Phylogeography of Japanese horse chestnut (<Emphasis Type="Italic">Aesculus turbinata</Emphasis>) in the Japanese Archipelago based on chloroplast DNA haplotypes

Japanese horse chestnut (Aesculus turbinata: Hippocastanaceae) is one of the typical woody plants that grow in temperate riparian forests in the Japanese Archipelago. To analyze the phylogeography of this plant in the Japanese Archipelago, we determined cpDNA haplotypes for 337 samples from 55 populations covering the entire distribution range. Based on 1,313 bp of two spacers, we determined ten haplotypes that are distinguished from adjacent haplotypes by one or two steps. Most of the populations had a single haplotype, suggesting low diversity. Spatial analysis of molecular variance suggested three obvious phylogeographic structures in western Japan, where Japanese horse chestnut is scattered and isolated in mountainous areas. Conversely, no clear phylogeographic structure was observed from the northern to the southern limit of this species, including eastern Japan, where this plant is more common. Rare and private haplotypes were also found in southwestern Japan, where Japanese horse chestnuts are distributed sparsely. These findings imply that western Japan might have maintained a relatively large habitat for A. turbinata during the Quaternary climatic oscillations, while northerly regions could not.     

αvβ3-Integrin-targeting lanthanide complex: synthesis and evaluation as a tumor-homing luminescent probe

The application of lanthanide complexes in the time-resolved fluorescence imaging of living cells has emerged in the last few decades, providing high-contrast images of cells through detection of the delayed emission. In the present study, we synthesized novel trivalent lanthanide complexes containing the cyclic peptide c(RGDfK) to visualize the α_vβ₃-integrin-expressing tumor cells. Conjugation of c(RGDfK) with the macrocyclic bipyridine ligand had little effect on the fluorescence properties of the complex, indicating that the coordinated lanthanide ion was well isolated from the peptide. Bright luminescence images of α_vβ₃-integrin-expressing U87-MG cells were successfully obtained by employing the probes.     

A wheat homolog of MOTHER OF FT AND TFL1 acts in the regulation of germination

Seed dormancy is an adaptive mechanism and an important agronomic trait. Temperature during seed development strongly affects seed dormancy in wheat (Triticum aestivum) with lower temperatures producing higher levels of seed dormancy. To identify genes important for seed dormancy, we used a wheat microarray to analyze gene expression in embryos from mature seeds grown at lower and higher temperatures. We found that a wheat homolog of MOTHER OF FT AND TFL1 (MFT) was upregulated after physiological maturity in dormant seeds grown at the lower temperature. In situ hybridization analysis indicated that MFT was exclusively expressed in the scutellum and coleorhiza. Mapping analysis showed that MFT on chromosome 3A (MFT-3A) colocalized with the seed dormancy quantitative trait locus (QTL) QPhs.ocs-3A.1. MFT-3A expression levels in a dormant cultivar used for the detection of the QTL were higher after physiological maturity; this increased expression correlated with a single nucleotide polymorphism in the promoter region. In a complementation analysis, high levels of MFT expression were correlated with a low germination index in T1 seeds. Furthermore, precocious germination of isolated immature embryos was suppressed by transient introduction of MFT driven by the maize (Zea mays) ubiquitin promoter. Taken together, these results suggest that MFT plays an important role in the regulation of germination in wheat.     

Expression profile of two storage-protein gene families in hexaploid wheat revealed by large-scale analysis of expressed sequence tags

To discern expression patterns of individual storage-protein genes in hexaploid wheat (Triticum aestivum cv Chinese Spring), we analyzed comprehensive expressed sequence tags (ESTs) of common wheat using a bioinformatics technique. The gene families for alpha/beta-gliadins and low molecular-weight glutenin subunit were selected from the EST database. The alignment of these genes enabled us to trace the single nucleotide polymorphism sites among both genes. The combinations of single nucleotide polymorphisms allowed us to assign haplotypes into their homoeologous chromosomes by allele-specific PCR. Phylogenetic analysis of these genes showed that both storage-protein gene families rapidly diverged after differentiation of the three genomes (A, B, and D). Expression patterns of these genes were estimated based on the frequencies of ESTs. The storage-protein genes were expressed only during seed development stages. The alpha/beta-gliadin genes exhibited two distinct expression patterns during the course of seed maturation: early expression and late expression. Although the early expression genes among the alpha/beta-gliadin and low molecular-weight glutenin subunit genes showed similar expression patterns, and both genes from the D genome were preferentially expressed rather than those from the A or B genome, substantial expression of two early expression genes from the A genome was observed. The phylogenetic relationships of the genes and their expression patterns were not correlated. These lines of evidence suggest that expression of the two storage-protein genes is independently regulated, and that the alpha/beta-gliadin genes possess novel regulation systems in addition to the prolamin box.     

Proteomic analysis of slow- and fast-twitch skeletal muscles

Skeletal muscles are composed of slow- and fast-twitch muscle fibers, which have high potential in aerobic and anaerobic ATP production, respectively. To investigate the molecular basis of the difference in their functions, we examined protein profiles of skeletal muscles using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis with pH 4-7 and 6-11 isoelectric focusing gels. A comparison between rat soleus and extensol digitorum longus (EDL) muscles that are predominantly slow- and fast-twitch fibers, respectively, showed that the EDL muscle had higher levels of glycogen phosphorylase, most glycolytic enzymes, glycerol 3-phosphate dehydrogenase, and creatine kinase; while the soleus muscle had higher levels of myoglobin, TCA cycle enzymes, electron transfer flavoprotein, and carbonic anhydrase III. The two muscles also expressed different isoforms of contractile proteins including myosin heavy and light chains. These protein patterns were further compared with those of red and white gastrochnemius as well as red and white quadriceps muscles. It was found that metabolic enzymes showed a concerted regulation dependent on muscle fiber types. On the other hand, expression of contractile proteins seemed to be independent of the metabolic characteristics of muscle fibers. These results suggest that metabolic enzymes and contractile proteins show different expression patterns in skeletal muscles.     

Characterization of oligosaccharide ligands expressed on SW1116 cells recognized by mannan-binding protein. A highly fucosylated polylactosamine type N-glycan

Mannan-binding protein (MBP) is a C-type serum lectin and activates complement through the lectin pathway when it binds to ligand sugars such as mannose, N-acetylglucosamine, and fucose on microbes. In addition, the vaccinia virus carrying the human MBP gene was shown to exhibit potent growth inhibitory activity toward human colorectal carcinoma, SW1116, cells in nude mice. We have proposed calling this activity MBP-dependent cell-mediated cytotoxicity (MDCC) (Ma, Y., Uemura, K., Oka, S., Kozutsumi, Y., Kawasaki, N., and Kawasaki, T. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 371-375). In this study, the MBP ligands on the surface of SW1116 cells were characterized. Initial experiments involving plant lectins and anti-Lewis antibodies as inhibitors of MBP binding to SW1116 cells indicated that fucose plays a crucial role in the interaction. Subsequently, Pronase glycopeptides were prepared from whole cell lysates, and oligosaccharides were liberated by hydrazinolysis. After being tagged by pyridylamination, MBP ligand oligosaccharides were isolated with an MBP affinity column, and then their sequences were determined by mass spectrometry and tandem mass spectrometry after permethylation, in combination with endo-beta-galactosidase digestion and chemical defucosylation. The MBP ligands were shown to be large, multiantennary N-glycans carrying a highly fucosylated polylactosamine type structure. At the nonreducing termini, Le(b)/Le(a) or tandem repeats of the Le(a) structure prevail, a substantial proportion of which are attached via internal Le(x) or N-acetyllactosamine units to the trimannosyl core. The structures characterized are unique and distinct from those of other previously reported tumor-specific carbohydrate antigens. It is concluded that MBP requires clusters of tandem repeats of the Le(b)/Le(a) epitope for recognition.