Separation and isolation of BTV dsRNA segments and viral proteins.

Bluetongue virus (BTV) genome contains ten double-stranded RNA segments. The sequence of the plus strand of each of the BTV genomic double-stranded RNAs is the same as that of its mRNA, which encodes for a single viral protein, except the smallest S4 segment which can encode for two nonstructural proteins, primarily for the release assistance of the viral progeny. The separation and isolation of each BTV dsRNA segment and viral protein have provided extensive data related to its viral infection, pathology, suppression of host cellular functions, and eventual apoptosis of the infected host cells. This cytoplasmic virus is also an animal killer that costs the U.S. livestock industry at least $125 million yearly. However, this virus has no known effect on humans. Thus, it is very safe to carry out investigation with the virus, preferably in a BSL-2 laboratory.

Generation and characterization of anti-alpha-enolase single-chain antibodies in chicken.

It was previously reported that up-regulation of alpha-enolase protein was detected in 65% of patients with non-small cell lung cancers (NSCLC). Moreover, a high titer of anti-alpha-enolase antibodies was developed in a smaller proportion (7.4%) of these patients than in non-tumor-associated patients and healthy subjects. In the present study, we characterized polyclonal and single-chain variable fragment (scFv) anti-alpha-enolase antibodies from immunized chickens. The E. coli-derived recombinant alpha-enolase protein was purified to its high homogenicity as verified by SDS-PAGE. After the 4th immunization, a high titer of specific polyclonal anti-alpha-enolase antibodies was elicited in immunized chickens and specifically recognized the purified human alpha-enolase antigen as determined by Western blot and ELISA. The expressed heavy and light chain variable genes (VH and VL) were isolated from spleen B cells and amplified to construct phage antibody libraries containing scFv molecules. After four rounds of panning selection, the scFv antibodies of randomly chosen clones were expressed and their binding specificity to alpha-enolase protein was verified using competitive ELISA, flow cytometry and immunofluorescence staining. Nucleotide sequence analysis from 10 alpha-enolase binding clones showed that 3 (30%) clones used identical heavy and light genes for scFv antibody expression, as represented by EnL5. Notably, amino acid changes in complementarity-determining regions (CDRs) were more frequently observed than those in framework regions (FRs) in all clones, indicating a strong affinity selection through mutations. All together, it is believed that these polyclonal and scFv IgY antibodies may be helpful in the development of molecular diagnostic and therapeutic agents for lung cancers in the future.

Taiwanese native plants inhibit matrix metalloproteinase-9 activity after ultraviolet B irradiation.

Medicinal plants have long been used as a source of therapeutic agents. They are thought to be important anti-aging ingredients in prophylactic medicines. The aim of this study was to screen extracts from Taiwanese plant materials for phenolic contents and measure the corresponding matrix metalloproteinase-9 (MMP-9) activity. We extracted biological ingredients from eight plants native to Taiwan (Alnus formosana, Diospyros discolor, Eriobotrya deflex, Machilus japonica, Pyrrosia polydactylis, Pyrus taiwanensis, Vitis adstricta, Vitis thunbergii). Total phenolic content was measured using the Folin-Ciocalteu method. MMP-9 activities were measured by gelatin zymography. The extracted yields of plants ranged from 3.7 % to 16.9 %. The total phenolic contents ranged from 25.4 to 36.8 mg GAE/g dry material. All of these extracts (except Vitis adstricta Hance) were shown to inhibit MMP-9 activity of WS-1 cell after ultraviolet B irradiation. These findings suggest that total phenolic content may influence MMP-9 activity and that some of the plants with higher phenolic content exhibited various biological activities that could serve as potent inhibitors of the ageing process in the skin. This property might be useful in the production of cosmetics.

Fisetin, morin and myricetin attenuate CD36 expression and oxLDL uptake in U937-derived macrophages.

Dietary flavonoid intake has been reported to be inversely associated with the incidence of coronary artery disease. To clarify the possible role of flavonoids in the prevention of atherosclerosis, we investigated the effects of some of these compounds, including fisetin, morin and myricetin, on the susceptibility of low-density lipoprotein (LDL) to oxidative modification and on oxLDL uptake in macrophages. The results demonstrated that fisetin had stronger inhibitory activity than the other two on inhibiting Cu(2+)-mediated LDL oxidation measured by thiobarbituric acid-reactive substances assay (TBARS), conjugated diene formation and electrophoretic mobility. The class B scavenger receptor, CD36, to which oxLDL binds, is present in atherosclerotic lesions. Treatment of U937-derived macrophages with myricetin (20 microM) significantly inhibited CD36 cell surface protein and mRNA expression (p<0.01). Fisetin, morin and myricetin (20 microM) also reduced the feed-forward induction of CD36 mRNA and surface protein expression by PPARgamma. The inhibition of CD36 by flavonols was mediated by interference with PPARgamma activation thus counteracting the deleterious autoamplification loop of CD36 expression stimulated by PPARgamma ligand. All three flavonols (10 and 20 microM) markedly decreased the uptake of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanide perchlorate (DiI)-labeled oxLDL uptake in U937-derived macrophages dose-dependently. Current evidences indicate that fisetin, morin and myricetin not only prevent LDL from oxidation but also block oxLDL uptake by macrophages at least in part through reducing CD36 gene expression on macrophages. In conclusion, flavonols may play a role in ameliorating atherosclerosis.

Determination of camptothecins in DMSO extracts of Nothapodytes foetida by direct injection capillary electrophoresis.

A rapid capillary electrophoresis procedure was developed for determining the anti-cancer components, camptothecins, in Nothapodytes foetida. The hydrophobic compound was extracted from plant tissue (ca. 1 mL of DMSO for 100 mg of dried plant tissue) with a water-miscible organic solvent, DMSO, at elevated temperature (60 degrees C). The extract was directly injected into the separation capillary (untreated fused silica, 34 cm in length, 75 microm i.d.) and analysed in MEKC mode (369 nm). Within 5 min of migration, camptothecins were successfully separated and quantified by adding organic modifiers to the running buffer (20% DMSO, 90 mm SDS in 10 mm borate buffer, pH 8.60). The linear dynamic range for camptothecin was from 5 to 400 microg/mL. This method was proven to be very suitable for monitoring the amount of camptothecins during the cultivation of the medicinal plant.

Chicken single-chain variable fragments against the SARS-CoV spike protein.

The major concern for severe acute respiratory syndrome (SARS), caused by the SARS-associated coronavirus (SARS-CoV), is the lack of diagnostic and therapeutic agents. Using a phage display technology in a chicken system, high-affinity monoclonal antibody fragments against the SARS-CoV spike protein were characterized. Ten truncated spike protein gene fragments were expressed in Escherichia coli cells. Following the immunization of chickens with these recombinant spike proteins, two single-chain variable fragment (scFv) antibody libraries were established with short or long linkers to contain 5x10(7) and 9x10(6) transformants, respectively. After four rounds of panning selection, the scFv antibodies of randomly chosen clones were demonstrated by Coomassie blue staining, and verified by western blot analysis. In a comparison of nucleotide sequences with the chicken germline gene, we found that all clones varied in the complementarity-determining regions, that two scFv antibodies reacted significantly with SARS-CoV-infected Vero cells, and that those two specific scFv antibodies recognized the same region of the spike protein spanning amino acid residues 750-1000. In conclusion, the results suggest that the chicken scFv phage display system can be a potential model for mass production of high-affinity antibodies against the SARS-CoV spike protein.

A dominant antigenic epitope on SARS-CoV spike protein identified by an avian single-chain variable fragment (scFv)-expressing phage.

Severe acute respiratory syndrome (SARS) is a newly emergent human disease, which requires rapid diagnosis and effective therapy. Among antibody sources, immunoglobulin Y (IgY) is the major antibody found in chicken eggs and can be used as an alternative to mammalian antibodies normally used in research and immunotherapy. In this study, phage-expressing chicken monoclonal scFv antibody was chosen and characterized with phage display antibody technology. Truncated fragments of SARS-CoV spike protein were cloned in pET-21 vector and expressed in BL-21 Escherichia coli (E. coli) cells. After purification, the purity of these recombinant spike proteins was examined on SDS-PAGE and their identity verified with Western blot analysis using anti-his antibodies and sera from convalescent stage SARS-CoV-infected patients. Using these bacteria-derived proteins to immunize chickens, it was found that polyclonal IgY antibodies in the egg yolk and sera were highly reactive to the immunogens, as shown by Western blot and immunocytochemical staining analysis. A phage displaying scFv library was also established from spleen B cells of immunized chicken with 5 x 10(7) clones. After four panning cycles, the eluted phage titer showed a 10-fold increase. In sequence analysis with chicken germline gene, five phage clones reacted, with large dissimilarities of between 31 and 62%, in the complementarity-determining regions, one dominant phage 4S1 had strong binding to fragment Se-e, located between amino acid residues 456-650 of the spike protein and this particular phage had significantly strong binding to SARS-CoV-infected Vero E6 cells. Based on the results, we conclude that generating specific scFv-expressing phage binders with the phage display system can be successfully achieved and that this knowledge can be applied in clinical or academic research.