Identification and characterization of nucleoplasmin 3 as a histone-binding protein in embryonic stem cells

Embryonic stem (ES) cells are thought to have unique chromatin structures responsible for their capacity for self-renewal and pluripotency. To examine this possibility, we sought nuclear proteins in mouse ES cells that specifically bind to histones using a pull-down assay with synthetic peptides of histone H3 and H4 tail domain as baits. Nuclear proteins preferentially bound to the latter. We identified 45 proteins associated with the histone H4 tail and grouped them into four categories: 10 chromatin remodeling proteins, five histone chaperones, two histone modification-related proteins, and 28 other proteins. mRNA expression levels of 20 proteins selected from these 45 proteins were compared between undifferentiated and retinoic acid (RA)-induced differentiated ES cells. All of the genes were similarly expressed in both states of ES cells, except nucleoplasmin 3 (NPM3) that was expressed at a higher level in the undifferentiated cells. NPM3 proteins were localized in the nucleoli and nuclei of the cells and expression was decreased during RA-induced differentiation. When transfected with NPM3 gene, ES cells significantly increased their proliferation compared with control cells. The present study strongly suggests that NPM3 is a chromatin remodeling protein responsible for the unique chromatin structure and replicative capacity of ES cells.     

Comparison of genetic population structure between two cyprinids, <Emphasis Type="Italic">Hemigrammocypris rasborella</Emphasis> and <Emphasis Type="Italic">Pseudorasbora pumila</Emphasis> subsp., in the Ise Bay basin,central Honshu,Japan

Two small cyprinid fishes, Hemigrammocypris rasborella and Pseudorasbora pumila subsp. (sensu Nakamura 1963), inhabit similar habitats and often occur sympatrically in the Ise Bay basin, central Honshu Island, Japan. Their genetic population structures were revealed, using sequence data from the mitochondrial cytochrome b gene, and then compared. Hemigrammocypris rasborella populations in the Ise Bay area formed a monophyletic group that has been isolated from eastern (Tenryu River) and western (Lake Biwa–Yodo River) populations at least for several hundred thousand years. Pseudorasbora pumila subsp., endemic to the Ise Bay area, was estimated to have become isolated from its sister subspecies, P. p. pumila, about 5 million years ago. Both H. rasborella and P. pumila subsp. had centers of genetic diversity around the Okazaki Plain in the eastern part of the basin and showed trans-bay distribution of haplotypes or haplotype groups. Their common population structure was explained by geological features in the Ise Bay area, in which a large paleo-river system developed in regression periods, suggesting gene flow among populations of each species in the mid to lower reaches of the paleo-river. Based on the estimated expansion or divergence time, however, not all populations experienced gene flow during the Last Glacial. In contrast to the maintenance of high genetic diversity in H. rasborella, almost all populations of P. pumila subsp. have lost mitochondrial DNA genetic diversity. This implies that effective population size of P. pumila subsp. tended to be smaller, probably because of differences in reproductive ecology, even though the two species have been exposed to similar environmental changes. For conservation of the two species, genetic and adaptive differentiation among local populations should be considered, and attention should be paid to inbreeding depression, especially in P. pumila subsp. An erratum to this article can be found at     

Addition of Aromatic Substrates Restores Trichloroethylene Degradation Activity in Pseudomonas putida F1

The rate of trichloroethylene (TCE) degradation by toluene dioxygenase (TDO) in resting cells of Pseudomonas putida F1 gradually decreased and eventually stopped within 1.5 h, as in previous reports. However, the subsequent addition of toluene, which is the principal substrate of TDO, resulted in its immediate degradation without a lag phase. After the consumption of toluene, degradation of TCE restarted at a rate similar to its initial degradation, suggesting that this degradation was mediated by TDO molecules that were present before the cessation of TCE degradation. The addition of benzene and cumene, which are also substrates of TDO, also caused restoration of TCE degradation activity: TCE was degraded simultaneously with cumene, and a larger amount of TCE was degraded after cumene was added than after toluene or benzene was added. But substrates that were expected to supply the cells with NADH or energy did not restore TCE degradation activity. This cycle of pseudoinactivation and restoration of TCE degradation was observed repeatedly without a significant decrease in the number of viable cells, even after six additions of toluene spread over 30 h. The results obtained in this study demonstrate a new type of restoration of TCE degradation that has not been previously reported.     

Adsorption study of metal ions onto crosslinked seaweed Laminaria japonica

An efficient and cost effective non-conventional adsorbent has been prepared from seaweed Laminaria japonica by crosslinking with epichlorohydrin. Its adsorption behavior for trivalent and divalent metal ions was studied and it was found to exhibit excellent selectivity towards several metal ions. As a typical example, binary mixture of Pb(II) and Zn(II) was studied by using a packed column, indicating that the Pb(II) ion can be easily separated from its mixture with a concentration factor of 74 times. The maximum adsorption capacity for Pb(II), Cd(II), Fe(III) was found to be 1.35, 1.1, 1.53 mol kg(-1), respectively, while 0.8 7 mol kg(-1) for both La(III) and Ce(III) from the single metal ion solution according to the adsorption isotherm. The obtained values are comparable to the commercially available synthetic chelating resins.     

Novel BK channel openers containing dehydroabietic acid skeleton: structure-activity relationship for peripheral substituents on ring C

A series of dehydroabietic acid (DHAA, 2) derivatives was synthesized and evaluated as BK channel openers in an assay system of CHO-K1 cells expressing hBKalpha channels. Systematic modifications of the peripheral functionality of ring C of DHAA showed that the introduction of a nitro or (thio)urea group in ring C greatly enhanced the BK channel-opening activity.     

Ionizing radiation downregulates ASPM, a gene responsible for microcephaly in humans

Microcephaly is a malformation associated with in utero exposed atomic bomb survivors and can be induced in mice by fetal exposure to ionizing radiation (IR). The pathogenesis of IR-induced microcephaly, however, has not been fully understood. Our analyses of high-coverage expression profiling (HiCEP) demonstrated that the abnormal spindle-like microcephaly associated gene (ASPM) was down-regulated in irradiated human diploid fibroblasts. ASPM was recently reported as the causative gene for MCPH-5, the most common type of congenital microcephaly in humans. Here, we show that the expression of the Aspm gene was significantly reduced by IR in various human and murine cells. Additionally, Aspm was found downregulated in the irradiated fetal mouse brain, particularly in the ventricular zones. A similar suppression was observed in the irradiated neurosphere cultures. This is the first report suggesting that the suppression of Aspm by IR could be the initial molecular target leading to the future microcephaly formation.     

Xenogenesis in teleost fish through generation of germ-line chimeras by single primordial germ cell transplantation

Primordial germ cells (PGCs) are the only cells in developing embryos with the potential to transmit genetic information to the next generation. PGCs therefore have the potential to be of value for gene banking and cryopreservation, particularly via the production of donor gametes with germ-line chimeras. Currently, it is not clear how many PGCs are required for germ-line differentiation and formation of gonadal structures. In the present study, we achieved complete germ-line replacement between two related teleost species, the pearl danio (Danio albolineatus) and the zebrafish (Danio rerio), with transplantation of a single PGC into each host embryo. We isolated and transplanted a single PGC into each blastula-stage, zebrafish embryo. Development of host germ-line cells was prevented by an antisense dead end morpholino oligonucleotide. In many host embryos, the transplanted donor PGC successfully migrated toward the gonadal anlage without undergoing cell division. At the gonadal anlage, the PGC differentiated to form one normally sized gonad rather than the pair of gonads usually present. Offspring were obtained from natural spawning of these chimeras. Analyses of morphology and DNA showed that the offspring were of donor origin. We extended our study to confirm that transplanted single PGCs of goldfish (Carassius auratus) and loach (Misgurnus anguillicaudatus) can similarly differentiate into sperm in zebrafish host embryos. Our results show that xenogenesis is realistic and practical across species, genus, and family barriers and can be achieved by the transplantation of a single PGC from a donor species.     

Polymorphism in Nucleotide Sequence of Mitochondrial Intergenic Region in Scleractinian Coral (Galaxea fascicularis)

A region of 826 bp that is unlikely to code for a protein, ribosomal RNA, or transfer RNA was identified between the cytochrome b and NADH–ubiquinone oxidoreductase chain 2 loci in the mitochondrial DNA of the scleractinian reef coral Galaxea fascicularis. Nucleotide sequences were determined in a part (625 bp) of this intergenic region in 95 individuals collected at 9 sites in the Ryukyu Archipelago in southwestern Japan. A total of 8 haplotypes were found, and a deletion of 290 bp was found in 3 of them. Significant differences were found in frequencies of the haplotypes at 3 sampling sites. The presence or absence of the deletion was highly correlated with the hard or soft morphotype. The deletion was found in the majority of hard-type colonies, but in a small fraction of soft-type individuals.     

Coaggregation facilitates interspecies hydrogen transfer between Pelotomaculum thermopropionicum and Methanothermobacter thermautotrophicus

A thermophilic syntrophic bacterium, Pelotomaculum thermopropionicum strain SI, was grown in a monoculture or coculture with a hydrogenotrophic methanogen, Methanothermobacter thermautotrophicus strain DeltaH. Microscopic observation revealed that cells of each organism were dispersed in a monoculture independent of the growth substrate. In a coculture, however, these organisms coaggregated to different degrees depending on the substrate; namely, a large fraction of the cells coaggregated when they were grown on propionate, but relatively few cells coaggregated when they were grown on ethanol or 1-propanol. Field emission-scanning electron microscopy revealed that flagellum-like filaments of SI cells played a role in making contact with DeltaH cells. Microscopic observation of aggregates also showed that extracellular polymeric substance-like structures were present in intercellular spaces. In order to evaluate the importance of coaggregation for syntrophic propionate oxidation, allowable average distances between SI and DeltaH cells for accomplishing efficient interspecies hydrogen transfer were calculated by using Fick's diffusion law. The allowable distance for syntrophic propionate oxidation was estimated to be approximately 2 mum, while the allowable distances for ethanol and propanol oxidation were 16 mum and 32 mum, respectively. Considering that the mean cell-to-cell distance in the randomly dispersed culture was approximately 30 mum (at a concentration in the mid-exponential growth phase of the coculture of 5 x 10(7) cells ml(-1)), it is obvious that close physical contact of these organisms by coaggregation is indispensable for efficient syntrophic propionate oxidation.     

Roles of aromatic residues in the structure and biological activity of the small cytokine,growth-blocking peptide (GBP)

Growth-blocking peptide (GBP) is a small (25 amino acids) insect cytokine with a variety of functions: controlling the larval development of lepidopteran insects, acting as a mitogen for various types of cultured cells, and stimulating insect blood cells. The aromatic residues of GBP (Phe-3, Tyr-11, and Phe-23) are highly conserved in the ENF peptide family found in lepidopteran insects. We investigated the relationship between the biological activities and structural properties of a series of GBP mutants, in which each of the three aromatic residues is replaced by a different residue. The results of the hemocytes-stimulating assays of GBP mutants indicated that Phe-3 is the key residue in this activity: Ala or Tyr replacement resulted in significant loss of the activity, but Leu replacement did not. The replacements of other aromatic residues hardly affected the activity. On the other hand, NMR analysis of the mutants suggested that Tyr-11 is a key residue for maintaining the core structure of GBP. Surprisingly, the Y11A mutant maintained its biological activity, although its native-like secondary structure was disordered. Detailed analyses of the (15)N-labeled Y11A mutant by heteronuclear NMR spectroscopy showed that the native-like beta-sheet structure of Y11A was induced by the addition of 2,2,2-trifluoroethanol. The results suggest that Y11A has a tendency to form a native-like structure, and this property may give the Y11A mutant native-like activity.