In this study, we show how the red spotted grouper nervous necrosis virus (RGNNV) causes loss of mitochondrial membrane potential and promotes host secondary apoptotic necrosis. RGNNV viral proteins such as protein alpha (42 kDa) and protein A (110 kDa) were quickly expressed between 12 h and 24 h postinfection (p.i.) in GL-av cells. Annexin V staining revealed that the NNV infection of GL-av cells induced phosphatidylserine (PS) externalization and development of bulb-like vesicles (bleb formation) at 24 h p.i. NNV infection also induced DNA fragmentation detectable by TUNEL assay between 12 h (8%) and 72 h (32%) p.i. Bongkrekic acid (1.6 microM; BKA) blocked permeability of the mitochondrial permeability transition pore, but cyclosporine A (CsA) did not block secondary necrosis. Finally, secondary necrotic cells were not engulfed by neighboring cells. Our data suggest that RGNNV induces apoptotic death via opening the mitochondrial permeability transition pore thereby triggering secondary necrosis in the mid-apoptotic phase.
DNA was isolated from the lipid-containing bacteriophage PRD1 and subjected to restriction endonuclease analysis. The total genome size is 14.7 kb. PRD1 DNA was resistant to cutting by fifteen restriction endonucleases with six base specificity. HaeII made thirty-seven cuts in the DNA, MboI made one cut, and MnlI made six cuts. DNA that was not treated with protease yielded two fewer fragments when treated with HaeII. Evidence is presented to indicate that the PRD1 DNA has protein at the ends of the DNA. The thirty-eight HaeII fragments were ordered using the ladder technique of Smith and Birnstiel (1976) on MboI and MnlI fragments of the genome. Clones of HaeII partial digests of PRD1 DNA in pBR322 were analyzed by HaeII digestion and were then assigned to specific regions of the genome by their HaeII fragment composition. A comparison of the marker rescue characteristics of the cloned DNA with the overall restriction fragment map generated a physical map of the genome. Some genes that have not been mapped because of a lack of mutants or leakiness at restrictive conditions were mapped by studying the in vitro protein synthesis of restriction endonuclease fragments.
Marine heterotrophic microalgal species which are potentially rich in docosahexaenoic acid (DHA, C22:6n-3) have been found in Taiwan; however, there was a lack of detailed analysis and characterization of these indigenous algae which is needed for the development of commercial applications. Hence, the objective of this study was to screen DHA-rich heterotrophic microalgae species indigenous to Taiwan for commercial purposes. Heterotrophic microalgae from a variety of marine habitats were isolated, cultivated, and then identified according to their 18S rRNA gene sequences and morphological characteristics. A comparison was made of their fatty acid profiles, fatty acid content, and amount of biomass. For the strain with highest DHA yield, the optimal growth conditions were determined in order to establish the best fermentation conditions for scale-up. In this study, 25 heterotrophic microalgal strains were successfully isolated from marine habitats around Taiwan. All of the isolated strains showed a close phylogenic relationship with the Thraustochytriaceae family according to their 18S rRNA gene sequences. GC/MS analysis discerned seven distinctive fatty acid profiles of these strains, with the production of eicosapentaenoic acid (C20:5n-3) ranging from 0.02 to 2.61 mg L(-1), and DHA ranging from 0.8 to 18.0 mg L(-1). An Aurantiochytrium strain BL10 with high DHA production was subsequently chosen for further manipulation. Under optimal growth conditions it could produce up to 59.0 g of dry biomass per liter of culture, with dry biomass containing 73% total fatty acid and 29% DHA, revealing BL10 as an excellent source of microbial DHA.
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