Characterization of the rRNA locus of Pfiesteria piscicida and development of standard and quantitative PCR-based detection assays targeted to the nontranscribed spacer

Pfiesteria piscicida is a heterotrophic dinoflagellate widely distributed along the middle Atlantic shore of the United States and associated with fish kills in the Neuse River (North Carolina) and the Chesapeake Bay (Maryland and Virginia). We constructed a genomic DNA library from clonally cultured P. piscicida and characterized the nontranscribed spacer (NTS), small subunit, internal transcribed spacer 1 (ITS1), 5.8S region, ITS2, and large subunit of the rRNA gene cluster. Based on the P. piscicida ribosomal DNA sequence, we developed a PCR-based detection assay that targets the NTS. The assay specificity was assessed by testing clonal P. piscicida and Pfiesteria shumwayae, 35 additional dinoflagellate species, and algal prey (Rhodomonas sp.). Only P. piscicida and nine presumptive P. piscicida isolates tested positive. All PCR-positive products yielded identical sequences for P. piscicida, suggesting that the PCR-based assay is species specific. The assay can detect a single P. piscicida zoospore in 1 ml of water, 10 resting cysts in 1 g of sediment, or 10 fg of P. piscicida DNA in 1 micro g of heterologous DNA. An internal standard for the PCR assay was constructed to identify potential false-negative results in testing of environmental sediment and water samples and as a competitor for the development of a quantitative competitive PCR assay format. The specificities of both qualitative and quantitative PCR assay formats were validated with >200 environmental samples, and the assays provide simple, rapid, and accurate methods for the assessment of P. piscicida in water and sediments.     

Glycosylation-dependent interactions of C-type lectin DC-SIGN with colorectal tumor-associated Lewis glycans impair the function and differentiation of monocyte-derived dendritic cells

Dendritic cells (DCs) are APCs that play an essential role by bridging innate and adaptive immunity. DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) is one of the major C-type lectins expressed on DCs and exhibits high affinity for nonsialylated Lewis (Le) glycans. Recently, we reported the characterization of oligosaccharide ligands expressed on SW1116, a typical human colorectal carcinoma recognized by mannan-binding protein, which is a serum C-type lectin and has similar carbohydrate-recognition specificities as DC-SIGN. These tumor-specific oligosaccharide ligands were shown to comprise clusters of tandem repeats of Lea/Leb epitopes. In this study, we show that DC-SIGN is involved in the interaction of DCs with SW1116 cells through the recognition of aberrantly glycosylated forms of Lea/Leb glycans on carcinoembryonic Ag (CEA) and CEA-related cell adhesion molecule 1 (CEACAM1). DC-SIGN ligands containing Lea/Leb glycans are also highly expressed on primary cancer colon epithelia but not on normal colon epithelia, and DC-SIGN is suggested to be involved in the association between DCs and colorectal cancer cells in situ by DC-SIGN recognizing these cancer-related Le glycan ligands. Furthermore, when monocyte-derived DCs (MoDCs) were cocultured with SW1116 cells, LPS-induced immunosuppressive cytokines such as IL-6 and IL-10 were increased. The effects were significantly suppressed by blocking Abs against DC-SIGN. Strikingly, LPS-induced MoDC maturation was inhibited by supernatants of cocultures with SW1116 cells. Our findings imply that colorectal carcinomas affecting DC function and differentiation through interactions between DC-SIGN and colorectal tumor-associated Le glycans may induce generalized failure of a host to mount an effective antitumor response.     

Production of biologically active human thioredoxin 1 protein in lettuce chloroplasts

The production of human therapeutic proteins in plants provides opportunities for low-cost production, and minimizes the risk of contamination from potential human pathogens. Chloroplast genetic engineering is a particularly promising strategy, because plant chloroplasts can produce large amounts of foreign target proteins. Oxidative stress is a key factor in various human diseases. Human thioredoxin 1 (hTrx1) is a stress-induced protein that functions as an antioxidant against oxidative stress, and overexpression of hTrx1 has been shown to suppress various diseases in mice. Therefore, hTrx1 is a prospective candidate as a new human therapeutic protein. We created transplastomic lettuce expressing hTrx1 under the control of the psbA promoter. Transplastomic plants grew normally and were fertile. The hTrx1 protein accumulated to approximately 1% of total soluble protein in mature leaves. The hTrx1 protein purified from lettuce leaves was functionally active, and reduced insulin disulfides. The purified protein protected mouse insulinoma line 6 cells from damage by hydrogen peroxide, as reported previously for a recombinant hTrx1 expressed in Escherichia coli. This is the first report of expression of the biologically active hTrx1 protein in plant chloroplasts. This research opens up possibilities for plant-based production of hTrx1. Considering that this expression host is an edible crop plant, this transplastomic lettuce may be suitable for oral delivery of hTrx1.     

Expression, purification and characterization of isoforms of peripheral stalk subunits of human V-ATPase

The vacuolar-type H+-ATPase (V-ATPase) is a multi-subunit proton pump that is involved in both intra- and extracellular acidification processes throughout human body. Subunits constituting the peripheral stalk of the V-ATPase are known to have several isoforms responsible for tissue/cell specific different physiological roles. To study the different interaction of these isoforms, we expressed and purified the isoforms of human V-ATPase peripheral stalk subunits using Escherichia coli cell-free protein synthesis system: E1, E2, G1, G2, G3, C1, C2, H and N-terminal soluble part of a1 and a2 isoforms. The purification conditions were different depending on the isoforms, maybe reflecting the isoform specific biochemical characteristics. The purified proteins are expected to facilitate further experiments to study about the cell specific interaction and regulation and thus provide insight into physiological meaning of the existence of several isoforms of each subunit in V-ATPase.     

ER stress-induced protein, VIGG, disturbs plant cation homeostasis, which is correlated with growth retardation and robustness to ER stress

VIGG is a putative endoplasmic reticulum (ER) resident protein induced by virus infection and ER stress, and is correlated with fruit quality in grapevine. The present study was undertaken to determine the biological function of VIGG in grapevine. Experiments using fluorescent protein-VIGG fusion protein demonstrated that VIGG is localized in ER and the ER targeting sequence is in the N-terminus. The overexpression of VIGG in Arabidopsis plant led to growth retardation. The rosette leaves of VIGG-overexpressing plants were smaller than those of the control plants and rolled at 42 days after seeding. VIGG-overexpressing plants revealed robustness to ER stress as well as the low expression of ER stress marker proteins, such as the luminal binding proteins. These characteristics of VIGG-overexpressing plants were supported by a microarray experiment that demonstrated the disruption of genes related to ER stress response and flowering, as well as cation mobility, in the plants. Finally, cation homeostasis in the plants was disturbed by the overexpression of VIGG. Taken together, these results suggest that VIGG may disturb cation homeostasis in plant, which is correlated with the robustness to ER stress and growth retardation.     

Establishment of a New Quality Control and Vaccine Safety Test for Influenza Vaccines and Adjuvants Using Gene Expression Profiling

We have previously identified 17 biomarker genes which were upregulated by whole virion influenza vaccines, and reported that gene expression profiles of these biomarker genes had a good correlation with conventional animal safety tests checking body weight and leukocyte counts. In this study, we have shown that conventional animal tests showed varied and no dose-dependent results in serially diluted bulk materials of influenza HA vaccines. In contrast, dose dependency was clearly shown in the expression profiles of biomarker genes, demonstrating higher sensitivity of gene expression analysis than the current animal safety tests of influenza vaccines. The introduction of branched DNA based-concurrent expression analysis could simplify the complexity of multiple gene expression approach, and could shorten the test period from 7 days to 3 days. Furthermore, upregulation of 10 genes, Zbp1, Mx2, Irf7, Lgals9, Ifi47, Tapbp, Timp1, Trafd1, Psmb9, and Tap2, was seen upon virosomal-adjuvanted vaccine treatment, indicating that these biomarkers could be useful for the safety control of virosomal-adjuvanted vaccines. In summary, profiling biomarker gene expression could be a useful, rapid, and highly sensitive method of animal safety testing compared with conventional methods, and could be used to evaluate the safety of various types of influenza vaccines, including adjuvanted vaccine.