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This study was conducted to examine the effects of alterations in water temperature (WT) on biochemical and immunological factors in the hemolymph of the abalones, Haliotis gigantea and H. discus discus. The abalone were exposed to various WT; 18, 20, 22, 24, 26 and for 96 hours. In biochemical factors, total-protein (TP), glucose, magnesium (Mg), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were not significant changes in hemolymph of H. gigantea and H. discus discus. But calcium was significantly increased by high WT (). In immunological factor, The phenoloxidase (PO) activity was decreased in hemolymph of H. gigantea and H. discus discus exposed to high temperature () compared to the control (P

Sclerostin, encoded by the Sost gene, is mainly produced by osteocytes in bone and antagonizes the Wnt/β-catenin signaling pathway, which is a requisite for bone formation. Currently, human anti-sclerostin antibodies are being tested in phase III clinical trials. In addition, serum sclerostin levels are reported to be associated with bone mineral density and fracture risk in normal individuals; however, the correlation between serum sclerostin and bone mass remains controversial. To study the effects of the continuous exposure of exogenous sclerostin on bone, a ΦC31 integrase system, which has the characteristics of site-specificity and efficiency, was applied for the delivery of the Sost gene in this study. We injected Sost-attB plasmid with or without ΦC31 integrase plasmid into the mouse tail vein using a hydrodynamic-based method. The site-specific integration of the Sost gene into the mouse genome was confirmed by examining a pseudo-attP site on the hepatic genomic DNA. Sclerostin was expressed in the hepatocytes, secreted into the blood flow, and maintained at high concentrations in the mice with both Sost-attB plasmid and ΦC31 integrase plasmid injections, which was observed by serial measurement. Moreover, the mice with long-term high levels of serum sclerostin showed trabecular bone loss on micro-CT analysis. Peripheral B cell populations were not affected. Our results suggested that sclerostin could be expressed in the liver and sustained successfully at high levels in the blood by using the ΦC31 integrase system, leading to trabecular bone loss. These findings may help to further ascertain the effects of sclerostin introduced exogenously on the skeleton.

Epithin/PRSS14, a type II transmembrane serine protease, plays critical roles in cancer metastasis. Previously, we have reported that epithin/PRSS14 undergoes ectodomain shedding in response to phorbol myristate acetate (PMA) stimulation. In this study, we show that transforming growth factor-β (TGF-β) induces rapid epithin/PRSS14 shedding through receptor mediated pathway in 427.1.86 thymoma cells. Tumor necrosis factor-α converting enzyme (TACE) is responsible for this shedding. Amino acid sequence encompassing the putative shedding cleavage site of epithin/PRSS14 exhibit strong homology to the cleavage site of l-selectin, a known TACE substrate. TACE inhibitor, TAPI-0 and TACE siRNA greatly reduced TGF-β-induced epithin/PRSS14 shedding. TGF-β treatment induces translocation of intracellular pool of TACE to the membrane where epithin/PRSS14 resides. These findings suggest that TGF-β induces epithin/PRSS14 shedding by mediating translocation of epithin/PRSS14 sheddase, TACE, to the membrane.

The tumor suppressor, QM has been cloned and characterized from eukaryotic organisms including humans, vertebrates, invertebrates, plants and yeast. However, no study on Pieris rapae QM (PrQM) has been reported to date. In this study, cDNA encoding a putative QM protein (PrQM) was obtained from expressed sequence tags (ESTs) of the Pieris rapae cDNA library. Phylogenetic analysis of PrQM showed high similarity of amino acid sequence with other putative homologues identified from Heliothis virescens (95%), Bombyx mori (92%), Plutella xylostella (92%), Drosophila melanogaster (89%) and Polyrhachis vicina (85%), indicating that QM is highly conserved among insects. Semi-quantitative PCR datasets revealed that PrQM transcripts are highly expressed in head, silk gland, integument and fat body, with pronounced expression observed during the egg stage. The coding region of the PrQM gene was cloned into the pET28 (+) expression vector and the recombinant protein purified by His-tag affinity chromatography was used for antibody production. Western blotting with the anti-PrQM antibody detected a single band corresponding to the expected molecular weight of both endogenous (26 kDa) and recombinant (29 kDa) PrQM. Furthermore, immunohistochemical and confocal microscopic analysis with the anti-PrQM antibody showed that the QM gene is highly expressed in the cytoplasm of fat body, gut and Malpighian tubules in virus-uninfected control larvae, but down-regulated at 4 days post Pieris rapae granulovirus (PiraGV) infection. These data show that PrQM is negatively regulated upon virus infection and suggests a putative immunomodulatory function.

The tumor suppressor, QM, has been cloned and characterized from various model organisms such as human, plant and invertebrates. Yet, it has not been seriously investigated for its role in conjunction with antiviral mechanisms involving innate insect immunity. From the expressed sequence tag (ESTs) project, conducted with larval cDNA library of cabbage butterfly, Pieris rapae, a partial fragment (718 bp) of QM homologue, termed PrQM containing 660 bp long open reading frame (ORF) encoding protein of 219 amino acids was identified. In silico analysis of PrQM ORF revealed the presence of ribosomal protein L10a/L10e type domain. Phylogenetic analysis of the P. rapae QM-like protein showed high amino acid sequence similarity with other PrQM polypeptides identified from Heliothis virescenes (95%), Plutella rapae (92%), Bombyx mori (92%), Drosophila melanogaster (89%), and Polyrhachis vicina (85%). The butterfly QM has the closest phylogenetic relationship to a moth (Hv) QM homologue. Further investigations revealed the expression of PrQM at all developmental stages, with pronounced presence at the egg stage. In addition, spatial pattern analysis indicated its high expression in the head, salivary gland, integument and fat body with visible presence in Malpighian tubule and gut. Time course expression studies conducted after immune-challenge with lipoteichoic acid (LTA) showed the induction of PrQM mRNA at 12 h and 24 h after challenge and also in response to granulovirus (GV). Results of this investigation therefore suggest possible role of QM-like proteins from Pieris rapae to be involved in innate antiviral immune responses. Further elucidation on the precise function of PrQM during antiviral immune responses by using RNA interference remains a viable research front.

It has been reported that QM may have antiviral function in invertebrate. However, it has not been seriously investigated whether insect QM plays a critical role in antiviral immunity against nucleopolyhedrosis virus (NPV). In this context, in silico identification and comparative analysis of Spodopetera exigua QM cDNA was conducted. As expected, it is highly conserved protein. To further examine whether entomopathogenic NPV up-regulates the level of SeQM mRNA, budded virus (AcMNPV-BV) was infected to the 3rd instar larvae. Real-time PCR analysis of SeQM in virus-infected larvae with poly I:C was conducted and potential significance of SeQM was discussed. It remains to elucidate the precise function of SeQM during antiviral immune responses by using RNA interference.