Analysis of food allergen structures and development of foods for allergic patients

Food allergy is one of the important health problems, and countermeasures are socially required. We have been undertaking studies on wheat allergens and their epitopes, and have developed a method for producing hypoallergenic wheat flour by enzymatic modification. The hypoallergenic products are now provided to patients. More noteworthy, by taking hypoallergenic cupcakes over a long period, more than half of patients are hyposensitized and become able to eat normal wheat products. This suggests that the hypoallergenic wheat flour can act as anti-allergenic via allergen-specific immunotolerance. This series of studies was followed by expansive research on food allergy: analysis of epitopes of bovine serum albumin (the major beef allergen), isolation and identification of inhibitory peptides for allergen absorption at the intestine, evaluation of hesperetin as an inhibitor of degranulation of mast cells, and the development of PCR detection methods for verifying allergen labeling and for identifying hidden allergic ingredients in processed foods.     

Overcoming hERG issues for brain-penetrating cathepsin S inhibitors: 2-cyanopyrimidines. Part 2

We describe here orally active and brain-penetrant cathepsin S selective inhibitors, which are virtually devoid of hERG K(+) channel affinity, yet exhibit nanomolar potency against cathepsin S and over 100-fold selectivity to cathepsin L. The new non-peptidic inhibitors are based on a 2-cyanopyrimidine scaffold bearing a spiro[3.5]non-6-yl-methyl amine at the 4-position. The brain-penetrating cathepsin S inhibitors demonstrate potential clinical utility for the treatment of multiple sclerosis and neuropathic pain.     

ClC-3-independent, PKC-dependent activity of volume-sensitive Cl channel in mouse ventricular cardiomyocytes.

Volume-sensitive outwardly rectifying (VSOR) Cl- channels are activated during osmotic swelling and involved in the subsequent volume regulation in most animal cells. To test the hypothesis that the ClC-3 protein is the molecular entity corresponding to the VSOR Cl- channel in cardiomyocytes, the properties of VSOR Cl- currents in single ventricular myocytes isolated from ClC-3-deficient (Clcn3(-/-)) mice were compared with those of the same currents in ClC-3-expressing wild-type (Clcn3(+/+)) and heterozygous (Clcn3(+/-)) mice. Basal whole-cell currents recorded under isotonic conditions in ClC-3-deficient and -expressing cells were indistinguishable. The biophysical and pharmacological properties of whole-cell VSOR Cl- currents in ClC-3-deficient cells were identical in ClC-3-expressing cells. The VSOR Cl- current density, which is an indicator of the plasmalemmal expression of functional channels, was essentially the same in cells isolated from these 3 types of mice and C57BL/6 mice. Activation of protein kinase C (PKC) by a phorbol ester was found to upregulate VSOR Cl- currents in ClC-3-deficient and -expressing cardiomyocytes. This effect is opposite to the reported downregulatory effect of PKC activators on ClC-3-associated Cl- currents. We thus conclude that functional expression of VSOR Cl- channels in the plasma membrane of mouse cardiomyocytes is independent of the molecular expression of ClC-3.     

Complex copulatory behavior and the proximate effect of genital and body size differences on mechanical reproductive isolation in the millipede genus Parafontaria

The role of species-specific genitalia in reproductive isolation is unclear. Males of the millipede genus Parafontaria use gonopods (modified eighth legs) charged with sperm from the genital openings of the second legs as intromittent organs. Males perform both preliminary and true intromission during mating. During preliminary intromission, a male attempts to insert his gonopods into the female genitalia before charging the gonopods with sperm. If this intromission is completed, it is followed by the ejaculation of sperm to the gonopods and true intromission for insemination. In two sympatric species of Parafontaria that lack effective precopulatory isolation, copulation was terminated without insemination because of preliminary intromission failure caused by mismatched genital and body sizes. Thus, mechanical isolation between these sympatric species resulted from morphological differentiation mediated by the obligatory preliminary intromission. These findings demonstrate the proximate importance of genital and body size differences for reproductive isolation within this genus of millipede.     

Outer arm fucosylation of N-glycans increases in sera of hepatocellular carcinoma patients

Serum glycans are promising markers for early-stage cancer detection, but the research remains challenging because low concentrations of serum glycoproteins are secreted from early-stage tumors. We have established an N-glycan profiling method using liquid chromatography electrospray ionization-mass spectrometry with high sensitive derivative, trimethyl(4-aminophenyl)ammonium chloride (TMAPA). The mass sensitivity of TMAPA-labeled oligosaccharides was enhanced more than 50 times compared with 2-aminopyridine (PA) labeled oligosaccharides, and the analytical period was significantly shortened compared with traditional HPLC 2D-mapping. Using this method, we found about 28 major N-linked oligosaccharides in human sera, and we investigated their alterations in patients who developed hepatocellular carcinoma (HCC). We found that outer arm fucosylation (attached GlcNAc via an alpha 1-3/4 linkage) in highly branched oligosaccharides increased significantly in sera of HCC patients. Normalizing the level of outer arm fucosylation by taking into account platelet concentration allowed us to distinguish more clearly between HCC and LC patients.     

Dioxin interferes in chromosomal positioning through the aryl hydrocarbon receptor

Each chromosome occupies its own-specific space called a ‘territory’ within the interphase nucleus, and the arrangement of chromosome territories (CTs) is important in epigenetic mechanisms. The molecular mechanism to determine the positioning of CTs, however, remains unknown. On the other hand, dioxin is known to be the typical environmental pollutant that affects a wide variety of biological events in many species. Here, we show that dioxin enlarges the minimum distance between chromosome 12 and chromosome 16 territories in human preadipocyte cells, and the alteration of chromosome positioning is canceled by an aryl hydrocarbon receptor (AhR) antagonist α-naphthoflavone. Thus, AhR may be a key molecule to regulate chromosome positioning. Our results suggest a novel effect of dioxin toxicity, and demonstrate a clue to reveal the novel molecular mechanism for the arrangement of CTs.     

Coupling between cyclooxygenase, terminal prostanoid synthase, and phospholipase A2.

We have recently shown that two distinct prostaglandin (PG) E(2) synthases show preferential functional coupling with upstream cyclooxygenase (COX)-1 and COX-2 in PGE(2) biosynthesis. To investigate whether other lineage-specific PG synthases also show preferential coupling with either COX isozyme, we introduced these enzymes alone or in combination into 293 cells to reconstitute their functional interrelationship. As did the membrane-bound PGE(2) synthase, the perinuclear enzymes thromboxane synthase and PGI(2) synthase generated their respective products via COX-2 in preference to COX-1 in both the -induced immediate and interleukin-1-induced delayed responses. Hematopoietic PGD(2) synthase preferentially used COX-1 and COX-2 in the -induced immediate and interleukin-1-induced delayed PGD(2)-biosynthetic responses, respectively. This enzyme underwent stimulus-dependent translocation from the cytosol to perinuclear compartments, where COX-1 or COX-2 exists. COX selectivity of these lineage-specific PG synthases was also significantly affected by the concentrations of arachidonate, which was added exogenously to the cells or supplied endogenously by the action of cytosolic or secretory phospholipase A(2). Collectively, the efficiency of coupling between COXs and specific PG synthases may be crucially influenced by their spatial and temporal compartmentalization and by the amount of arachidonate supplied by PLA(2)s at a moment when PG production takes place.     

Fabrication of wool keratin sponge scaffolds for long-term cell cultivation.

Wool keratin sponge scaffolds were fabricated by lyophilization of an aqueous wool keratin solution after controlled freezing. Freezing at -20 degrees C for 3 days was needed for the preparation of stable sponges, which did not show significant changes against heat treatment at 60 degrees C for several hours. Scanning electron microscopic observation revealed that the wool keratin sponges had a homogeneously porous microstructure, the pore size was approximately equal to 100 microm. At 1 h from seeding, the adhesion of cells was observed and at 1 day, cells spread on the sponge surface. Rapid cell growth on the sponge (doubling time: 29.0 h) was observed for at least 7 days, as well as on a commercially available plastic culture dish (doubling time: 27.4 h). At long-term (23-43 days) cultivation, cells were constantly counted to be approximately 4.2-7.4 million per sponge (1 cm in diameter). The maximum cell number was 7.4 million, approximately 37 times higher than on the same area dish. Living cells on the sponge were observed at 23-43 days by SEM observation and no abnormal morphology of the cells was observed. These results show that wool keratin sponges are useful scaffolds for long-term and high-density cell cultivation.     

Tree shape, forest structure and diversity of drosophilid community: Comparison between boreal and temperate birch forests

Some models, based on the latitudinal variation in sun angle distribution, predict that trees at high latitudes have narrowly conical crowns and constitute simple-layered forests, whereas trees at low latitudes have shallowly dome-shaped and form more structurally complex multilayered forests. There is a hypothesis that structurally complex habitats can harbor potentially more species than simple ones. In this study, we examined latitudinal correlations between tree shape, forest structure and diversity in drosophilid communities, comparing boreal and cool-temperate forests. We selected secondary birch forests with a common canopy tree species, white birch (Betula platyphylla Sukatchev), as study sites. The crown shape of white birch tended to be spherical in the cool-temperate forest, but narrowly conical in the boreal forest. The foliage structure differed between the two forests. The cool-temperate forest was characterized by a clearly two-layered structure, whereas foliage in the boreal forest was less clearly stratified, being distributed somewhat continuously from the ground to the canopy at lower densities. The structural complexity expressed by foliage height diversity was greater in the cool-temperate forest than in the boreal forest. Various measures of drosophilid diversity were higher in the cool-temperate forest than in the boreal forest, probably resulting from the impoverishment of the canopy subcommunity in the boreal forest. Thus, a physical environmental factor (i.e. the angle of solar inclination) could be a potentially important factor in structuring latitudinal patterns of sylvan animal communities through changes in plant structure at the individual and community levels.     

Genetic identification of two distinct DNA polymerases, DnaE and PolC, that are essential for chromosomal DNA replication in Staphylococcus aureus

We isolated and characterized temperature-sensitive mutants for two genes, dnaE and polC, that are essential for DNA replication in Staphylococcus aureus. DNA replication in these mutants had a slow-stop phenotype when the temperature was shifted to a non-permissive level. The dnaE gene encodes a homolog of the alpha-subunit of the DNA polymerase III holoenzyme, the replicase essential for chromosomal DNA replication in Escherichia coli. The polC gene encodes PolC, another catalytic subunit of DNA polymerase, which is specifically found in gram-positive bacteria. The wild-type dnaE or polC gene complemented the temperature-sensitive phenotypes of cell growth and DNA replication in the corresponding mutant. Single mutations resulting in amino-acid exchanges were identified in the dnaE and polC genes of the temperature-sensitive mutants. The results indicate that these genes encode two distinct DNA polymerases which are both essential for chromosomal DNA replication in S. aureus. The number of viable mutant cells decreased at non-permissive temperature, suggesting that inactivation of DnaE and PolC has a bactericidal effect and that these enzymes are potential targets of antibiotics.