Characterization of the Rana grylio virus 3 beta-hydroxysteroid dehydrogenase and its novel role in suppressing virus-induced cytopathic effect

The 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) isoenzymes play a key role in cellular steroid hormone synthesis. Here, a 3 beta-HSD gene homolog,was cloned from Rana grylio virus (RGV), a member of family Iridoviridae. RGV 3 beta-HSD gene has 1068 bp, encoding a 355 aa predicted protein. Transcription analyses showed that RGV 3 beta-HSD gene was transcribed immediate-early during infection from an initiation site 19 nucleotides upstream of the translation start site. Confocal microscopy revealed that the 3 beta-HSD-EGFP fusion protein was exclusively colocalized with the mitochondria marker (pDsRed2-Mito) in EPC cells. Upon morphological observation and MTT assay, it was revealed that overexpression of RGV 3 beta-HSD in EPC cells could apparently suppress RGV-induced cytopathic effect (CPE). The present studies indicate that the RGV immediate-early 3 beta-HSD gene encodes a mitochondria-localized protein, which has a novel role in suppressing virus-induced CPE. All these suggest that RGV 3 beta-HSD might be a protein involved in host-virus interaction. @ 2006 Elsevier Inc. All rights reserved.

Suppression of zebrafish VEGF gene by cytomegalovirus promoter-driven short hairpin constructs induces vascular development defects and down regulation NRP1 expression

The usage of RNA interference for gene knockdown in zebrafish through expression of the small interfering RNA mediators from DNA vectors has created a lot of excitement in the research community. In this work, the ability of human cytomegalovirus immediate early promoter (CMV promoter)-driven short hairpin RNA (shRNA) expression vector to induce shRNA against vascular endothelial growth factor (VEGF) gene in zebrafish was tested, and its effects on VEGF-mediated vasculogenesis and angiogenesis were evaluated. Altogether four vectors targeting various locations of VEGF gene were constructed, and pSI-V4 was proven to be the most effective one. Microinjection of pSI-V4 into the zebrafish embryos resulted in defective vascular formation and down regulation of VEGF expression. In situ hybridization analysis indicated that silencing VEGF gene expression by pSI-V4 resulted in down regulation of neuropilin-1 (NRP1), a potent VEGF receptor. Knockdown of VEGF expression by morpholino gave the same result. This provided evidence that the VEGF-mediated angiogenesis in zebrafish was in part dependent on NRP1 expression. The results contributed to a better understanding of molecular mechanisms of cardiovascular development and provided a potential promoter for making inducible knockdown in zebrafish.

VEGF gene silencing by cytomegalovirus promoter driven ShRNA expression vector results in vascular development defects in zebrafish

Zebrafish has been generally considered as an excellent model in case of drug screening, disease model establishment, and vertebrate embryonic development study. In this work, the ability of human cytomegalovirus immediate early promoter (CMV promoter)-driven short hairpin RNA (shRNA) expression vector to induce shRNA against VEGF gene in zebrafish was tested, and its effect on vascular development was assed, too. Using RT-qPCR, blood vessel staining, and in situ hybridization, we confirmed certain transcriptional activity and down regulation of gene expression by the vector. In situ hybridization analysis indicated selective inhibition of NRP1 expression in the VEGF gene loss of function model, which might imply in turn that VEGF could not only activate endothelial cells directly but also could contribute to stimulating angiogenesis in vivo by a mechanism that involved up-regulation of its cognate receptor expression in zebrafish. This contributed to a better understanding of molecular mechanisms of cardiovascular development. The system improved the success rate in making inducible knockdown and widened the possibilities for better therapeutic targets in zebrafish.

Construction and analysis of an experimental Streptococcus iniae DNA vaccine

Streptococcus iniae is a severe aquaculture pathogen that can also infect humans and animal. A putative secretory antigen, Slat 0, was identified from a pathogenic S. iniae strain by in vivo-induced antigen technology. Using turbot as an animal model, the immunoprotective effect of Sia10 was examined as a DNA vaccine in the form of plasmid pSia10, which expresses sia10 under the cytomegalovirus immediate-early promoter. In fish vaccinated with pSia10, transcription of sia10 was detected in muscle, liver, spleen, and kidney at 7, 14, 21, 28, 35, 42, and 49 days post-vaccination. In addition, production of Sia10 protein was also detected in the muscle tissues of pSia10-vaccinated fish. Fish vaccinated with pSia10 exhibited a relative percent survival (RPS) of 73.9% and 92.3%, respectively, when challenged with high and low doses (producing a cumulative mortality of 92% and 52%, respectively, in the control groups) of S. iniae. Immunological and transcriptional analyses showed that vaccination with pSia10(i) induced much stronger chemiluminescence response and significantly higher levels of nitric oxide production and acid phosphatase activity in head kidney macrophages; (ii) caused the production of specific serum antibodies, which afforded apparent immunoprotection when transferred passively into naive fish; and (iii) upregulated the expression of the genes encoding proteins that are possibly involved in both innate and adaptive immune responses. Taken together, these results indicated that pSia10 is an effective vaccine candidate and may be used in the control of S. iniae infection in aquaculture. (C) 2010 Elsevier Ltd. All rights reserved.

Vortex motion in the early stages of unsteady flow around a circular cylinder

In this paper, processes in the early stages of vortex motion and the development of flow structure behind an impulsively-started circular cylinder at high Reynolds number are investigated by combining the discrete vortex model with boundary layer theory, considering the separation of incoming flow boundary layer and rear shear layer in the recirculating flow region. The development of flow structure and vortex motion, particularly the formation and development of secondary vortex and a pair of secondary vortices and their effect on the flow field are calculated. The results clearly show that the flow structure and vortices motion went through a series of complicated processes before the symmetric main vortices change into asymmetric: development of main vortices induces secondary vortices; growth of the secondary vortices causes the main vortex sheets to break off and causes the symmetric main vortices to become “free” vortices, while a pair of secondary vortices is formed; then the vortex sheets, after breaking off, gradually extend downstream and the structure of a pair of secondary vortices becomes relaxed. These features of vortex motion look very much like the observed features in some available flow field visualizations. The action of the secondary vortices causes the main vortex sheets to break off and converts the main vortices into free vortices. This should be the immediate cause leading to the instability of the motion of the symmetric main vortices. The flow field structure such as the separation position of boundary layer and rear shear layer, the unsteady pressure distributions and the drag coefficient are calculated. Comparison with other results or experiments is also made.

PIXE and ICP-AES analysis of early glass unearthed from Xinjiang (China)

Early glasses (about 1066 BC-220 AD) unearthed from Xinjiang of China were chemically characterized by using PIXE and ICP-AES. It was found that these glasses were basically attributed to PbO-BaO-SiO2 system, K2O-SiO2 system, Na2O-CaO-SiO2 system and Na2O-CaO-PbO-SiO2 system. The results from the cluster analysis showed that some glasses had basically similar recipe and technology. The PbO-BaO-SiO2 glass and the K2O-SiO2 glass were thought to come from the central area and the south of ancient China, respectively. The part of the Na2O-CaO-SiO2 glass (including the Na2O-CaO-PbO-SiO2 glass) might be imported from Mesopotamia, while the other part might be locally produced. (c) 2005 Elsevier B.V. All rights reserved.