Characterization of Epstein-Barr virus (EBV)-positive NK cells isolated from hydroa vacciniforme-like eruptions

Recently, the involvement of Epstein-Barr virus (EBV) in hydroa vacciniforme (HV)-like eruptions has been suggested. To elucidate the role of EBV in this disease, we isolated EBV-infected cell clones from peripheral blood mononuclear cells (PBMC) and the skin lesions of a patient with HV-like eruptions; cells isolated from PBMC were designated SNK-12, and those from the eruption SNK-11. Both cells expressed CD16, CD56, and HLA-DR and had germline configurations of the T-cell receptor and the immunoglobulin genes, indicating that the cell clones were of NK cell lineage. The analysis of EBV terminal repeats indicated that the cells were monoclonal, had identical clonality, and originated from EBV-positive cells in the PBMC and eruption. Both clones expressed EBNA-1, but not EBNA-2. Although LMP-1 was weakly detected in SNK-11, no LMP-1 was detected in SNK-12. Interestingly, EBV-infected cells required less IL-2 for in vitro growth in the later phase of this disease and this appeared to correlate with the expression of LMP-1, suggesting that the proliferative capacity of the EBV-positive NK cells increased during the time course of the disease, and LMP-1 expression might be responsible for that. This is the first report of the isolation of EBV-infected cells from the skin lesions of HV-like eruptions and strongly suggests that the HV-like eruption in the patient was caused by clonal NK cells with latent EBV infection.     

The number of species occurring in a sample of a biotic community and its connections with species-abundance relationship and spatial distribution

Connections among species-abundance (i-m _i ), species-frequency (i-F _i ), and species-sample size (S _n -n) relationships were examined on the basis of the mapping data of a natural forest in Thailand. The spatial distribution of individual trees without any discrimination of species was nearly random. Provided that the spatial distribution of each species was random, thei-m _i and thei-F _i relationship was reconstructed from each other in terms of the total number of species (S) and the total number of individuals (N) in the data. The number of species (S _n ) in a subsample consisting ofn individuals was then obtained from thei-F _i relationship. Logarithm ofS _n increased with logn and showed a convex curve through the origin. The values of diversity indices based onN andS(orn andS _n ) were affected by sample size. These trends were further examined on the basis of 944 data sets of biotic communities and three mathematical models of anS-N relationship. The properties of species-area relation were discussed in the light of these results.     

Inference of S-system models of genetic networks using a cooperative coevolutionary algorithm

MOTIVATION: To resolve the high-dimensionality of the genetic network inference problem in the S-system model, a problem decomposition strategy has been proposed. While this strategy certainly shows promise, it cannot provide a model readily applicable to the computational simulation of the genetic network when the given time-series data contain measurement noise. This is a significant limitation of the problem decomposition, given that our analysis and understanding of the genetic network depend on the computational simulation. RESULTS: We propose a new method for inferring S-system models of large-scale genetic networks. The proposed method is based on the problem decomposition strategy and a cooperative coevolutionary algorithm. As the subproblems divided by the problem decomposition strategy are solved simultaneously using the cooperative coevolutionary algorithm, the proposed method can be used to infer any S-system model ready for computational simulation. To verify the effectiveness of the proposed method, we apply it to two artificial genetic network inference problems. Finally, the proposed method is used to analyze the actual DNA microarray data.     

A novel,potent, and orally active VLA-4 antagonist with good aqueous solubility: trans-4-[1-[[2-(5-Fluoro-2-methylphenylamino)-7-fluoro-6-benzoxazolyl]acetyl]-(5S)-[methoxy(methyl)amino]methyl-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylic acid

We have carried out the optimization of substituents at the C-3 or the C-5 position on the pyrrolidine ring of VLA-4 antagonist 3 with 2-(phenylamino)-7-fluorobenzoxazolyl moiety for the purpose of improving in vivo efficacy while maintaining good aqueous solubility. As a result, we successfully increased in vitro activity in the presence of 3% human serum albumin and achieved an exquisite lipophilic and hydrophilic balance of compounds suitable for oral administrative regimen. The modification resulted in the identification of zwitterionic compound 7n with (5S)-[methoxy(methyl)amino]methylpyrrolidine, which significantly alleviated bronchial hyper-responsiveness to acetylcholine chloride at 12.5 mg/kg, p.o. in a murine asthma model and showed favorable aqueous solubility (JP1, 89 μg/mL; JP2, 462 μg/mL). Furthermore, this compound showed good oral bioavailability (F = 54%) in monkeys.     

Development of an HPLC Method with an ODS Column to Determine Low Levels of Aspartame Diastereomers in Aspartame

α-L-Aspartyl-D-phenylalanine methyl ester (L, D-APM) and α-D-aspartyl-L-phenylalanine methyl ester (D, L-APM) are diastereomers of aspartame (N-L-α-Aspartyl-L-phenylalanine-1-methyl ester, L, L-APM). The Joint FAO/WHO Expert Committee on Food Additives has set 0.04 wt% as the maximum permitted level of the sum of L, D-APM and D, L-APM in commercially available L, L-APM. In this study, we developed and validated a simple high-performance liquid chromatography (HPLC) method using an ODS column to determine L, D-APM and D, L-APM in L, L-APM. The limits of detection and quantification, respectively, of L, D-APM and D, L-APM were found to be 0.0012 wt% and 0.004 wt%. This method gave excellent accuracy, repeatability, and reproducibility in a recovery test performed on five different days. Moreover, the method was successfully applied to the determination of these diastereomers in commercial L, L-APM samples. Thus, the developed method is a simple, useful, and practical tool for determining L, D-APM and D, L-APM levels in L, L-APM.     

Effects of free proline accumulation in petunias under drought stress

Petunias (Petunia hybrida cv. 'Mitchell') accumulate free proline (Pro) under drought-stress conditions. It is therefore believed that Pro acts as an osmoprotectant in plants subjected to drought conditions. Petunia plants were transformed by Delta(1)-pyrroline-5-carboxylate synthetase genes (AtP5CS from Arabidopsis thaliana L. or OsP5CS from Oryza sativa L.). The transgenic plants accumulated Pro and their drought tolerance was tested. The Pro content amounted to 0.57-1.01% of the total amino acids in the transgenic plants, or 1.5-2.6 times that in wild-type plants grown under normal conditions. The transgenic plant lines tolerated 14 d of drought stress, which confirms that both P5CS transgenes had full functionality. Exogenous L-Pro treatment caused the plants to accumulate Pro; plants treated with 5 mM L-Pro accumulated up to 18 times more free Pro than untreated plants. Exogenous L-Pro restricted the growth of wild-type petunias more than that of Arabidopsis plants. The capacity for free Pro accumulation might depend on the plant species. The growth of petunia plants was influenced not only by the Pro concentration in the plants, but by the ratio of the Pro content to the total amino acids, because the growth of the transgenic petunia plants appeared normal.     

Mechanistic studies on the intramolecular one-electron transfer between the two flavins in the human endothelial NOS reductase domain

The object of this study was to clarify the mechanism of electron transfer in the human endothelial nitric oxide synthase (eNOS) reductase domain using recombinant eNOS reductase domains; the FAD/NADPH domain containing FAD- and NADPH-binding sites and the FAD/FMN domain containing FAD/NADPH-, FMN-, and a calmodulin-binding sites. In the presence of molecular oxygen or menadione, the reduced FAD/NADPH domain is oxidized via the neutral (blue) semiquinone (FADH(*)), which has a characteristic absorption peak at 520 nm. The FAD/NADPH and FAD/FMN domains have high activity for ferricyanide, but the FAD/FMN domain has low activity for cytochrome c. In the presence or absence of calcium/calmodulin (Ca(2+)/CaM), reduction of the oxidized flavins (FAD-FMN) and air-stable semiquinone (FAD-FMNH(*)) with NADPH occurred in at least two phases in the absorbance change at 457nm. In the presence of Ca(2+)/CaM, the reduction rate of both phases was significantly increased. In contrast, an absorbance change at 596nm gradually increased in two phases, but the rate of the fast phase was decreased by approximately 50% of that in the presence of Ca(2+)/CaM. The air-stable semiquinone form was rapidly reduced by NADPH, but a significant absorbance change at 520 nm was not observed. These findings indicate that the conversion of FADH(2)-FMNH(*) to FADH(*)-FMNH(2) is unfavorable. Reduction of the FAD moiety is activated by CaM, but the formation rate of the active intermediate, FADH(*)-FMNH(2) is extremely low. These events could cause a lowering of enzyme activity in the catalytic cycle.     

Rapid Screening of a Novel Arrayed Medaka (Oryzias latipes) Cosmid Library

Medaka (Oryzias latipes) has many advantages for genetic and developmental studies. With recent advances in the genome analyses of other species, rapid accumulation of resources for medaka genomics is expected. In this study, we generated an arrayed medaka cosmid library from the HNI inbred strain, carrying a 40-kb insert on average. The library consists of approximately 120,000 clones with a 6-fold genomic coverage. Cosmid clones can be screened within 2 days using standard polymerase chain reaction. Considering the advantage of the cosmid insert size and the compact genome size of the medaka, this library provides a powerful tool for future genome analyses.     

Human neuronal nitric oxide synthase can catalyze one-electron reduction of adriamycin: role of flavin domain

We have analyzed the mechanism of one-electron reduction of adriamycin (Adr) using recombinant full-length human neuronal nitric-oxide synthase and its flavin domains. Both enzymes catalyzed aerobic NADPH oxidation in the presence of Adr. Calcium/calmodulin (Ca(2+)/CaM) stimulated the NADPH oxidation of Adr. In the presence or absence of Ca(2+)/CaM, the flavin semiquinone radical species were major intermediates observed during the oxidation of the reduced enzyme by Adr. The FAD-NADPH binding domain did not significantly catalyze the reduction of Adr. Neither the FAD semiquinone (FADH*) nor the air-stable semiquinone (FAD-FMNH*) reacted rapidly with Adr. These data indicate that the fully reduced species of FMN (FMNH(2)) donates one electron to Adr, and that the rate of Adr reduction is stimulated by a rapid electron exchange between the two flavins in the presence of Ca(2+)/CaM. Based on these findings, we propose a role for the FAD-FMN pair in the one-electron reduction of Adr.     

ADAPTATIONS TO TEMPERATE CLIMATES AND EVOLUTION OF OVERWINTERING STRATEGIES IN THE DROSOPHILA MELANOGASTER SPECIES GROUP

The Drosophila melanogaster species group is considered to have originated in the tropics and only recently invaded temperate habitats. The temperate species of this group that were studied here may be subdivided into the warm-temperate species (D. lutescens and D. rufa) and the cool-temperate species (species of the auraria complex). The warm-temperate species were more cold-hardy than were their tropical relatives (D. takahashii or D. melanogaster) at the larval and imaginal stages, and the cool-temperate species were more cold-hardy than the warm-temperate species, although only at the imaginal stage. However, these species showed little or no intraspecific variation in cold-hardiness, in spite of great variation in winter temperature within the species' ranges. It is assumed that cold-hardiness is one of the main factors restricting their distributions at high latitudes and that it is the key for evolution of the warm- and cool-temperate species from their subtropical or warm-temperate ancestors. Both warm- and cool-temperate species had photoperiodically controlled reproductive diapause. In the cool-temperate species, the development of cold-hardiness was affected by diapause, but diapause had little or no effect on cold-hardiness in the warm-temperate species. Critical daylengths and the diapause rates varied from species to species according to variation in their overwintering plans and also varied geographically in consequence of their adaptation to local climates. Species showed different responses to temperature in preimaginal and ovarian development. These differences are considered to reflect adaptation to different environmental temperatures.