Novel inhibition mechanism of Bacillus cereus sphingomyelinase by beryllium fluoride

Phosphate analogs have been known to inhibit competitively various phosphatases and phospholipase C and D. We found for the first time that only beryllium fluoride (BeF(x)) among the phosphate analogs studied inhibits Bacillus cereus sphingomyelinase (SMase) activity. The active inhibitory species proved to be not BeF(3)(-) but BeF(2) by the measurement of SMase activity and of (19)F NMR spectroscopy in the presence of a fixed concentration of BeCl(2) and different concentrations of NaF, although both the species have been reported for other kinds of enzymes. The result of kinetic experiment also indicated that the BeF(x) binds in the vicinity of the essential binding site for the substrate and that the Mg(2+) binding to SMase is essential for the binding of BeF(x) to the enzyme.     

Identification and characterization of a protostome homologue of peropsin from a jumping spider

Peropsin, a member of the opsin family, has characteristics of two functionally distinct opsin-groups, that is, amino acid residues conserved among opsins for light-sensing and a retinal-photoisomerase-like molecular property. Although such a bilateral feature of peropsin seems to be important for understanding the diversity of the opsin family, previous studies have been limited to higher deuterostome, vertebrate and amphioxus peropsins. Here, we report a protostome peropsin homologue from a jumping spider. We found a spider opsin that shares amino acid homology and conserved amino acid residues with known peropsins. The spider opsin-based pigment heterologously expressed in cultured cells exhibited photoisomerase-like isomerization characteristics and a bistable nature. Based on the characteristics of both the amino acid homology and its photochemical properties, we concluded that the spider opsin is the first protostome peropsin homologue. These results show that peropsin existed before the deuterostome–protostome split like other members of the opsin family. In addition, the spider peropsin was localized to non-visual cells in the retina, and fluorescence from reduced retinal chromophore was also observed in the region where peropsin was localized. These findings provide the first demonstration that the peropsin can form a photosensitive pigment in vivo and underlie non-visual function.     

Moxibustion activates host defense against herpes simplex virus type I through augmentation of cytokine production

Moxibustion is a technique used in traditional oriental medicine, the aim of which is to cure and/or prevent illness by activating a person's ability for self‐healing. In this study, we assessed how moxibustion would affect the immune system and whether it would augment protective immunity. Mice were treated with moxibustion at Zusanli (ST36) acupoints; we analyzed mortality and cytokine activity in sera after infection with herpes simplex virus type 1 (HSV‐1), and cytokine gene expression in the skin and the spleen without a virus challenge. Our study demonstrates that pretreatment of BALB/c mice with moxibustion resulted in a marked increase in the survival rate after infection with lethal doses of HSV‐1, and elevated serum levels of IL‐1β and IFN‐γ on days 1 and 6 post‐infection with HSV‐1. Semi‐quantitative RT‐PCR assay showed that moxibustion treatment augmented the expression of IL‐1α, IL‐1β, IL‐6, universal‐IFN‐α, MIP‐1α, and TNF‐α mRNA in the skin, and IL‐1α, IL‐1β, IL‐12p40, IL‐15, u‐IFN‐α, MIP‐1α, and TNF‐α mRNA in the spleen. Moreover, moxibustion induces augmentation of natural killer cell activity. Collectively, our study demonstrates that moxibustion activates protective responses against HSV‐1 infection through the activation of cytokine production including IFN, and of NK cells.     

Dispersal of yellow phase Japanese eels <Emphasis Type="Italic">Anguilla japonica</Emphasis> after recruitment in the Kojima Bay-Asahi River system,Japan

The density, size and age distribution were investigated for 233 eels, Anguilla japonica, sampled in fresh and brackish water areas of the Kojima Bay-Asahi River system, Okayama, Japan, to evaluate the possible patterns of dispersal of eels that recruit to this area. Migratory histories of 183 eels were categorized into 5 types depending on the Sr and Ca concentrations in their otoliths: (1) brackish water residents (74 fish, 40.4%), which settled in saline water and remained until capture; (2) freshwater residents (46 fish, 25.1%), which settled in freshwater and remained until capture; (3) upstream shifters (3 fish, 1.6%), which settled in saline water and moved upstream into freshwater; (4) downstream shifters (53 fish, 29.0%), which settled in freshwater and moved downstream into saline water; (5) multiple habitat shifters (7 fish, 3.8%), which shifted their habitats between freshwater and saline water more than twice. For eels captured in the brackish water area, fish density decreased with distance in the downstream direction, while the size and age of eels increased. For eels captured in the freshwater area, size and age were greater than those in the upper-most brackish site. These observations suggest that eels in this system initially accumulate in the lower reaches of the river and then disperse in both upstream and downstream directions following their growth.     

Survival and behavioral characteristics of amphidromous goby larvae of Sicyopterus japonicus (Tanaka, 1909) during their downstream migration

To understand the ecology and environmental tolerances of newly hatched larvae of the amphidromous fish Sicyopterus japonicus during their downstream migration, the salinity tolerance of eggs, 0-15 day old larvae, and adults, and the temperature tolerance, specific gravity and phototaxis of hatched larvae were examined. Tolerances of adults were measured as survival after a 24 h challenge in freshwater (FW), brackish water (1/3 SW) and seawater (SW). The survival rate of adult S. japonicus was 100% in FW and 1/3 SW, while none survived in SW. Hatching success of eggs (30 eggs each) was significantly higher in FW (mean: 73%) and 1/3 SW (73%) than in SW (19%). Tolerance of newly hatched larvae to salinity and temperature was investigated in different combinations of salinities (FW, 1/3 SW and SW) and temperatures (18, 23 and 28 °C). Larval survival was significantly different in each salinity and temperature. Survival rate was significantly higher in 1/3 SW than in FW and higher in SW than in FW at 23 °C and 28 °C. At the latter part of the experiment, there was no survival in FW and at 28 °C. Survival was higher in lower temperatures, but larval development did not occur in FW. Specific gravity of newly hatched larvae was 1.036 at 28 °C and 1.034 at 23 °C. When exposed to a light source on one side of an aquarium, larval distribution was not affected. Our results indicated larval S. japonicus are more adapted to brackish water and seawater than freshwater, while the adults and eggs are more adapted to freshwater and brackish water than seawater. This is consistent with their amphidromous life history with growth and spawning occurring in freshwater and the larval stage utilizing marine habitats.     

Enhanced activation of T lymphocytes by urease-deficient recombinant bacillus Calmette-Guérin producing heat shock protein 70-major membrane protein-II fusion protein

To activate naive T cells convincingly using Mycobacterium bovis bacillus Calmette-Guérin (BCG), recombinant BCG (BCG-D70M) that was deficient in urease, expressed with gene encoding the fusion of BCG-derived heat shock protein (HSP) 70 and Mycobacterium leprae-derived major membrane protein (MMP)-II, one of the immunodominant Ags of M. leprae, was newly constructed. BCG-D70M was more potent in activation of both CD4(+) and CD8(+) subsets of naive T cells than recombinant BCGs including urease-deficient BCG and BCG-70M secreting HSP70-MMP-II fusion protein. BCG-D70M efficiently activated dendritic cells (DCs) to induce cytokine production and phenotypic changes and activated CD4(+) T cells even when macrophages were used as APCs. The activation of both subsets of T cells was MHC and CD86 dependent. Pretreatment of DCs with chloroquine inhibited both surface expression of MMP-II on DCs and the activation of T cells by BCG-D70M-infected APCs. The naive CD8(+) T cell activation was inhibited by treatment of DCs with brefeldin A and lactacystin so that the T cell was activated by TAP- and proteosome-dependent cytosolic cross-priming pathway. From naive CD8(+) T cells, effector T cells producing perforin and memory T cells having migration markers were produced by BCG-D70M stimulation. BCG-D70M primary infection in C57BL/6 mice produced T cells responsive to in vitro secondary stimulation with MMP-II and HSP70 and more efficiently inhibited the multiplication of subsequently challenged M. leprae than vector control BCG. These results indicate that the triple combination of HSP70, MMP-II, and urease depletion may provide a useful tool for inducing better activation of naive T cells.     

Genetic dissection of vasculitis, myeloperoxidase-specific antineutrophil cytoplasmic autoantibody production, and related traits in spontaneous crescentic glomerulonephritis-forming/Kinjoh mice

The spontaneous crescentic glomerulonephritis-forming/Kinjoh (SCG/Kj) mouse is a model of human crescentic glomerulonephritis and vasculitis associated with the production of the myeloperoxidase (MPO)-specific antineutrophil cytoplasmic autoantibody (MPO-ANCA). Although the disease is mediated initially by mutation of the Fas gene (lpr), SCG/Kj mice also have non-Fas predisposing genetic factors. To define these factors, genome-wide quantitative trait locus (QTL) mapping was performed on female (B(6)x SCG/Kj) F(2) intercross mice. Fourteen non-Fas QTLs were identified. QTLs of glomerulonephritis were located on chromosomes 1, 10, 13, 16, and 17, vasculitis on chromosomes 1 and 17, splenomegaly on chromosome 1, hypergammaglobulinemia on chromosomes 1, 2, 4, 6, 7, 11, 13, and 17, antinuclear Ab on chromosomes 1, 8, 10, and 12, and MPO-ANCA production on chromosomes 1 and 10. Significant QTLs derived from SCG/Kj on chromosomes 1, 2, 7, and 13 were designated Scg-1 to Scg-5, respectively, and those derived from B(6) on chromosomes 4, 6, 17, and 10 were designated Sxb-1 to Sxb-4, respectively. Two loci linked to MPO-ANCA production on chromosomes 1 and 10 were designated Man-1 and Man-2 (for MPO-ANCA), respectively. Although both Scg-1 and Scg-2 were on chromosome 1 and shared several functions, it was of interest that aberrant MPO-ANCA production was exclusively controlled by Man-1, the centromeric half region of the Scg-2 chromosomal segment. We also examined the epistatic effects between the lpr mutation and non-Fas susceptibility genes. QTLs are discussed in relation to previously described loci, with emphasis on their candidate genes.     

The assembly and maintenance of heterochromatin initiated by transgene repeats are independent of the RNA interference pathway in mammalian cells

A role for the RNA interference (RNAi) pathway in the establishment of heterochromatin is now well accepted for various organisms. Less is known about its relevance and precise role in mammalian cells. We previously showed that tandem insertion of a 1,000-copy inducible transgene into the genome of baby hamster kidney (BHK) cells initiated the formation of an extremely condensed chromatin locus. Here, we characterized the inactive transgenic locus as heterochromatin, since it was associated with heterochromatin protein 1 (HP1), histone H3 trimethylated at lysine 9, and cytosine methylation in CpG dinucleotides. Northern blot analysis did not detect any transgene-derived small RNAs. RNAi-mediated Dicer knockdown did not disrupt the heterochromatic transgenic locus or up-regulate transgene expression. Moreover, neither Dicer knockdown nor overexpression of transgene-directed small interfering RNAs altered the bidirectional transition of the transgenic locus between the heterochromatic and euchromatic states. Interestingly, tethering of HP1 to the transgenic locus effectively induced transgene silencing and chromatin condensation in a Dicer-independent manner, suggesting a role for HP1 in maintaining the heterochromatic locus. Our results suggest that the RNAi pathway is not required for the assembly and maintenance of noncentromeric heterochromatin initiated by tandem transgene repeats in mammalian cells.     

A role of kinase inactive ZAP-70 in altered peptide ligand stimulated T cell activation

T cell activation signals induced by altered peptide ligands (APLs) are different from those induced by the original agonistic peptide. The characteristics of the former are partial phosphorylation of TCR-zeta and no tyrosine-phosphorylation of zeta-associated protein-70 (ZAP-70). To analyze further those signaling pathways, we introduced a dominant negative (DN) form of ZAP-70 into a human CD4(+) T cell clone in which fully and partially agonistic peptide ligands have been well characterized. We found that some over-expressed partially agonistic ligands (OPALs) induced T cell responses without tyrosine-phosphorylation and kinase activation of ZAP-70. However, those responses were inhibited in T cells expressing DN ZAP-70, which could associate with partially phosphorylated TCR-zeta. In OPAL-stimulated T cells, PLC-gamma1 was phosphorylated and it was suppressed by DN ZAP-70 expression, suggesting that the ZAP-70-TCR-zeta association mediates the activation of PLC-gamma1 leading to T cell responses even in the absence of kinase activation of ZAP-70.