Ploidy chimeras induced in haploid sporophytes of <Emphasis Type="Italic">Osmunda claytoniana</Emphasis> and <Emphasis Type="Italic">Osmunda japonica</Emphasis>

Haploid sporophytes of Osmunda claytoniana (2n = x = 22) were apogamously produced from calli when cultivated on a hormone-free medium. Flow cytometric analysis showed that ploidy chimeras were spontaneously produced in a haploid sporophyte of O. claytoniana and those of O. japonica that were obtained in the previous study. In the haploid sporophyte of O. claytoniana, a diploid pinnule and a partially diploid terminal segment were produced in a haploid pinna. In O. japonica, a haploid sporophyte yielded a diploid pinna in a haploid frond, and another haploid sporophyte yielded a diploid pinnule in a haploid pinna. Diploid chimeras were large in size and could be readily distinguished from other haploid parts of the fronds. It is likely that the chimeras were produced clonally from a single diploid cell that established chromosome doubling.     

Discovery of spiropiperidine-based potent and selective Orexin-2 receptor antagonists

To generate novel human Orexin-2 Receptor (OX2R) antagonists, a spiropiperidine based scaffold was designed and a SAR study was carried out. Compound 4f possessed the highest OX2R antagonistic activity with an IC(50) value of 3nM with 450-fold selectivity against Orexin-1 Receptor (OX1R).     

Discovery of potent, selective, orally active benzoxazepine-based Orexin-2 receptor antagonists

During our efforts to identify a series of potent, selective, orally active human Orexin-2 Receptor (OX2R) antagonists, we elucidated structure-activity relationship (SAR) on the 7-position of a benzoxazepine scaffold by utilizing Hammett σ(p) and Hansch-Fujita π value as aromatic substituent constants. The attempts led to the discovery of compound 1m, possessing good in vitro potency with over 100-fold selectivity against OX1R, good metabolic stability in human and rat liver microsome, good oral bioavailability in rats, and in vivo antagonistic activity in rats by oral administration.     

Outer membrane vesicles of Porphyromonas gingivalis elicit a mucosal immune response

We previously reported that mutation of galE in Porphyromonas gingivalis has pleiotropic effects, including a truncated lipopolysaccharide (LPS) O-antigen and deglycosylation of the outer membrane protein OMP85 homolog. In the present study, further analysis of the galE mutant revealed that it produced little or no outer membrane vesicles (OMVs). Using three mouse antisera raised against whole cells of the P. gingivalis wild type strain, we performed ELISAs to examine the reactivity of these antisera with whole cells of the wild type or the galE mutant. All three antisera had significantly lower reactivity against the galE mutant compared to wild type. OMVs, but not LPS, retained the immunodominant determinant of P. gingivalis, as determined by ELISAs (with wild type LPS or OMVs as antigen) and absorption assays. In addition, we assessed the capacity of OMVs as a vaccine antigen by intranasal immunization to BALB/c mice. Synthetic double-stranded RNA polyriboinosinic polyribocytidylic acid [Poly (I∶C)], an agonist of Toll-like receptor 3 (TLR3), was used as the mucosal adjuvant. Vaccination with OMV elicited dramatically high levels of P. gingivalis-specific IgA in nasal washes and saliva, as well as serum IgG and IgA. In conclusion, the OMVs of P. gingivalis have an important role in mucosal immunogenicity as well as in antigenicity. We propose that P. gingivalis OMV is an intriguing immunogen for development of a periodontal disease vaccine.     

Ethnic differences in CYP2C9*2 (Arg144Cys) and CYP2C9*3 (Ile359Leu) genotypes in Japanese and Israeli populations

CYP2C9 is a major P450 2C enzyme, which hydroxylates about 16% of drugs that are in current clinical use and contributes to the metabolism of a number of clinically important substrate drugs such as warfarin. Ethnic differences in the genetic variation of CYP2C9 have been reported, and might be related to the frequencies of adverse reactions to drugs metabolized by CYP2C9 in different ethnic groups. In the present study, ethnic differences in the CYP2C9*2 and CYP2C9*3 allele distribution in Japanese and Israeli populations were evaluated using a newly developed oligonucleotide based DNA array (OligoArray(R)). The population studied consisted of 147 Japanese and 388 Israeli donors (100 Ashkenazi Jews, 99 Yemenite Jews, 100 Moroccan Jews and 89 Libyan Jews). The CYP2C9*2 [Arg144Cys (416 C>T), exon 3] and CYP2C9*3 [Ile359Leu (1061 A>C), exon 7] genotypes were determined using an OligoArray(R). The accuracy of genotyping by the OligoArray(R) was verified by the fluorescent dye-terminator cycle sequencing method. A Hardy-Weinberg test indicated equilibrium (chi(2)<3.84 is Hardy-Weinberg) in all populations. The CYP2C9*2 genotype (CC/CT+TT) was absent in Japanese (1/0) (OR 0.02), and its frequency was significant in Libyan Jews (0.697/0.303) (OR 2.13; 95% CI 1.07-4.24) compared with Ashkenazi Jews (0.83/0.17), Yemenite Jews (0.899/0.101), and Moroccan Jews (0.81/0.19). The frequencies of CYP2C9*3 genotype (AA/AC+CC) was significantly lower in Japanese (0.986/0.014) (OR 0.08), and was higher in Libyan Jews (0.652/0.348) (OR 3.03; 95% CI 1.5-6.1) and Moroccan Jews (0.77/0.23) (OR 1.69; 95% CI 0.62-3.48) compared with those in Ashkenazi Jews (0.85/0.15) and Yemenite Jews (0.849/0.151). Thus, the CYP2C9*2 (Arg144Cys) and CYP2C9*3 (Ile359Leu) variants were rare in the Japanese population, and showed different frequencies in the four Jewish ethnic groups examined.     

Promotive effect of macrophage colony-stimulating factor on long-term engraftment of murine hematopoietic stem cells

Large ex vivo expansion of hematopoietic stem cells (HSCs) sufficient for use in clinical applications has not been achieved, although the influence of some cytokines including SCF, IL-11, Flt3-L, and TPO for this purpose has been reported. We present evidence for an indirect effect of macrophage colony-stimulating factor (M-CSF) on expansion of murine HSCs. Fresh Lin(-/low) cells were isolated from Ly5.1 mouse bone marrow and cultured with or without M-CSF in the presence of SCF + IL-11 + Flt3-L or SCF + IL-11 + TPO for 6 days. The expanded cells were harvested and transplanted into lethally irradiated Ly5.2 recipients with competitor cells. Culture of Lin(-/low) cells with M-CSF significantly enhanced long-term engraftment. When the more enriched HSC populations of Lin(-/low) c-Kit(+) Sca-1(+) cells were used as a source of HSCs, such a promotive effect was not observed, in agreement with negative expression of the M-CSF receptor (c-Fms). However, co-culture with Lin(-/low) c-Fms(+) resulted in a significant increase of long-term engraftment. These results suggested that M-CSF is an indirect stimulator for ex vivo expansion of HSCs in the presence of SCF, IL-11, Flt3-L, and TPO. These observations provide new directions for ex vivo expansion and insight into new engraftment regulation through M-CSF signaling.     

Interpreting expression profiles of cancers by genome-wide survey of breadth of expression in normal tissues

A critical and difficult part of studying cancer with DNA microarrays is data interpretation. Besides the need for data analysis algorithms, integration of additional information about genes might be useful. We performed genome-wide expression profiling of 36 types of normal human tissues and identified 2503 tissue-specific genes. We then systematically studied the expression of these genes in cancers by reanalyzing a large collection of published DNA microarray datasets. We observed that the expression level of liver-specific genes in hepatocellular carcinoma (HCC) correlates with the clinically defined degree of tumor differentiation. Through unsupervised clustering of tissue-specific genes differentially expressed in tumors, we extracted expression patterns that are characteristic of individual cell types, uncovering differences in cell lineage among tumor subtypes. We were able to detect the expression signature of hepatocytes in HCC, neuron cells in medulloblastoma, glia cells in glioma, basal and luminal epithelial cells in breast tumors, and various cell types in lung cancer samples. We also demonstrated that tissue-specific expression signatures are useful in locating the origin of metastatic tumors. Our study shows that integration of each gene's breadth of expression (BOE) in normal tissues is important for biological interpretation of the expression profiles of cancers in terms of tumor differentiation, cell lineage, and metastasis.     

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.