Molecular cloning and characterization of PTEN in the orange-spotted grouper (Epinephelus coioides).

PTEN is a key tumor suppressor gene that can play a regulatory role in the cellular proliferation, survival and apoptosis. In this study, the full-length PTEN (EcPTEN) was obtained, containing a 5'UTR of 745 bp, an ORF of 1269 bp and a 3'UTR of 106 bp. The EcPTEN gene encoded a polypeptide of 422 amino acids with an estimated molecular mass of 49.14 KDa and a predicted isoelectric point (pI) of 6.34. The deduced amino acid sequence analysis showed that EcPTEN comprised the conserved residues and the characteristic domains known to the critical functionality of PTEN. qRT-PCR analysis revealed that EcPTEN mRNA was broadly expressed in all the examined tissues, while the highest expression level was observed in liver, followed by the expression in blood, kidney, spleen, heart, gill, muscle and intestine. The groupers challenged with Vibrio alginolyticus showed a sharp increase of EcPTEN mRNA expression in immune tissues. In addition, western blotting analysis confirmed that the up-regulation of EcPTEN protein expression was steadily induced in liver. Subcellular localization analysis indicated that EcPTEN was localized in both nucleus and cytoplasm. Overexpression of EcPTEN can activate the apoptotic cascade and abrogate NF-kB, AP-1, Stat3 and Myc promoter activity in Hela cells. These results indicated that EcPTEN harboring highly-conserved domains with a close sequence similarity to those of PTP superfamily may disrupt the mammalian signalings and play a regulatory role in the apoptotic process.

The GRIM-19 plays a vital role in shrimps' responses to Vibrio alginolyticus.

GRIM-19 (gene associated with retinoid-interferon-induced mortality 19), a novel cell death regulatory gene, plays important roles in cell apoptosis, mitochondrial respiratory chain and immune response. It has been reported to interact physically with STAT3 and inhibit STAT3-dependent signal transduction. In this study, a new GRIM-19 gene, which is a 789-bp gene encoding a 149 amino acids protein, is identified and characterized from Litopenaeus vannamei. The tissue distribution patterns showed that LvGRIM-19 was widely expressed in all examined tissues, with the highest expression in muscle. Quantitative real-time PCR revealed that LvGRIM-19 was down-regulated in hepatopancreas after infection with the Vibrio alginolyticus. Knockdown of LvGRIM-19 by RNA interference resulted in a lower mortality of L. vannamei under V. alginolyticus infection, as well as an enhancement in the protein expression of STAT gene and JAK gene. V. alginolyticus infection caused an increase apoptotic cell ratio and ROS production of L. vannamei, while LvGRIM-19 silenced shrimps showed significantly lower than GFP group. Our results suggest that the GRIM-19 plays a vital role in shrimps' responses to V. alginolyticus. Interferenced LvGRIM-19 treatment during V. alginolyticus infection could increase 12 h survival rate, which might indicated that LvGRIM-19 is closely related to death of shrimps.

Molecular cloning, expression of a galectin gene in Pacific white shrimp Litopenaeus vannamei and the antibacterial activity of its recombinant protein.

Galectins play crucial roles in innate immune responses in invertebrate by recognizing and eliminating microinvaders. In this study, a cDNA encoding a galectin in the Pacific white shrimp (L. vannamei) was identified and characterized. A recombinant variant of this lectin, rLvgalectin, was expressed in the model organism P. pastoris and its expression was confirmed by Western blot. Biochemical assays indicated that the recombinant protein antibacterial rLvgalectin activity and was expressed in all of the organism's tested tissues Injection of the bacterium V. alginolyticus into L. vannamei induced hemocytes upregulation of Lvgalectin. The recombinant Lvgalectin protein (rLvgalectin) could bind various microorganism including Gram-positive bacteria, Gram-negative bacteria and yeast. And it revealed antimicrobial activity against the test Gram-positive bacteria, Gram-negative bacteria, but did not inhibit the growth of fungus Pichia pastoris. Moreover, rLvgalectin could significantly enhance the clearance activity of V. alginolyticus in vivo. In vivo challenge experiments showed that the recombinant rLvgalectin protein can significantly reduce the mortalities of V. alginolyticus injection. Furthermore, Compared to their wild-type counterparts, Lvgalectin-silenced shrimp exhibited increased mortality and hemocyte apoptosis, decreased bacterial clearance ability and total hemocyte counts, and stronger expression of Lvp53, LvproPO, LvPEN3, and LvCrustin following V. alginolyticus challenge. Taken together, these results suggest that galectin is important in the innate immune response of shrimp to pathogens infection.

Essential roles of Cdc42 and MAPK in cadmium-induced apoptosis in Litopenaeus vannamei.

Cadmium, one of the most toxic heavy metals in aquatic environments, has severe effects on marine invertebrates and fishes. The MAPK signaling pathway plays a vital role in stress responses of animals. The mitogen-activated protein kinase (MAPK) signaling pathway plays a vital role in animals' stress responses, including mediation of apoptosis induced by the Rho GTPase Cdc42. However, there is limited knowledge about its function in shrimps, although disorders exacerbated by environmental stresses (including heavy metal pollution) have caused serious mortality in commercially cultured shrimps. Thus, we probed roles of Cdc42 in Litopenaeus vannamei shrimps (LvCdc42) during cadmium exposure by inhibiting its expression using dsRNA-mediated RNA interference. The treatment successfully reduced expression levels of MAPKs (including p38, JNK, and ERK). Cadmium exposure induced significant increases in expression levels of LvCdc42 and MAPKs, accompanied by reductions in total hemocyte counts (THC) and increases in apoptotic hemocyte ratios and ROS production. However, all of these responses were much weaker in LvCdc42-suppressed shrimps, in which mortality rates were higher than in controls. Our results suggest that the MAPK pathway plays a vital role in shrimps' responses to Cd(2+). They also indicate that LvCdc42 in shrimps participates in its regulation, and thus plays key roles in ROS production, regulation of apoptosis and associated stress responses.

Effects of temperature acclimation on body mass and energy budget in the Chinese bulbul Pycnonotus sinensis.

Chinese bulbuls (Pycnonotus sinensis) are small passerine birds that inhabit areas of central, southern and eastern China. Previous observations suggest that free-living individuals of this species may change their food intake in response to seasonal changes in ambient temperature. In the present study, we randomly assigned Chinese bulbuls to either a 30℃ or 10℃ group, and measured their body mass (BM), body temperature, gross energy intake (GEI), digestible energy intake (DEI), and the length and mass of their digestive tracts over 28 days of acclimation at these temperatures. As predicted, birds in the 30℃ group had lower body mass, GEI and DEI relative to those in the 10℃ group. The length and mass of the digestive tract was also lower in the 30℃ group and trends in these parameters were positively correlated with BM, GEI and DEI. These results suggest that Chinese bulbuls reduced their absolute energy demands at relatively high temperatures by decreasing their body mass, GEI and DEI, and digestive tract size.