Expression of a synthetic neutralizing epitope of porcine epidemic diarrhea virus fused with synthetic B subunit of <Emphasis Type="Italic">Escherichia coli</Emphasis> heat labile enterotoxin in rice endosperm

Epitopes often require co-delivery with adjuvant and targeting proteins to enable recognition by the immune system, and this approach may also increase the efficacy of the antigen. In this study, we assess and describe the ability of transgenic rice plants to express a fusion protein consisting of the B-subunit of the Escherichia coli heat-labile enterotoxin (LTB) and a synthetic core-neutralizing epitope (COE) of porcine epidemic diarrhea virus (PEDV), inducing an enteric disease that is seen most predominantly in piglets. Both components of the fusion proteins were detected with Western blot analysis. The fusion protein was determined to assemble into pentamers, as was evidenced by its ability to bind to GM1 gangliosides, and evidenced an average level of expression in a transgenic rice endosperm. This indicates that the expression system of the plant is capable of generating a sizable amount of antigen, possibly allowing for the successful development of an edible vaccine.     

Sinomenine inhibits primary CD4+ T-cell proliferation via apoptosis

Sinomenine is an active component isolated from Sinomenium acutum and is widely used as an immunosuppressive drug for treating autoimmune diseases. CD4(+) T-cell population plays a key role in adaptive immune response and is related to some autoimmune diseases. In this study, we investigated the possible immunosuppressive effect of sinomenine on CD4(+) T cells and its underlying mechanism. Our data demonstrated that sinomenine remarkably suppressed the proliferation of CD4(+) T cells, blocked the cell cycle progression from G0/G1 phase to S plusG2/M phases. Finally, the immunosuppressive activity elicited by sinomenine in CD4(+) primary lymphocytes was found to be largely accounted for by caspase 3-dependent cells apoptosis. Sinomenine did not significantly alter the expression of bcl-2 in activated CD4(+) primary T cells, suggesting that bcl-2 might not be involved in sinomenine-induced T cells apoptosis. In sum, this study proposes a novel mechanism for the immunosuppressive function of sinomenine on primary mouse CD4(+) T cells.     

Structural and expression analyses of two vitellogenin genes in the carp, Cyprinus carpio

We cloned and sequenced two vitellogenin (vg) cDNAs of the carp, Cyprinus carpio, using a cDNA library constructed from estradiol-17β (E₂)-treated livers. One was a novel, longer 5000 bp-long cDNA termed vg-B2 encoding 1624 amino acids in a single open reading frame. The other was a shorter cDNA (vg-B1), identical to that registered previously as carp vg cDNA in the international nucleotide sequence database. The deduced amino acid sequences of these two molecules were well-aligned with known vertebrate Vgs sharing common characteristics such as N-terminal lipovitellin I (LVI), phosvitin (PV) and C-terminal lipovitellin II (LVII). The novel Vg-B2 bore a highly conserved GL/ICG motif within the LVII region, in contrast to the shorter Vg-B1 that has a truncated C-terminal and lacks the β-component within the LVII region including the GL/ICG motif. Both vg-B2 and vg-B1 genes were expressed in the livers of females and E₂-injected males. Western blot analysis using anti-Vg and anti-vitellin (Vn) antisera demonstrated that both Vg-B2 and Vg-B1 were detected as polypeptides with an estimated molecular mass of 180 kDa and 160 kDa, respectively, in the blood of females and E₂-injected males. The results suggest the potential utilization of these genes as sensitive xenoestrogenic markers.     

Computational protein design: structure, function and combinatorial diversity

Computational protein design has blossomed with the development of methods for addressing the complexities involved in specifying the structure, sequence and function of proteins. Recent applications include the design of novel functional membrane and soluble proteins, proteins incorporating non-biological components and protein combinatorial libraries.     

Enhancement of algicidal activity by immobilization of algicidal bacteria antagonistic to Stephanodiscus hantzschii (Bacillariophyceae)

AIMS: Enhancement of algicidal activity by immobilization of algicidal bacteria antagonistic to Stephanodiscus hantzschii. METHODS AND RESULTS: In laboratory studies, A diatom-lysing bacterium, Pseudomonas fluorescens HYK0210-SK09 showed strong algicidal activity against S. hantzschii, but a natural mesocosm study revealed that this bacterium failed to fully control natural blooms of Stephanodiscus at the low water temperatures that favour these blooms. Here, we sought to develop an effective immobilization strategy for enhancing the algicidal activity of HYK0210-SK09 in the natural setting. Bacterium HYK0210-SK09 was immobilized with various carriers including agar, alginate, polyurethane and cellulose sponge. The bacterial cells immobilized with cellulose sponge (CIS) induced more rapid and complete lysis of S. hantzschii than other carriers, and had a higher packing ability than polyurethane. Furthermore, CIS-immobilized cells showed higher lysis of S. hantzschii at the same concentrations as that of free cells (< or =1 x 10(7) cells ml(-1)), and had especially strong algicidal activity at the low temperatures (<10 degrees C). Based on these laboratory studies, we assessed the possible application of HYK0210-SK09 cells in the field by performing a mesocosm study during the winter season. The CIS-immobilized cells with species-specific activity towards the genera Stephanodiscus showed extremely high algicidal activity (up to 95%) against a bloom of Stephanodiscus hantzschii even at low water temperatures, because of high cell packing and subsequent cell protection against low temperatures and predators, whereas free cells showed negligible algicidal activities under these conditions. CONCLUSION: Immobilizing cells of HYK0210-SK09 in CIS foam, rather than in the other matrices tested, could achieve more efficient control of Stephanodiscus blooms and showed a significant algicidal activity on in vitro and in vivo blooms, even at low water temperature. SIGNIFICANCE AND IMPACT OF THE STUDY: Collectively, these results indicate that CIS of algicidal bacteria may form an important strategy for effective management of Stephanodiscus blooms at low water temperatures.     

COOH-terminal association of human smooth muscle calcium channel Ca(v)1.2b with Src kinase protein binding domains: effect of nitrotyrosylation

The carboxyl terminus of the calcium channel plays an important role in the regulation of calcium entry, signal transduction, and gene expression. Potential protein-protein interaction sites within the COOH terminus of the L-type calcium channel include those for the SH3 and SH2 binding domains of c-Src kinase that regulates calcium currents in smooth muscle. In this study, we examined the binding sites involved in Src kinase-mediated phosphorylation of the human voltage-gated calcium channel (Ca(v)) 1.2b (hCav1.2b) and the effect of nitrotyrosylation. Cotransfection of human embryonic kidney (HEK)-293 cells with hCa(v)1.2b and c-Src resulted in tyrosine phosphorylation of the calcium channel, which was prevented by nitration of tyrosine residues by peroxynitrite. Whole cell calcium currents were reduced by 58 + 5% by the Src kinase inhibitor PP2 and 64 + 6% by peroxynitrite. Nitrotyrosylation prevented Src-mediated regulation of the currents. Glutathione S-transferase fusion protein of the distal COOH terminus of hCa(v)1.2b (1809-2138) bound to SH2 domain of Src following tyrosine phosphorylation, while binding to SH3 required the presence of the proline-rich motif. Site-directed mutation of Y(2134) prevented SH2 binding and resulted in reduced phosphorylation of hCa(v)1.2b. Within the distal COOH terminus, single, double, or triple mutations of Y(1837), Y(1861), and Y(2134) were constructed and expressed in HEK-293 cells. The inhibitory effects of PP2 and peroxynitrite on calcium currents were significantly reduced in the double mutant Y(1837-2134F). These data demonstrate that the COOH terminus of hCa(v)1.2b contains sites for the SH2 and SH3 binding of Src kinase. Nitrotyrosylation of these sites prevents Src kinase regulation and may be importantly involved in calcium influx regulation during inflammation.     

红豆杉科白豆杉属植物的分类学研究

白豆杉属Pseudotaxus现包括2种,即白豆杉P.chienii(Cheng)Cheng和李氏白豆杉P.lianaJ.Silba,二者之间的区别在于叶片宽度不同。本文对来自浙江、江西、湖南和广西等地的白豆杉属植物标本进行了观察和测量。尽管t检验分析表明来自浙江的标本与来自江西、湖南和广西的标本在叶片宽度平均数上的确存在显著差异,但是,散点图分析却显示叶片宽度变异是连续的,这与J.Silba的观察不同,因此,将P.lianaJ.Silba作为P.chienii(Cheng)Cheng的异名处理。