pathways. Detailed analyses further suggest that antisense transcripts are independently regulated with respect to their sense transcripts, and diverse mechanisms might be potentially involved in the biogenesis and degradation of antisense RNAs. Taken together, antisense transcription may have profound impacts on global gene regulation in S. pombe.

PKC signaling regulates drug resistance of the fungal pathogen Candida albicans via circuitry comprised of Mkc1, calcineurin, and Hsp90.

Fungal pathogens exploit diverse mechanisms to survive exposure to antifungal drugs. This poses concern given the limited number of clinically useful antifungals and the growing population of immunocompromised individuals vulnerable to life-threatening fungal infection. To identify molecules that abrogate resistance to the most widely deployed class of antifungals, the azoles, we conducted a screen of 1,280 pharmacologically active compounds. Three out of seven hits that abolished azole resistance of a resistant mutant of the model yeast Saccharomyces cerevisiae and a clinical isolate of the leading human fungal pathogen Candida albicans were inhibitors of protein kinase C (PKC), which regulates cell wall integrity during growth, morphogenesis, and response to cell wall stress. Pharmacological or genetic impairment of Pkc1 conferred hypersensitivity to multiple drugs that target synthesis of the key cell membrane sterol ergosterol, including azoles, allylamines, and morpholines. Pkc1 enabled survival of cell membrane stress at least in part via the mitogen activated protein kinase (MAPK) cascade in both species, though through distinct downstream effectors. Strikingly, inhibition of Pkc1 phenocopied inhibition of the molecular chaperone Hsp90 or its client protein calcineurin. PKC signaling was required for calcineurin activation in response to drug exposure in S. cerevisiae. In contrast, Pkc1 and calcineurin independently regulate drug resistance via a common target in C. albicans. We identified an additional level of regulatory control in the C. albicans circuitry linking PKC signaling, Hsp90, and calcineurin as genetic reduction of Hsp90 led to depletion of the terminal MAPK, Mkc1. Deletion of C. albicans PKC1 rendered fungistatic ergosterol biosynthesis inhibitors fungicidal and attenuated virulence in a murine model of systemic candidiasis. This work establishes a new role for PKC signaling in drug resistance, novel circuitry through which Hsp90 regulates drug resistance, and that targeting stress response signaling provides a promising strategy for treating life-threatening fungal infections.

The inoculum effect and band-pass bacterial response to periodic antibiotic treatment.

The inoculum effect (IE) refers to the decreasing efficacy of an antibiotic with increasing bacterial density. It represents a unique strategy of antibiotic tolerance and it can complicate design of effective antibiotic treatment of bacterial infections. To gain insight into this phenomenon, we have analyzed responses of a lab strain of Escherichia coli to antibiotics that target the ribosome. We show that the IE can be explained by bistable inhibition of bacterial growth. A critical requirement for this bistability is sufficiently fast degradation of ribosomes, which can result from antibiotic-induced heat-shock response. Furthermore, antibiotics that elicit the IE can lead to 'band-pass' response of bacterial growth to periodic antibiotic treatment: the treatment efficacy drastically diminishes at intermediate frequencies of treatment. Our proposed mechanism for the IE may be generally applicable to other bacterial species treated with antibiotics targeting the ribosomes.

HAMSTER OVIDUCTIN ENHANCES TYROSINE PHOSPHORYLATION OF SPERM PROTEINS DURING CAPACITATION

Capacitation is essential for fertilization of ovulated oocytes. Capacitation is correlated with activation of a signal transduction pathway leading to protein tyrosine phosphorylation, an essential prerequisite for fertilization. Oviductin has been shown to bind to the acrosomal cap and the equatorial segment region of the sperm head. In light of findings reported in previous studies, we hypothesized that estrus stage-specific oviductin (EOV) enhances tyrosine phosphorylation. Immunofluorescent detection by light and confocal microscopy and immunogold labeling by electron microscopy and surface replica techniques were used to localize tyrosine phosphorylated proteins to the equatorial segment region and midpiece after incubation in medium in the presence or absence of EOV. In the presence of EOV, an increase in tyrosine phosphorylation in the equatorial segment region was observed as early as 5 minutes after incubation. On prolonging incubation in medium containing EOV immunostaining further increased, indicative of increased levels of tyrosine phosphorylation of sperm proteins as capacitation proceeds. Regardless of the presence or absence of EOV, phosphotyrosine expression was observed along the tail, specifically at the midpiece. However, this reactivity was enhanced in the presence of EOV. Western blot analysis of NP-40 extractable and non-extractable sperm proteins confirmed these observations. NP-40 extractable sperm proteins (25, 37, 44kDa) and non-extractable sperm proteins (70, 83, 90kDa) showed increased intensity when sperm were capacitated in the presence of EOV after 5-, 60-, 120- and 180-minutes of capacitation. Mass spectrophotometric analysis identified enolase, ATP-specific succinyl CoA, succinate CoA ligase, zona pellucida binding protein, heat shock protein 90, aconitase and hexokinase as proteins that undergo enhancement in tyrosine phosphorylation in the presence of EOV. The proteins identified are known to be involved in specific functions including cellular metabolism, molecular chaperoning and normal sperm development. In summary, the present investigation has provided new evidence showing that sperm capacitated in vitro in the presence of EOV display an enhanced expression of tyrosine phosphorylation compared to sperm incubated in capacitating medium alone. These results indicate that inclusion of oviductin in media used for in vitro fertilization (IVF) may improve success rates of IVF by enhancing the signaling pathways involved in sperm capacitation.