Infection and propagation of lymphocystis virus isolated from the cultured flounder Paralichthys olivaceus in grass carp cell lines

The causative agent of lymphocystis disease that frequently occurs in cultured flounder Paralichthys olivaceus in China is lymphocystis virus (LV). In this study, 13 fish cell lines were tested for their susceptibility to LV. Of these, 2 cell lines derived from the freshwater grass carp Ctenopharyngodon idellus proved susceptible to the LV, and 1 cell line, GCO (grass carp ovary), was therefore used to replicate and propagate the virus. An obvious cytopathic effect (CPE) was first observed in cell monolayers at 1 d post-inoculation, and at 3 d this had extended to about 75% of the cell monolayer. However, no further CPE extension was observed after 4 d. Cytopathic characteristics induced by the LV were detected by Giemsa staining and fluorescence microscopic observation with Hoechst 33258 staining. The propagated virus particles were also observed by electron microscopy. Ultrastructure analysis revealed several distinct cellular changes, such as chromatin compaction and margination, vesicle formation, cell-surface convolution, nuclear fragmentation and the occurrence of characteristic 'blebs' and cell fusion. This study provides a detailed report of LV infection and propagation in a freshwater fish cell line, and presents direct electron microscopy evidence for propagation of the virus in infected cells. A possible process by which the CPEs are controlled is suggested.

Purification and characterization of White Spot Syndrome Virus (WSSV) produced in an alternate host: crayfish, Cambarus clarkii

Penaeid shrimp is the natural host of White Spot Syndrome Virus (WSSV) that can cause high mortality in the infected hosts. Attempts to obtain sufficient amounts of purified intact WSSV for characterization have been unsuccessful. Using crayfish, Cambarus clarkii as a proliferation system, a large amount of infectious WSSV was reproduced and intact WSSV viral particles were purified with a new isolation medium by ultra-centrifugation. Purified WSSV particles were very sensitive to organic solvents and the detergent, Triton X-100. The size of the rod-shape, somewhat elliptical, intact WSSV was 110-130 x 260-350 mm with a long, tail-like envelope extension. The naked viral nucleocapsid was about 80 x 350 nm, and it possessed 15 spiral and cylindrical helices composed of 14 globular capsomers along its long axis, and a 'ring' structure at one terminus. Distinct WSSV genome DNA patterns were obtained when the purified genomic dsDNA of WSSV was digested with five different restriction enzymes (HindIII, XhoI, B(BamHI, SalI, and SacI). In addition, at least 13 major and distinct protein bands could be observed when purified intact WSSV viruses were separated by SDS-PAGE followed by Coomassie Brilliant R-250 staining. The estimated molecular weights of these proteins were 190, 84, 75, 69, 68, 58, 52, 44, 28, 27.5, 23, 19, and 16 kD, respectively. Both the 44 and 190 kD proteins were easily removed if the hemolymph from the: WSSV infected crayfish was transiently treated with 1%, Triton X-100 before it was subjected to gradient centrifugation, indicating that both of them are located on the surface of the viral envelope. These characteristics are consistent with WSSV isolated from the penaeid shrimp. (C) 2001 Elsevier Science B.V. All rights reserved.

Adenovirus-mediated wild-type P53 Transfer radiosensitizes H1299 cells to subclinical-dose carbon-ion irradiation through the restoration of p53 function

To determine whether adenovirus-mediated wild-type p53 transfer after radiotherapy could radiosensitize non-small-cell lung cancer (NSCLC) cells to subclinical-dose carbon-ion beam (C-beam), H1299 cells were exposed to a C-beam or -ray and then infected with 5 MOI of AdCMV-p53 or GFP (C-beam or -ray with p53 or GFP).Cell cycle was detected by flow cytometric analysis. The apoptosis was examined by a fluorescent microscope with DAPI staining. DNA fragmentation was monitored by the TUNEL assay. P53 mRNA was detected by reverse-transcriptase polymerase chain reaction. The expression of p53, MDM2, and p21 was monitored by Western blot. Survival fractions were determined by colony-forming assay. The percentages of G1-phase cells in C-beam with p53 increased by 8.2%–16.0%, 5.2%–7.0%, and 5.8%–18.9%, respectively, compared with C-beam only, -ray with p53, or p53 only. The accumulation of G2-phase cells in C-beam with p53 increased by 5.7%–8.9% and 8.8%–14.8%, compared with those in -ray with p53 or p53 only, respectively. The percentage of apoptosis for C-beam with p53 increased by 7.4%–19.1%, 5.8%–11.7%, and 5.2%–19.2%, respectively, compared with C-beam only, -ray with p53, or p53 only. The level of p53 mRNA in C-beam with p53 was significantly higher than that in p53 only. The expression level of p53 and p21 in C-beam with p53 was significantly higher than that in both C-beam with GFP and p53 only. The survival fractions for C-beam with p53 were significantly less than those for the other groups (p 0.05). The data suggested that AdCMV-p53 transfer could more efficiently radiosensitize H1299 cells to subclinical-dose C-beam irradiation through the restoration of p53 function.

Cell cycle and apoptosis alteration of human hepatocarcinoma cells by subclinical-dose C-12(6+)-beam irradiation

Objective The purpose of this study is to investigate the effect of subdinical-dose C-12(6+)-beam irradiation on cell cycle and cell apoptosis in hepatocarcinoma cells. Materials and methods The HepG(2) cells were exposed to 0-2.0 Gy of either the C-12(6+) beam or a gamma-ray. Cell survival was detected by clonogenic assay. Cell cycle was determined by flow-cytometry analysis. The apoptosis was monitored by fluorescence microscope with DAPI staining. p53 and p21 expression were detected by Western blot. Results The G(0)/G(1) cells in the irradiated groups were significantly more than those in the control (P<0.05). The C-12(6+)-ion irradiation had a greater effect on the cell cycle of HepG(2) cells (including promoting G(1)-phase and G(2)-phase arrest) than gamma-ray irradiation. The apoptotic cells induced by C-12(6+) beam were significantly more numerous than those induced by gamma-ray (P<0.05). The carbon ions had a stronger effect on p53 and p21 expression than the gamma-ray irradiation. The survival fractions for cells irradiated by C-12(6+) beam were significantly smaller than those irradiated by gamma-ray (P<0.05).

Highly efficient separation of dsDNA fragments on glass chips by using an ultralow viscosity sieving matrix

A novel protocol has been established to separate dsDNA fragments with high efficiency on glass chips by using an ultralow viscosity sieving matrix with added glucose. Low-molecular-weight hydroxypropylmethylcellulose (HPMC), with a viscosity nearly equivalent to that of water, was used to electrophoretically separate fluorescent inter-calator-labeled double-stranded DNA (dsDNA) fragments on microfluidic glass chips. In comparison with conventional sieving protocols, low-molecular-weight HPMC as sieving matrix could result in reduced running cost and analysis time, in addition to a comparable separation efficiency of dsDNA fragments. In this paper, the addition of glucose was investigated to enhance the separation of DNA in the lowest viscosity polymer evaluated. The effect of staining dye and field strength were also evaluated. At an applied electric field strength of 200 V/cm, satisfactory resolution of the PBR322/HaeIII DNA marker could be achieved within 4 min by using 2% HPMC-5 with 6% glucose added. Coelectrophoresing PCR product along with phiX174/HaeIII DNA sizing marker was also demonstrated by using the ultralow viscosity HPMC-5 solution on a glass chip.

One step to detect the latent fingermarks with gold nanoparticles

A simple and environment friendly chemical route for detecting latent fingermarks by one-step single-metal nanoparticles deposition method (SND) was achieved successfully on several non-porous items. Gold nanoparticles (AuNPs) synthesized using sodium borohydride as reducing agent in the presence of glucose, were used as working solution for latent fingermarks detection. The SND technique just needs one step to obtain clear ridge details in a wide pH range (2.5-5.0), whereas the standard multi-metal deposition (MMD) technique requires six baths in a narrow pH range (2.5-2.8). The SND is very convenient to detect latent fingermarks in forensic scene or laboratory for forensic operators. The SND technique provided sharp and clear development of latent fingermarks, without background staining, dramatically diminished the bath steps.

Preparation and Bioactivity of the Composite of PLGA and Hydroxyapatite Nanocrystals Surface-grafted with L-lactic Acid Oligomer

The hydroxyapatite (HA) nanocrystals of 100-200 nm in length and 20-30 nm in width were hydrothermally synthesized by the reaction of phosphoric acid and calcium hydroxide. Lactic acid oligomer surface grafted HA(op-HA) nanoparticles were obtained by oligomeric lactic acid with a certain molecular weight grafting onto the HA surface to form a Ca carboxylate bond in the absence of any catalyst. The op-HA was further blended with poly(lactide-co-glycolide) (PLGA) to prepare the nanocomposite of op-HA/PLGA. FTIR, TGA, ESEM and EDX were used to analyze grafting reaction, the graft ratio of op-HA, surface topography and calcium deposition of the composites, respectively. The rabbit osteoblasts were seeded and cultured on the surface of composites in vitro. The cell morphology, adhesion, proliferation and gene expression were evaluated with FITC staining, NIH image J software and the analysis of real-time PCR, respectively. The results show that the graft ratio of op-HA is 8.3% (mass fraction). The op-HA/PLGA nanocomposite possessed more suitable surface properties, including roughness and plenty of calcium and phosphor. It exhibited better cell adhesion, spreading and proliferation of rabbit osteoblasts, compared to pure PLGA.

Preparation, Surface Properties and Biological Activity of the Composite of Poly (lactide-co-glycolide) and Bioglass Nanoparticles Surface Grafted with Poly(L-lactide)

SiO2-CaO-P2O5 gel bioglass (BG) nanoparticles with the diameter of 40 nm were synthesized by sol-gel approach. The surface of BG nanoparticles was grafted through the ring-open polymerization of the L-lactide to yield poly (L-lactide) (PLLA) grafted gel particle (PLLA-g-BG). The PLLA-g-BG was further blended with poly(lactide-co-glycolide) (PLGA) to prepare the nanocomposites of PLLA-g-BG/PLGA with the various blend ratios of two phases. PLLA-g-BG accounted 10%, 20% and 40% in the composite, respectively. TGA, ESEM and EDX were used to analyze the graft ratio of PLLA-g-BG, the dispersion of nano-particles and the surface elements of the composites respectively. The rabbit osteoblasts were seeded and cultured on the thin films of composites in vitro. The cell adhesion, spreading and growth of osteoblasts were analyzed with FITC staining, NIH Image J software and MTT assay. The change of cell cycle was monitored by flow cytometry (FCM). The results demonstrated that the Surface modification of BG with PLLA could significantly improve the dispersing of the particles in the matrix of PLGA. The nanocomposite with 20% PLLA-g-BG exhibited superior surface properties, including roughness and plenty of silicon, calcium and phosper, to enhance the adhesion, spreading and proliferation of osteoblasts.

Synthesis and characterization of photo-cross-linked hydrogels based on biodegradable polyphosphoesters and poly(ethylene glycol) copolymers

Novel biodegradable hydrogels by photo-cross-linking macromers based on polyphosphoesters and poly(ethylene glycol) (PEG) are reported. Photo-cross-linkable macromers were synthesized by ring-opening polymerization of the cyclic phosphoester monomer 2-(2-oxo-1,3,2-dioxaphospholoyloxy) ethyl methacrylate (OPEMA) using PEG as the initiator and stannous octoate as the catalyst. The macrorners were characterized by H-1 NMR, Fourier transform infrared spectroscopy, and gel permeation chromatography measurements. The content of polyphosphoester in the macromer was controlled by varying the feed ratio of OPEMA to PEG. Hydrogels were fabricated by exposing aqueous solutions of macromers with 0.05% (w/w) photoinitiator to UV light irradiation, and their swelling kinetics as well as degradation behaviors were evaluated. The results demonstrated that cross-linking density and pH values strongly affected the degradation rates. The macromers was compatible to osteoblast cells, not exhibiting significant cytotoxicity up to 0.5 mg/mL. "Live/dead" cell staining assay also demonstrated that a large majority of the osteoblast cells remained viable after encapsulation into the hydrogel constructs, showing their potential as tissue engineering scaffolds.

Direct patterning of rhodamine 6G molecules on mica by dip-pen nanolithography

Dip-pen nanolithography (DPN) has been developed to pattern monolayer film of various molecules on suitable substrate through the controlled movement of ink-coated atomic force microscopy (AFM) tip, which makes DPN a potentially powerful tool for making the functional nanoscale devices. In this paper, the direct patterning of rhodamine 6G on mica by dip-pen nanolithography was demonstrated. R6G features patterned on the mica was successfully achieved with different tip movement which can be programmed by Nanoscript(TM) language. From the AFM image of R6G patterns, we know that R6G molecule is flatly binding to the mica surface through electrostatic interaction, thus stable R6G nanostructures could be formed on mica. The influence of translation speed and contact time on DPN was discussed. The method can be extended to direct patterning of many other organic molecules, and should open many opportunities for miniaturized optical device and site-specific biological staining.

The integrative expression of GUS gene driven by FCP promoter in the seaweed Laminaria japonica (Phaeophyta)

In this study, the background activity of beta-glucuronidase (GUS) was analyzed histochemically and fluorometrically in the negative control of Laminaria japonica (Phaeophyta) thalli, showing low level of activity. GUS gene transformation without selectable gene in L. japonica was performed using four different promoters, i.e., Cauliflower mosaic virus 35S promoter (CaMV35S) from cauliflower mosaic virus, ubiquitin promoter (UBI) from maize, adenine-methyl transfer enzyme gene promoter (AMT) from virus in green alga Chlorella, and fucoxanthin chlorophyll a/c-binding protein gene promoter (FCP) from diatom Phaeodactylum tricornutum. The GUS transient activity was determined fluorometrically after bombarding sliced parthenogenetic sporophytes explants, and it was found that the activity resulting from CaMV35S and FCP promoters (in 114.3 and 80.6 pmol MU min(-1) (mg protein)(-1), respectively) was higher than for the other two promoters. The female gametophytes were bombarded and regenerated parthenogenetic sporophytes. FCP was the only promoter that resulted in detectable GUS chimeric expression activity during histochemical staining and polymerase chain reaction. Results of Southern blot showed that GUS gene was integrated with the L. japonica genome.

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.

Establishment of a micro-particle bombardment transformation system for Dunaliella saliva

In this study, we chronicle the establishment of a novel transformation system for the unicellular marine green alga, Dunaliella salina. We introduced the CaMV35S promoter-GUS construct into D. saliva with a PDS1000/He micro-particle bombardment system. Forty eight h after transformation, via histochemical staining, we observed the transient expression of GUS in D. salina cells which had been bombarded under rupture-disc pressures of 450 psi and 900 psi. We observed no GUS activity in either the negative or the blank controls. Our findings indicated that the micro-particle bombardment method constituted a feasible approach to the genetic transformation of D. salina. We also conducted tests of the cells' sensitivity to seven antibiotics and one herbicide, and our results suggested that 20 mu g/ ml of Basta could inhibit cell growth completely. The bar gene, which encodes for phosphinothricin acetyltransferase and confers herbicide tolerance, was introduced into the cells via the above established method. The results of PCR and PCR-Southern blot analyses indicated that the gene was successfully integrated into the genome of the transformants.

Cortactin mediated morphogenic cell movements during zebrafish (Danio rerio) gastrulation

Cell migration is essential to direct embryonic cells to specific sites at which their developmental fates are ultimately determined. However, the mechanism by which cell motility is regulated in embryonic development is largely unknown. Cortactin, a filamentous actin binding protein, is an activator of Arp2/3 complex in the nucleation of actin cytoskeleton at the cell leading edge and acts directly on the machinery of cell motility. To determine whether cortactin and Arp2/3 mediated actin assembly plays a role in the morphogenic cell movements during the early development of zebrafish, we initiated a study of cortactin expression in zebrafish embryos at gastrulating stages when massive cell migrations occur. Western blot analysis using a cortactin specific monoclonal antibody demonstrated that cortactin protein is abundantly present in embryos at the most early developmental stages. Immunostaining of whole-mounted embryo showed that cortactin immunoreactivity was associated with the embryonic shield, predominantly at the dorsal side of the embryos during gastrulation. In addition, cortactin was detected in the convergent cells of the epiblast and hypoblast, and later in the central nervous system. Immunofluorescent staining with cortactin and Arp3 antibodies also revealed that cortactin and Arp2/3 complex colocalized at the periphery and many patches associated with the cell-to-cell junction in motile embryonic cells. Therefore, our data suggest that cortactin and Arp2/3 mediated actin polymerization is implicated in the cell movement during gastrulation and perhaps the development of the central neural system as well.

Programmed cell death in Laminaria japonica (Phaeophyta) tissues infected with alginic acid decomposing bacterium

TdT-mediated dUTP-biotin nick end labeling (TUNEL) is a sensitive and valid method for detecting DNA cleavage in programmed cell death (PCD). Using this method, DNA cleavage was observed in Laminaria japonica sporophytic tissues, which were infected with alginic acid decomposing bacterium. It was found that DNA cleavage occurred 5 min after the infection, the fragments with 3'-OH groups of cleaved nuclear DNA increased with time of infection and spread from the infection site. Although no typical DNA ladder (200 bp/ 180 bp) was detected by routine agarose gel electrophoresis, the cleavage of nuclear DNA fragments of 97 similar to 48.5 kb could be detected by pulsed field gel electrophoresis (PFGE). By using CaspGLOW(TM) fluorescein active caspase-3 staining method, caspase-3 activity has been detected in response to the infection of alginic acid decomposing bacterium. Our results are similar to the observations in hypersensitive response (HR) of higher plant, suggesting that the rapid cell death of L. japonica infected by alginic acid decomposing bacterium might be involved in PCD, and indicating that the occurrence of PCD is an active defense process against the pathogen's infection.