Molecular characterization of ENU mouse mutagenesis and archives

The large-scale mouse mutagenesis with ENU has provided forward-genetic resources for functional genomics. The frozen sperm archive of ENU-mutagenized generation-1 (G1) mice could also provide a "mutant mouse library" that allows us to conduct reverse genetics in any particular target genes. We have archived frozen sperm as well as genomic DNA from 9224 G1 mice. By genome-wide screening of 63 target loci covering a sum of 197 Mbp of the mouse genome, a total of 148 ENU-induced mutations have been directly identified. The sites of mutations were primarily identified by temperature gradient capillary electrophoresis method followed by direct sequencing. The molecular characterization revealed that all the identified mutations were point mutations and mostly independent events except a few cases of redundant mutations. The base-substitution spectra in this study were different from those of the phenotype-based mutagenesis. The ENU-based gene-driven mutagenesis in the mouse now becomes feasible and practical.     

Antinociceptive effect of 7-hydroxymitragynine in mice: Discovery of an orally active opioid analgesic from the Thai medicinal herb Mitragyna speciosa

Mitragynine is an indole alkaloid isolated from the Thai medicinal plant Mitragyna speciosa. We previously reported the morphine-like action of mitragynine and its related compounds in the in vitro assays. In the present study, we investigated the opioid effects of 7-hydroxymitragynine, which is isolated as its novel constituent, on contraction of isolated ileum, binding of the specific ligands to opioid receptors and nociceptive stimuli in mice. In guinea-pig ileum, 7-hydroxymitragynine inhibited electrically induced contraction through the opioid receptors. Receptor-binding assays revealed that 7-hydroxymitragynine has a higher affinity for micro-opioid receptors relative to the other opioid receptors. Administration of 7-hydroxymitragynine (2.5-10 mg/kg, s.c.) induced dose-dependent antinociceptive effects in tail-flick and hot-plate tests in mice. Its effect was more potent than that of morphine in both tests. When orally administered, 7-hydroxymitragynine (5-10 mg/kg) showed potent antinociceptive activities in tail-flick and hot-plate tests. In contrast, only weak antinociception was observed in the case of oral administration of morphine at a dose of 20 mg/kg. It was found that 7-hydroxymitragynine is a novel opioid agonist that is structurally different from the other opioid agonists, and has potent analgesic activity when orally administered.     

Different In Vivo Effects of HIV-1 Immunodominant Epitope-Specific Cytotoxic T Lymphocytes on Selection of Escape Mutant Viruses

HIV-1 escape mutants are well known to be selected by immune pressure via HIV-1-specific cytotoxic T lymphocytes (CTLs) and neutralizing antibodies. The ability of the CTLs to suppress HIV-1 replication is assumed to be associated with the selection of escape mutants from the CTLs. Therefore, we first investigated the correlation between the ability of HLA-A*1101-restricted CTLs recognizing immunodominant epitopes in vitro and the selection of escape mutants. The result showed that there was no correlation between the ability of these CTLs to suppress HIV-1 replication in vitro and the appearance of escape mutants. The CTLs that had a strong ability to suppress HIV-1 replication in vitro but failed to select escape mutants expressed a higher level of PD-1 in vivo, whereas those that had a strong ability to suppress HIV-1 replication in vitro and selected escape mutants expressed a low level of PD-1. Ex vivo analysis of these CTLs revealed that the latter CTLs had a significantly stronger ability to recognize the epitope than the former ones. These results suggest that escape mutations are selected by HIV-1-specific CTLs that have a stronger ability to recognize HIV-1 in vivo but not in vitro.HIV-1-specific cytotoxic T lymphocytes (CTLs) have an important role in the control of HIV-1 replication during acute and chronic phases of an HIV-1 infection (5, 28, 33). On the other hand, HIV-1 can escape from the host immune system by various mechanisms. These may include the appearance of HIV-1 carrying escape mutations in its immunodominant CTL epitopes as well as Nef-mediated downregulation of HLA class I molecules. There is a growing body of evidence for the former mechanism, i.e., that CTLs targeting immunodominant HIV-1 epitopes select escape mutants in chronically HIV-1-infected individuals (18, 20, 36), whereas the latter mechanism was proved by demonstrating that HIV-1-specific CTLs fail to kill Nef-positive-HIV-1-infected CD4⁺ T cells but effectively kill Nef-defective-HIV-1-infected ones or that they suppress the replication of Nef-defective HIV-1 much more than that of Nef-positive HIV-1 (12, 13, 42, 45).It is speculated that HIV-1 immunodominant epitope-specific CTLs have the ability to suppress HIV-1 replication and effectively select escape mutants. However, the correlation between this ability of the CTLs and the appearance of escape mutants is still unclear, because it is not easy to evaluate the ability of HIV-1-specific CTLs to exert a strong immune pressure in vivo. To examine this ability, most previous studies measured the number of HIV-1-specific CTLs or CD8⁺ T cells and the CTL activity against target cells prepulsed with the epitope peptide or those infected with HIV-1 recombinant vaccinia virus (6, 7, 23, 46). However, the results obtained from such experiments do not reflect the ability of the CTLs to exert immune pressure in vivo. We and other groups previously utilized an assay to directly evaluate the ability of the CTLs to suppress HIV-1 replication in vitro (1, 17, 18, 42, 43). This assay may be better for evaluation of immune pressure by HIV-1-specific CTLs than other assays, because the ability of the CTLs to suppress HIV-1 replication is directly measured in cultures of HIV-1-infected CD4⁺ T cells incubated with HIV-1-specific CTL clones. But it still remains unknown whether this assay reflects immune pressure in vivo.In the present study, we investigated whether HIV-1-specific CTLs having a strong ability to suppress HIV-1 replication could positively select escape mutants. Since HLA-A*1101 is known to be an HLA allele relatively associated with a slow progression to AIDS (32), it is speculated that some HLA-A*1101-restricted CTLs would have a strong ability to suppress HIV-1 replication in vitro. Therefore, we first focused on 4 well-known HLA-A*1101-restricted CTL epitopes in the present study. We investigated the frequency of CTLs specific for these epitopes in chronically HIV-1-infected individuals, the ability of these CTLs to suppress HIV-1 replication in vitro, and whether the escape mutants were selected by the CTLs. Furthermore, we analyzed the expression of Programmed Death-1 (PD-1) on these CTLs ex vivo and antigen recognition of them.     

Chirality of camphor derivatives by density functional theory

Infrared (IR) and vibrational circular dichroism (VCD) spectra of chiral camphor, camphorquinone and camphor-10-sulfonic acid (CSA), known as standard compounds for electronic circular dichroism (ECD) spectroscopy, are measured and their vibrational frequencies, infrared intensities, and rotational strengths are calculated using density functional theory (DFT). The observed IR and VCD spectra of chiral camphor and camphorquinone in carbon tetrachloride solution are reproduced by the DFT calculations, but those of CSA are not. DFT calculations of hydration models, where an anionic CSA specifically binds a few water molecules, are carried out. The average of the simulated VCD spectra in the hydration models is more consistent with the observed spectra. In addition, the wavelengths and dipole and rotational strengths for chiral camphor, camphorquinone, anionic CSA, and the hydration models were calculated by time-dependent DFT. In the region of 280-300 nm, the calculated wavelengths of the ECD bands for chiral camphor and camphorquinone coincide with the observed wavelengths that have been reported, and the calculated wavelengths for the hydration models are closer to the observed wavelengths reported than are those calculated for chiral anionic CSA. Consequently, the analysis combined with VCD and ECD spectroscopy using DFT calculations can elucidate the chirality of optically active molecules, even in an aqueous solution.     

Ordered assembly of Sld3, GINS and Cdc45 is distinctly regulated by DDK and CDK for activation of replication origins

Initiation of chromosome DNA replication in eukaryotes is tightly regulated through assembly of replication factors at replication origins. Here, we investigated dependence of the assembly of the initiation complex on particular factors using temperature-sensitive fission yeast mutants. The psf3-1 mutant, a GINS component mutant, arrested with unreplicated DNA at the restrictive temperature and the DNA content gradually increased, suggesting a defect in DNA replication. The mutation impaired GINS complex formation, as shown by pull-down experiments. Chromatin immunoprecipitation assays indicated that GINS integrity was required for origin loading of Psf2, Cut5 and Cdc45, but not Sld3. In contrast, loading of Psf2 onto origins depended on Sld3 and Cut5 but not on Cdc45. These results suggest that Sld3 functions furthest upstream in initiation complex assembly, followed by GINS and Cut5, then Cdc45. Consistent with this conclusion, Cdc7-Dbf4 kinase (DDK) but not cyclin-dependent kinase (CDK) was required for Sld3 loading, whereas recruitment of the other factors depended on both kinases. These results suggest that DDK and CDK regulate distinct steps in activation of replication origins in fission yeast.     

Capacity of dental pulp differentiation after tooth transplantation

Under pathological conditions, dental pulp elaborates both bone and dentin matrix in which the contribution of periodontal tissue cannot be excluded. This study has aimed to clarify the capability of dental pulp to deposit bone matrix in an auto-graft experiment by using (1) immunohistochemistry for 5-bromo-2′-deoxyuridine (BrdU) and nestin and (2) histochemistry for tartrate-resistant acid phosphatase (TRAP). Following the extraction of the molars of 3-week-old mice, the roots and pulp floor were resected and immediately transplanted into the sublingual region. On Days 5–7, tubular dentin formation commenced next to the pre-existing dentin at the pulp horn in which nestin-positive odontoblast-like cells were arranged. Up until Day 14, bone-like tissue formation occurred in the pulp chamber in which intense TRAP-positive cells appeared. These results suggest that odontoblast- and osteoblast-lineage cells reside in the dental pulp. Overall, specific dental pulp regeneration should provide fundamental knowledge for the realization of human tooth regeneration in the near future.This work was supported in part by a grant from MEXT to promote the 2001-multidisciplinary research project (in 2001–2005), KAKENHI (B) (no. 16390523 to H.O.) from MEXT, and the Japan-Korea Joint Research Project from JSPS and KOSEF (no. F01-2005-000-10212-0).     

Profile of rhythmic gene expression in the livers of obese diabetic KK-A(y) mice

Although a number of genes expressed in most tissues, including the liver, exhibit circadian regulation, gene expression profiles are usually examined only at one scheduled time each day. In this study, we investigated the effects of obese diabetes on the hepatic mRNA levels of various genes at 6-h intervals over a single 24-h period. Microarray analysis revealed that many genes are expressed rhythmically, not only in control KK mice but also in obese diabetic KK-A(y) mice. Real-time quantitative PCR verified that 19 of 23 putative circadianly expressed genes showed significant 24-h rhythmicity in both strains. However, obese diabetes attenuated these expression rhythms in 10 of 19 genes. More importantly, the effects of obese diabetes were observed throughout the day in only two genes. These results suggest that observation time influences the results of gene expression analyses of genes expressed circadianly.     

The relationship between cell proliferation and differentiation and mapping of putative dental pulp stem/progenitor cells during mouse molar development by chasing BrdU-labeling

Human dental pulp contains adult stem cells. Our recent study demonstrated the localization of putative dental pulp stem/progenitor cells in the rat developing molar by chasing 5-bromo-2’-deoxyuridine (BrdU)-labeling. However, there are no available data on the localization of putative dental pulp stem/progenitor cells in the mouse molar. This study focuses on the mapping of putative dental pulp stem/progenitor cells in addition to the relationship between cell proliferation and differentiation in the developing molar using BrdU-labeling. Numerous proliferating cells appeared in the tooth germ and the most active cell proliferation in the mesenchymal cells occurred in the prenatal stages, especially on embryonic Day 15 (E15). Cell proliferation in the pulp tissue dramatically decreased in number by postnatal Day 3 (P3) when nestin-positive odontoblasts were arranged in the cusped areas and disappeared after postnatal Week 1 (P1W). Root dental papilla included numerous proliferating cells during P5 to P2W. Three to four intraperitoneal injections of BrdU were given to pregnant ICR mice and revealed slow-cycling long-term label-retaining cells (LRCs) in the mature tissues of postnatal animals. Numerous dense LRCs postnatally decreased in number and reached a plateau after P1W when they mainly resided in the center of the dental pulp, associating with blood vessels. Furthermore, numerous dense LRCs co-expressed mesenchymal stem cell markers such as STRO-1 and CD146. Thus, dense LRCs in mature pulp tissues were believed to be dental pulp stem/progenitor cells harboring in the perivascular niche surrounding the endothelium.     

Endophytic fungi associated with leaves of Betulaceae in Japan

Diversity and species composition of endophytic fungi on leaves of 11 tree species in Betulaceae were studied, with reference to climatic, tree species, and seasonal variations. A total of 186 fungal isolates were obtained from 190 leaves collected in a subalpine forest, a cool temperate forest, and a subtropical forest in Japan, and were divided into 46 operational taxonomic units (OTUs) according to the base sequences of D1-D2 region of large subunit rDNA. The 2 most frequent OTUs were Muscodor sp. and Nemania sp. in Xylariaceae, followed by Gnomonia sp., Glomerella acutata , Apiosporopsis sp., Asteroma sp., and Cladosporium cladosporioides . The similarities of OTU composition in endophytic fungal assemblages on leaves of Betulaceae were generally low among the forests of different climatic regions. Fungal OTU compositions were relatively similar between 2 Betula species in the subalpine forest, whereas 7 tree species in the cool temperate forest were divided into 3 groups according to the similarity of endophytic fungal assemblages on the leaves, with 4 Carpinus species assigned into 2 of the 3 groups. The similarity of endophytic fungal assemblages between August and October was relatively high in the subalpine forest, whereas the seasonal changes were generally greater (i.e., the similarities among sampling dates were lower) in the cool temperate forest.