Cationic Lipid Bilayer Coated Gold Nanoparticles-mediated Transfection of Mammalian Cells

Here, we demonstrated dimethyldioctadecylammonium bromide (DODAB), a cationic lipid, bilayer coated Au nanoparticles (AuNPs) could efficiently deliver two types of plasmid DNA into human embryonic kidney cells (HEK 293) in the presence of serum. The transfection efficiency of AuNPs was about five times higher than that of DODAB. The interaction of AuNPs with DNA was characterized with dye intercalation assay and agarose gel electrophoresis. The morphology of the complex of AuNPs with DNA was observed with scanning electron microscope (SEM). The intracellular trafficking of the complex was monitored with transmission electron microscope (TEM).

Membrane Deformability and Membrane Tension of Single Isolated Mitochondria

Mitochondria dynamics is crucial to many biological processes such as mitochondria fusion and fission, which is highly correlated to the mechanics of single mitochondria. However, the mechanobiological coupling of mitochondria has been poorly understood. Here membrane deformability and membrane tension of individual mitochondria isolated from MtDsRed labeled human embryonic T-Rex-293 kidney cells were measured using a micropipette aspiration assay. The results demonstrated that membrane deformation of isolated mitochondria exhibited an elastic transition phase followed by an equilibrium phase, and mitochondrial membrane tension was proportional to the area compressibility. It was also indicated that mitochondrial membrane deformability was significantly affected by physical chemical factors such as osmotic pressure or pH value, and was further correlated to mitochondrial functionality in different respiratory states and Ca2+ regulation. These findings provide a new insight into understanding the mechanical regulation of mitochondrial physiology.

The biological effects of rainbow trout (Oncorhynchus mykiss) recombinant interleukin-8

In this report, recombinant interieukin-8 (rIL-8) was produced and its activity tested for the first time in fish. The rainbow trout rIL-8 was produced in Escherichia coli and purified using a 6xHis tag at the N-terminus. The rIL-8 induced a dose-dependent migration of head kidney leukocytes at concentrations from 0.1 to 10 ng/ml, with a peak response at 1 ng/ml. Trout rIL-8 also had a significant effect on superoxide production by head kidney cells, with maximal, activity at 0.1 and 1 ng/ml. When injected intraperitoneally into trout, rIL-8 had a clear effect on total leukocyte number in the peritoneal cavity, with increasing doses (up to 5 mu g) eliciting more cells. Of three leukocyte types distinguished, neutrophils were the dominant cell type, especially at higher rIL-8 concentrations. In contrast, the proportion of macrophages and lymphocytes decreased with rIL-8 administration, suggesting that they were not attracted at the same rate as neutrophils. (C) 2007 Elsevier Ltd. All rights reserved.

Enhanced resistance to Aeromonas hydrophila infection and enhanced phagocytic activities in human lactoferrin-transgenic grass carp (Ctenopharyngodon idellus)

Human lactoferrin (hLF) is an iron-binding protein with antimicrobial and immunomodulatory activities. hLF cDNA was transferred into grass carp via electroporated sperm. The production of transgenic fish was as high as 55% tinder the best parameters. 2(11) pulses and 20-min incubation. The expression of the transgene was demonstrated by the detection of hLF mRNA by RT-PCR. We also investigated the response of G(0) transgenic grass carp to Aeromonas hydrophila infection. Serum lysozyme activities (P>0.05) and phagocytic activities of kidney cells (P<0.05) were measured in transgenic individuals. The transgenic fish not only cleared A. hydrophila significantly faster than the control carp (P<0.05), but also showed enhanced phagocytic activities. The result shows that hLF has immunomodulatory activities in hLF-transgenic grass carp. The transgenic grass carp exhibited enhanced immunity to A. hydrophila infection. These results reveal that the mechanisms of disease resistance are different between hLF-transgenic plants and hLF-transgenic grass carp. (C) 2004 Elsevier B.V. All rights reserved.

Embryonic and genetic manipulation in fish

Fishes, the biggest and most diverse community in vertebrates are good experimental models for studies of cell and developmental biology by many favorable characteristics. Nuclear transplantation in fish has been thoroughly studied in China since 1960s. Fish nuclei of embryonic cells from different genera were transplanted into enucleated eggs generating nucleo-cytoplasmic hybrids of adults. Most importantly, nuclei of cultured goldfish kidney cells had been reprogrammed in enucleated eggs to support embryogenesis and ontogenesis of a fertile fish. This was the first case of cloned fish with somatic cells. Based on the technique of microinjection, recombinant MThGH gene has been transferred into fish eggs and the first batch of transgenic fish were produced in 1984. The behavior of foreign gene was characterized and the onset of the foreign gene replication occurred between the blastula to gastrula stages and random integration mainly occurred at later stages of embryogenesis. This eventually led to the transgenic mosaicism. The MThGH-transferred common carp enhanced growth rate by 2-4 times in the founder juveniles and doubled the body weight in the adults. The transgenic common carp were more efficient in utilizing dietary protein than the controls. An "all-fish" gene construct CAgcGH has been made by splicing the common carp beta-actin gene (CA) promoter onto the grass carp growth hormone gene (gcGH) coding sequence. The CAgcGH-transferred Yellow River Carp have also shown significantly fast-growth trait. Combination of techniques of fish cell culture, gene transformation with cultured cells and nuclear transplantation should be able to generate homogeneous strain of valuable transgenic fish to fulfil human requirement in 21(st) century.

Detection of constitutive heterodimerization of the integrin Mac-1 subunits by fluorescence resonance energy transfer in living cells

Macrophage differentiation antigen associated with complement three receptor function (Mac-1) belongs to beta(2) subfamily of integrins that mediate important cell-cell and cell-extracellular matrix interactions. Biochemical studies have indicated that Mac-1 is a constitutive heterodimer in vitro. Here, we detected the heterodimerization of Mac-1 subunits in living cells by means of two fluorescence resonance energy transfer (FRET) techniques (fluorescence microscopy and fluorescence spectroscopy) and our results demonstrated that there is constitutive heterodimerization of the Mac-1 subunits and this constitutive heterodimerization of the Mac-1 subunits is cell-type independent. Through FRET imaging, we found that heterodimers of Mac-1 mainly localized in plasma membrane, perinuclear, and Golgi area in living cells. Furthermore, through analysis of the estimated physical distances between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) fused to Mac-1 subunits, we suggested that the conformation of Mac-1 subunits is not affected by the fusion of CFP or YFP and inferred that Mac-1 subunits take different conformation when expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293T cells, respectively. (c) 2006 Elsevier Inc. All rights reserved.

Ontogeny of IgM-producing cells in the mandarin fish Siniperca chuatsi identified by in situ hybridisation

The ontogeny of IgM-producing cells was studied in juvenile mandarin fish Simperca chuatsi, an important fish in China's aquaculture sector. The IgM-producing cells were localised through in situ hybridisation with a probe complementary to the Ig mu-chain in lymphoid-related tissues, including head kidney, spleen, thymus, intestine and gills. In head kidney, transcripts of Ig mu were first detected at 20 days post-hatching (dph) with a few positive signals. and the number of IgM-producing cells increased obviously from 39 dph onwards. At 136 dph, a large amount of positive cells were observed in the entire organ with clusters of these cells located around the blood vessels. In spleen, IgM-producing cells were found from 26 dph onwards, followed by an increase until 67 dph: clusters of positive cells were also detected around blood vessels at 102 dph. In thymus, IgM-producing cells were first observed at 39 dph; thereafter, no obvious increase was detected until 78 dph. The positive cells in thymus were distributed mainly in the outer zone of thymus. A few IgM-producing cells were still observed in thymus of 1-year-old mandarin fish. IgM-producing cells were not detected in the intestine until 87 dph, with several discrete positively stained cells distributed in the lamina propria. IgM-producing cells, scattered mainly in primary gill filaments around blood vessels, were detected in gills from 90 dph. As in other teleosts, these results indicated that the head kidney appears to be the primary organ for IgM production in mandarin fish, and IgM-producing cells exist in all organs examined in the present study, implying their lymphoid role in fish. In addition, it is suggested that vaccination after 20 dph may be much more effective in mandarin fish. (C) 2009 Elsevier B.V. All rights reserved.

Detection of serum and mucosal antibody production and antibody secreting cells (ASCs) in large yellow croaker (Pseudosciaena crocea) following vaccination with Vibrio harveyi via different routes

The kinetics of mucosal and serum antibody response is well as antibody secreting cells (ASCs) production were studied in large yellow croaker following vaccination with inactivated Vibrio harveyi by different routes: oral administration. intraperitoneal (IP) injection and immersion. Indirect ELISA was used to measure the antibody level in serum and cutaneous mucus, and ELISPOT was used to monitor the ASCs derived from gill, blood and head kidney. The data demonstrated that IP injection resulted in the highest antibody levels in the systemic circulation, whereas immersion induced significant antibody levels in mucous. As for the ASCs response, IP injection induced high numbers of ASCs in the head kidney and blood; oral intubation only induced a slight ASCs response in the head kidney: immersion induced a much stronger ASCs response in the gill. These results indicate that mucosal antibodies following immersion immunization are independent of a systemic response and more sensitive, since it could be triggered earlier than serum antibodies. The mucosal antibodies following IP injection immunization may depend oil a systemic immune response. The protective effects of the three vaccination methods were compared by challenging with live V. harveyi. Survival of the three groups of vaccinated fish varied front 40 to 60%. while 100% mortality was found in control fish. Compared with IP and oral vaccination, immersion stimulated higher specific antibody titers in the mucosal system and achieved similar protection, so it is in effective and efficient method for immunizing a large number of fish against V harveyi (C) 2008 Elsevier B.V. All rights reserved.