Molecular cloning and characterization of two isoforms of cyclophilin A gene from Venerupis philippinarum

Cyclophilin A (CypA) is a ubiquitously distributed intracellular protein belonging to the immunophilin family, which is recognized as the cell receptor for the potent immunosuppressive drug cyclosporine A. In the present study, two isoforms of cyclophilin A gene (named as VpCypA1 and VpCypA2) were isolated and characterized from Venerupis philippinarum by RACE approaches. Both VpCypA1 and VpCypA2 possessed all conserved features critical for the fundamental structure and function of CypA, indicating that the two isoforms of cyclophilin A should be new members of CypA family. The expression of VpCypA2 mRNA in haemocytes was significantly up-regulated and the highest expression level was detected at 96 h post-infection with 7.7-fold increase compared with that in the blank group. On the contrary, the relative expression level of VpCypA1 mRNA was down-regulated rapidly at 6 h post-infection and reached 0.4-fold of the control group. They exhibited different expression profile and identical effect of immune modulation, which might suggest the two VpCypA isoforms exert their function in a manner of synergy. These results provide valuable information for further exploring the roles of cyclophilin A in the immune responses of V. philippinarum.     

Molecular cloning and characterization of SOCS-2 from Manila clam Ruditapes philippinarum

Suppressor of cytokine signaling (SOCS) family members are key regulators of immunological homeostasis. In this study, we have discovered the SOCS-2 member from Manila clam Ruditapes philippinarum and further analyzed its immune responses against lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (poly I:C). Amino acid sequence of RpSOCS-2 consists of cytokine inducible SRC homology 2 (SH2) and SOCS box domains similar to vertebrate SOCS counterparts. It has the highest amino acid identity (41%) with Pacific oyster (Crassostrea gigas) SOCS-2 and showed close evolutional relationship with disk abalone (Haliotis discus discus) SOCS-2. Tissue specific expression results showed that RpSOCS-2 was constitutively expressed in all examined tissues with the highest level in gill tissue of un-challenged clams. RpSOCS-2 mRNA expression was up-regulated by LPS and poly I:C challenge in gills. Discovery of RpSOCS-2 homologue and expression analysis would support for understanding evolutional relationships and their role in innate immune responses in mollusks, respectively.     

Resistance of the Manila clam (Venerupis philippinarum) to infection with Mikrocytos mackini

Manila clams (Venerupis philippinarum) challenged in laboratory trials via bath exposure proved to be resistant to infections with Mikrocytos mackini (protistan parasite of unknown taxonomic affiliation), while Pacific oysters (Crassostrea gigas) challenged simultaneously using identical conditions developed infections. Although M. mackini was detected by a nucleic acid pathogen specific (PCR) assay in 10-30% of the challenged V. philippinarum that were sampled soon after exposure (0-48 h, n = 40), all of the subsequent V. philippinarum (n = 62) sampled 9-17 weeks post-exposure tested negative for M. mackini by PCR assay. Prevalence of infection for the exposed C. gigas (n = 100) during this same period ranged from 50% to 100% by PCR assay. Infection was confirmed in the oysters (58%, n = 60) by a digoxigenin-labelled DNA probe designed to detect M. mackini by in situ hybridization, but M. mackini was not found in any of the exposed Manila clams (n = 63) using this technique.     

Molecular cloning, characterization and expression of a novel jasmonate-dependent defensin gene from Ginkgo biloba

A novel defensin gene was isolated from Ginkgo biloba. The full-length cDNA of G. biloba defensin (designated as Gbd) was 534bp. The cDNA contained a 240-bp open reading frame encoding an 80-amino acid protein of 5.68 kDa with a potential 30 aa signal peptide. The putative GbD mature protein showed striking similarity to other plant defensins, representing low molecular size antimicrobial polypeptides. Eight cysteine sites conserved in plant defensins were also found in GbD at similar positions. Three-dimensional structure modeling showed that GbD strongly resembled defensin from tobacco (NaD1) and consisted of an alpha-helix and a triple-strand antiparallel beta-sheet that were stabilized by four intramolecular disulfide bonds, implying GbD may have functions similar to NaD1. The genomic DNA gel blot indicated that Gbd belonged to a multigene family. Expression analysis revealed that Gbd was up-regulated by wounding and methyl jasmonate treatments, suggesting that Gbd is potentially involved in plant resistance or tolerance to pathogens during wounding.     

Mineral phase in shell repair of Manila clam Venerupis philippinarum affected by brown ring disease

The mineral phase of shell repair in the Manila clam Venerupis philippinarum affected by brown ring disease (BRD) was characterised at various scales and at various stages of shell repair by confocal Raman microspectrometry and scanning electron microscopy. Spherulitic and quadrangular aragonite microstructures associated with polyene pigments were clearly observed. Von Kossa staining showed that at the beginning of shell repair, hemocytes are filled with insoluble calcium carbonate salts in all fluids and then are transported toward the extrapallial fluids and the repair sites. Our analyses suggest that after a Vibrio tapetis attack and BRD deposit some clams rapidly cover the deposit, resulting in a modification in the microstructure, which could be produced by the participation of both the mantle and hemocytes.     

Characterization of a novel molluscan MyD88 family protein from manila clam, Ruditapes philippinarum

Myeloid differentiation factor 88 (MyD88) is a universal adaptor protein which is required for signal transduction of TLR/IL-1R family. In this study, a novel molluscan MyD88 family member protein (named as RpMyD88) was identified from manila clam, Ruditapes philippinarum. It was identified using BLAST algorithm from GS-FLX? sequencing data. The cDNA of RpMyD88 consists of 1416 bp open reading frame (ORF) encoding 471 amino acid residues. The RpMyD88 contains death domain and Toll/interleukin-1 receptor (TIR) domain which are typical features of MyD88 family proteins. The predicted amino acid sequence of RpMyD88 shares 27% identity with scallop MyD88. The expression level of RpMyD88 mRNA was investigated in healthy and challenged clams by quantitative real-time RT-PCR. The RpMyD88 gene expression is ubiquitous in all selected tissues. The RpMyD88 mRNA was strongly expressed in hemocyte, gill and mantle. In contrast, it was weakly expressed in siphon, foot and adductor muscle. RpMyD88 was up-regulated in gill and hemocyte after immune challenge with both Vibrio tapetis and LPS challenge. All results considered, sequence characterization, comparison and gene expression data suggesting that MyD88-dependent signaling pathway is presence in manila clam and RpMyD88 plays an important role in innate immune response against bacteria.     

Purification and antibacterial characterization of a novel isoform of the Manila clam lectin (MCL-4) from the plasma of the Manila clam, Ruditapes philippinarum

In many bivalve molluscs, lectins are present in the hemolymph and are thought to be important for internal host defense mechanisms. For this study, we purified a novel isoform of the Manila clam lectin (designated MCL-4) from the plasma of the Manila clam, Ruditapes philippinarum, using affinity chromatography and gel filtration. Native PAGE results showed that the MCL-4 consisted of 70 kDa protein. MCL-4 was found to be composed of 58-kDa and 43-kDa bands when examined using SDS-PAGE under reducing and non-reducing conditions. The native MCL-4 was revealed as a 147 kDa molecular mass protein by gel filtration. The purified MCL-4 agglutinates calcium-dependently in the erythrocytes of sheep and rabbit, but not in cells of the three species of marine bacteria tested. However, the phagocytic ability of the R. philippinarum hemocytes for the MCL-4-opsonized Vibrio tubiashii cells was significantly greater than that for the BSS-treated bacterial cells. Addition of purified MCL-4 markedly suppressed Alteromonas haloplanktis growth. These results suggest that MCL-4, because of its opsonizing and bacteriostatic properties, might contribute to the host defense mechanisms against invading microorganisms in R. philippinarum.     

Purification and characterization of a novel anticancer peptide derived from Ruditapes philippinarum

The aim of this study was to purify a novel peptide from Ruditapes philippinarum and investigate its anticancer activities. For the aim, eight proteases were applied for enzymatic hydrolysis. α-Chymotrypsin hydrolyzates, which showed clearly superior cytotoxicity activity on prostate cancer cells, were further purified using a flow filtration system and consecutive chromatographic methods. Finally, a novel anticancer peptide was purified, and the sequence was identified as Ala-Val-Leu-Val-Asp-Lys-Gln-Cys-Pro-Asp at N-terminal. The peptide from R. philippinarum effectively induced apoptosis on prostate, breast and lung cancer cells but not on normal liver cells. This is the first report of an anticancer peptide purified from the hydrolyzates of R. philippinarum.     

Cloning and localization of MCdef, a defensin from Manila clams (Ruditapes philippinarum)

A defensin-like peptide was previously detected in hemocytes of Manila clams (Ruditapes philippinarum). In the current study, we cloned and characterized this defensin, designated MCdef. Cloning produced a full-length gene sequence of 201 bp predicted to encode a 66-amino-acid precursor protein maturing to a 44-amino-acid residue. Amino acid sequence analysis showed that MCdef is similar to defensins from marine mollusks and ticks. Phylogenetic analysis suggested that MCdef is closely related to defensins from Mytilus galloprovincialis (Mediterranean mussel) and Crassostrea gigas (Pacific cupped oyster). The three-dimensional structure of MCdef was modeled using the solution structure of C. gigas defensin as a template. With the exception of three variable loop areas, the modeled structure of MCdef was identical to that of C. gigas defensin. MCdef antiserum was raised against a synthetic MCdef peptide and verified by Western blotting using recombinant MCdef. RT-PCR analysis demonstrated high levels of MCdef mRNA in hemocytes and adductor, foot, gill, mantle, palp, and siphon tissues of Vibrio tapetis-infected Manila clams, whereas in V. tapetis-uninfected Manila clams, the level of MCdef mRNA was low in adductor, palp, and siphon tissues and even lower in the other tested tissues. Immunohistochemical analysis revealed high MCdef expression was detected in the gill, the mantle, and the digestive tubules of the diverticulum of V. tapetis-infected Manila clams. Minimum inhibitory concentration (MIC) of the purified rMCdef was determined. MCdef showed highest activity against Streptococcus iniae and Staphylococcus aureus.     

Molecular cloning and characterization of Scotch pine defensin 2

A 252 bp cDNA fragment that corresponds to defensin 2 (PsDef2) was amplified from a cDNA library from seven-day plantlets of Pinus sylvestris L. This fragment encodes a protein that consists of 83 amino acid residues. The protein contains an N-terminal signal peptide, which includes 33 amino acid residues. A mature form of defensin 2 of Scotch pine contains a gamma-thionine domain and it is also characterized by specific conservative residues that are common to all plant defensins.     

First report of three protozoan parasites (a haplosporidian, Marteilia sp. and Marteilioides sp.) from the Manila clam, Venerupis (=Ruditapes) philippinarum in Japan

Recently, natural stocks of the Manila clam, Venerupis (=Ruditapes) philippinarum, have been drastically reduced in Japan. To clarify the reason for this decline in number, clams were sampled monthly from Yamaguchi and processed for histological observations, during which three protozoan parasites were discovered. Transmission electron microscopy revealed that these parasites were unidentified haplosporidian in the connective tissues, Marteilia sp. in the digestive gland and Marteilioides sp. in the oocytes. Histopathological observations suggest that Marteilia sp. and Marteilioides sp. were not pathogenic to the host. However, infection with a haplosporidian may have a negative impact on the clams. The prevalence of these parasites was low and further investigations should be undertaken to clarify their taxonomic status and establish any pathogenicity to clams.     

Molecular evolution and recombination in gender-associated mitochondrial DNAs of the Manila clam Tapes philippinarum

Doubly uniparental inheritance (DUI) provides an intriguing system for addressing aspects of molecular evolution and intermolecular recombination of mitochondrial DNA. For this reason, a large sequence analysis has been performed on Tapes philippinarum (Bivalvia, Veneridae), which has mitochondrial DNA heteroplasmy that is consistent with a DUI. The sequences of a 9.2-kb region (containing 29 genes) from 9 individuals and the sequences of a single gene from another 44 individuals are analyzed. Comparisons suggest that the two sex-related mitochondrial genomes do not experience a neutral pattern of divergence and that selection may act with varying strength on different genes. This pattern of evolution may be related to the long, separate history of M and F genomes within their tissue-specific "arenas." Moreover, our data suggest that recombinants, although occurring in soma, may seldom be transmitted to progeny in T. philippinarum.     

Isolation and characterization of microsatellite markers for the clam Ruditapes philippinarum and cross-species amplification with the clam Ruditapes variegate

Ruditapes philippinarum and R. variegate are commercially important shellfish in Korea. In order to understand the processes and organization of genetic diversity and genetic resources for the sustainable management of fisheries resources we developed 13 primer pairs for microsatellite loci in R. philippinarum and tested their cross-amplification in R. variegate. Twelve primers amplified in both species. Nine loci were polymorphic in R. philippinarum and eight in R. variegate, each with nine to 26 alleles per locus. Unbiased expected heterozygosity levels varied from 0.73 to 0.94 in R. philippinarum. All polymorphic loci possessed species-specific alleles. No linkage disequilibrium was found. Results indicated that these microsatellite were highly polymorphic and will be useful for conservation genetics of both species.     

Molecular cloning and characterization of a cDNA encoding a novel antibacterial peptide, defensin, from the mulberry longicorn beetle, Apriona germari.

A full-length cDNA clone with high homology (62% mature peptide sequence identity) to an Acalolepta luxuriosa antibacterial gene, possessing a conserved cysteine-stabilized alphabeta motif, was cloned by screening an Apriona germari cDNA library. This gene (AgCRP) had a total length of 360 bp with an open reading frame of 207 bp, and encoded a predicted peptide of 69 amino acid residues. The mature AgCRP peptide was 27 amino acid residues long and had a cysteine-stabilized alphabeta motif of C...CXXXC...C...CXC consensus sequence, similar to insect defensins. Northern blot analysis revealed that the AgCRP exhibited fat body-specific expression and was up-regulated by wounding, bacterial or fungal challenge.     

Transcriptional regulation of selenium-dependent glutathione peroxidase from Venerupis philippinarum in response to pathogen and contaminants challenge

Glutathione peroxidases (GPx) are key enzymes in the antioxidant systems of living organisms by catalyzing the reduction of peroxides to non-reactive products. In the present study, the full-length cDNA encoding a selenium-dependent GPx was identified from Venerupis philippinarum (designated as VpSe-GPx), and the spatial and temporal expression patterns post-Vibrio anguillarum, heavy metals and benzo[a]pyrene (B[a]P) challenge were also investigated. VpSe-GPx possessed all the conserved features critical for the fundamental structure and function of glutathione peroxidase. The VpSe-GPx mRNA was found to be most abundantly expressed in hepatopancreas. Vibrio challenge could significantly up-regulate the mRNA expression of VpSe-GPx, and the highest expression level was detected at 24 h post-infection with 6.5-fold increase compared with that in the control group. For heavy metals exposure, the expression of VpSe-GPx was significantly induced by 20, 40 μg L(-1) Cd and 10, 20 μg L(-1) Cu but depressed by 10 μg L(-1) Cd and 40 μg L(-1) Cu. With regards to B[a]P exposure, the expression of VpSe-GPx mRNA was significantly induced at 48 and 96 h post challenge. All these results suggested that VpSe-GPx was potentially involved in mediating the immune response and antioxidant defense in V. Philippinarum.     

Molecular characterization of a novel beta-glucuronidase from Scutellaria baicalensis georgi

We cloned a gene encoding Scutellaria beta-glucuronidase (sGUS) that is involved in the initiation of H(2)O(2) metabolism in skullcap (Scutellaria baicalensis). This gene consists of a 1581-nucleotide open reading frame, the deduced amino acid sequence of which contains an ATP/GTP binding site and a leucine zipper motif. sGUS has apparent similarity to the heparan sulfate-metabolizing beta-glucuronidase heparanase but no homology to family 2 beta-glucuronidases. In addition, neither the family 2 glycosylhydrolase signature nor family 2 acid-base catalyst was found in this enzyme. These results suggested that sGUS does not belong to the family 2 beta-glucuronidases. We modified several residues predicted to act as the acid-base or nucleophilic residue of sGUS by site-directed mutagenesis. Mutations at Glu(212) or Glu(329) resulted in much lower k(cat)/K(m) values in the mutants as compared with the wild-type enzyme, indicating that these are the acid-base and nucleophilic residues of the active site, respectively. Moreover, similar site-directed mutagenesis confirmed that Tyr(281) is also involved in the beta-glucuronidase activity. The amino acid sequences of small regions containing these active site residues were conserved in heparanases. As sGUS has various structural characteristics in common with heparanase, we concluded that sGUS and heparanase belong to the same new family.