Effect of maillard reaction on allergenicity of scallop tropomyosin

Scallop tropomyosin (TM), the major allergen of shellfish, was prepared from adductor muscles and reacted with four reducing sugars to investigate the effect of the Maillard reaction on the allergenicity of TM. The IgE-binding ability of TM increased significantly with the progress of the reaction with glucose, ribose, and maltose, but not with maltotriose. The allergenicity was enhanced at the early stage of the Maillard reaction, and the trend of the effect depended on the type of reducing sugar used. 2,4,6-Trinitrobenzenesulfonic acid treatment of the lysine residues in TM showed that the protein surface charge resulting from the Maillard reaction had no effect on the enhancement of the allergenicity. Thus, the change in the allergenicity would be closely related to the structural change caused by the Maillard reaction.     

Isolation of lignin–carbohydrate bonds in wood. Model experiments and preliminary application to pine wood

A novel method for analysis of benzylic ether type lignin–carbohydrate bonds has been developed by using model compounds. Four diastereomers of model compound 3-(4-hydroxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-3-(methyl -d-glucopyranoside-6-O-yl)-1-propanol (GGMGP), were ozonized in acetic acid/water/methanol 16:3:1 for 1h at 0°C. The product from ozonation of each diastereomer was saponified and the corresponding -etherified tetronic acid (TAMGP) was isolated using ion exchange chromatography. Minor amounts of methyl -d-glucopyranoside (MGP) and small amounts of a gluconic acid etherified with tetronic acid (TAGLCA), tetronic acid, gluconic acid, and glyceric acid were detected in the product mixture of ozonated benzylic ether type model compounds. The results suggest that a benzyl ether bond between lignin and carbohydrate is rather stable during the ozone treatment. Acid treatments with sulfuric acid or trifluoroacetic acid of the derived TAMGP led to cleavage of the glucosidic bond but only a small amount of products (tetronic acid and glucose) resulting from cleavage of the -ether bond were formed. The successful chemical treatments were used for studies of benzylic ether bonds in Japanese red pine. The results suggest the presence of benzylic ether bonds to polysaccharides in the wood.Part of this paper was presented at the 10th International Symposium of Wood and Pulping Chemistry (ISWPC), Yokohama, 1999; the 11th ISWPC, Nice, 2001; and as a rapid comunication in J Wood Sci (2000) 46:263–265     

In Vitro Antitumor Activity of Stellettin B,a Triterpene from Marine Sponge Jaspis stellifera,on Human Glioblastoma Cancer SF295 Cells

Stellettin B was isolated from marine sponge Jaspis stellifera. In vitro antitumor activities were investigated on 39 human cancer cell lines. Stellettin B exhibited highly potent inhibition against the growth of a human glioblastoma cell line SF295, with a GI50 of 0.01 μM. In contrast, stellettin B showed very weak inhibitory activity on normal cell lines including HMEC, RPTEC, NHBE and PrEC, with GI50s higher than 10 μM, suggesting its relatively selective cytotoxicity against human cancer cells compared to normal human cell lines. We then focused on the antitumor activity of this compound on SF295 cells. Flow cytometric analysis indicated that stellettin B induced apoptosis in SF295 cells in a concentration-dependent manner. Further study indicated that stellettin B increased the production of ROS, the activity of caspase 3/7, as well as the cleavage of PARP, each of which is known to be involved in apoptosis. To investigate the molecular mechanism for cell proliferation inhibition and apoptosis induction, effect on the phosphorylation of several signal proteins of PI3K/Akt and RAS/MAPK pathways was examined. Stellettin B inhibited the phosphorylation of Akt potently, with no activity on p-ERK and p-p38, suggesting that inhibition of PI3K/Akt pathway might be involved in the antiproliferative and apoptosis-inducing effect. However, homogenous time-resolved fluorescence (HTRF) assay indicated that stellettin B did not inhibit PI3K activity, suggesting that the direct target might be signal protein upstream of Akt pathway other than PI3K.     

Expression and localization of tight junction-related proteins in adult rat pituitary stem/progenitor cell niches

Pituitary endocrine cells are supplied by Sox2-expressing stem/progenitor cells in the anterior lobe of the adult pituitary gland. These SOX2-positive cells are maintained in two types of microenvironments (niches): the marginal cell layer (MCL)-niche and the parenchymal-niche. Recently, we isolated dense SOX2-positive cell clusters from the parenchymal-niche by taking advantage of their resistance to protease treatment as parenchymal stem/progenitor cell (PS)-clusters. In the present study, by analyzing these isolated PS-clusters, we attempted to identify novel structural characteristics of pituitary stem/progenitor cell niches. Quantitative real-time PCR showed that tight junction-related genes were distinctly expressed in the isolated PS-clusters. Immunocytostaining showed that the tight junction molecules, ZO-1 and occludin, were localized in the apical membrane facing the pseudo-follicle-like structure of the isolated PS-clusters regardless of the expression of S100β, which distinguishes the sub-population of SOX2-positive cells. Furthermore, immunohistochemistry of the pituitary glands of adult rats clearly demonstrated that ZO-1 and occludin were densely present in the parenchymal-niche encircling the pseudo-follicle, while they were observed in the apical membrane in the MCL-niche facing the residual lumen. Collectively, these tight junction-related proteins might be involved in the architecture and maintenance of the plasticity of pituitary stem/progenitor cell niches.     

Diagnostic value of recombinant nanoluciferase fused Toxoplasma gondii antigens in Luciferase-linked Antibody Capture Assay (LACA) for Toxoplasma infection in pigs

Toxoplasmosis is a widespread protozoan zoonosis. Since ingesting undercooked meat harboring Toxoplasma gondii cyst is considered one of the major transmission routes to humans, the screening of T. gondii in meat-producing animals can reduce the risk of food-borne toxoplasmosis in humans. Among serological diagnostic methods, Luciferase-linked Antibody Capture Assay (LACA) has been found to be a promising platform with high sensitivity and specificity. In this study, we aimed to evaluate recombinant nanoluciferase fused-T. gondii antigens (rNluc-GRA6, rNluc-GRA7, rNluc-GRA8 and rNluc-BAG1) for their potential use in LACA for pigs. As a result, the sensitivity of GRA6-, GRA7-, GRA8- and BAG1-LACA were 70.0%, 80.0%, 80.0% and 30.0% with specificity 87.0%, 81.5%, 74.1% and 50.0%, respectively. The cocktail LACA using a mixture of rNluc-GRA6, rNluc-GRA7 and rNluc-GRA8 indicated higher sensitivity (90.0%) and a similar specificity (96.3%) in comparison with the commercial ELISA kit. Compared to the Dye-Test as a reference test, cocktail LACA showed strong agreement (kappa value=0.811) when we assessed pig sera collected at the slaughterhouse. In addition, we also successfully established the rapid LACA format for the detection of Toxoplasma infection in pigs (called Rapid-LACA) in which the test could be performed within 30 min. In Rapid-LACA, the protein A pre-coated/blocked plates could be preserved at −30°C, 4°C or room temperature conditions for at least two months without compromising on the quality of assay.