'Working' cardiomyocytes exhibiting plateau action potentials from human placenta-derived extraembryonic mesodermal cells

The clinical application of cell transplantation for severe heart failure is a promising strategy to improve impaired cardiac function. Recently, an array of cell types, including bone marrow cells, endothelial progenitors, mesenchymal stem cells, resident cardiac stem cells, and embryonic stem cells, have become important candidates for cell sources for cardiac repair. In the present study, we focused on the placenta as a cell source. Cells from the chorionic plate in the fetal portion of the human placenta were obtained after delivery by the primary culture method, and the cells generated in this study had the Y sex chromosome, indicating that the cells were derived from the fetus. The cells potentially expressed 'working' cardiomyocyte-specific genes such as cardiac myosin heavy chain 7beta, atrial myosin light chain, cardiac alpha-actin by gene chip analysis, and Csx/Nkx2.5, GATA4 by RT-PCR, cardiac troponin-I and connexin 43 by immunohistochemistry. These cells were able to differentiate into cardiomyocytes. Cardiac troponin-I and connexin 43 displayed a discontinuous pattern of localization at intercellular contact sites after cardiomyogenic differentiation, suggesting that the chorionic mesoderm contained a large number of cells with cardiomyogenic potential. The cells began spontaneously beating 3 days after co-cultivation with murine fetal cardiomyocytes and the frequency of beating cells reached a maximum on day 10. The contraction of the cardiomyocytes was rhythmical and synchronous, suggesting the presence of electrical communication between the cells. Placenta-derived human fetal cells may be useful for patients who cannot supply bone marrow cells but want to receive stem cell-based cardiac therapy.     

Human Misato regulates mitochondrial distribution and morphology

Misato of Drosophila melanogaster and Saccharomyces cerevisiae DML1 are conserved proteins having a homologous region with a part of the GTPase family that includes eukaryotic tubulin and prokaryotic FtsZ. We characterized human Misato sharing homology with Misato of D. melanogaster and S. cerevisiae DML1. Tissue distribution of Misato exhibited ubiquitous distribution. Subcellular localization of the protein studied using anti-Misato antibody suggested that it is localized to the mitochondria. Further experiments of fractionating mitochondria revealed that Misato was localized to the outer membrane. The transfection of Misato siRNA led to growth deficiencies compared with control siRNA transfected HeLa cells, and the Misato-depleted HeLa cells showed apoptotic nuclear fragmentation resulting in cell death. After silencing of Misato, the filamentous mitochondrial network disappeared and fragmented mitochondria were observed, indicating human Misato has a role in mitochondrial fusion. To examine the effects of overexpression, COS-7 cells were transfected with cDNA encoding EGFP-Misato. Its overexpression resulted in the formation of perinuclear aggregations of mitochondria in these cells. The Misato-overexpressing cells showed low viability and had no nuclei or a small and structurally unusual ones. These results indicated that human Misato has a role(s) in mitochondrial distribution and morphology and that its unregulated expression leads to cell death.     

Molecular phylogeography of two sibling species of <Emphasis Type="Italic">Eurema</Emphasis> butterflies

The common yellow butterfly Eurema hecabe is widely distributed in East Asia, and is one of the most burdensome species for taxonomists due to the numerous geographic and seasonal wing colour patterns. Moreover, within this species, individuals with a yellow wing fringe that occur in temperate regions of Japan (Y type) proved to be biologically different from others that occur widely in subtropical regions of Japan and all over East Asia (B type). To unveil the genetic variation within and between the two types, a total of 50 butterflies collected at 18 geographic localities in East Asia were examined for nucleotide sequence variation of three mitochondrial regions: cytochrome c oxidase subunit I (COI), cytochrome c oxidase subunit III (COIII) and NADH dehydrogenase subunit 5 (ND5). In addition, they were also examined for infection status with the endosymbiotic bacteria Wolbachia. The three mitochondrial sequences consistently showed that (i) Y type and B type were highly divergent, (ii) nucleotide variation within B type was very small although sampled from a geographically wide range, and (iii) a weak association existed between mitochondrial DNA haplotypes and Wolbachia infection status.     

Recombinant sea urchin flagellar adenylate kinase

Adenylate kinase (AK) is localized in sea urchin sperm flagella and embryonic cilia. To investigate sea urchin Strongylocentrotus purpuratus AK (SpAK) enzymatic characteristics, the full-length recombinant protein of 130 kDa (SpAKr) and each of its three catalytic domains were expressed in Escherichia coli. Although the full-length SpAK had high enzymatic activity, each of the three catalytic domains had no activity. The Km for ATP synthesis from ADP was 0.23 mM and the Vmax was 4.51 mumol ATP formed per minute per milligram of protein. The specific AK inhibitor, Ap5A, blocks SpAKr enzymatic activity with an IC50 of 0.53 microM. The pH optimum for SpAKr is 8.1, as compared to 7.7 for the natural SpAK. Calcium inhibits SpAKr activity in a dose-dependent manner. Although SpAKr has three cAMP-dependent protein kinase phosphorylation sites, and can be phosphorylated in vitro, the enzymatic kinetics after phosphorylation are not significantly altered. SpAK and Chlamydomonas flagellar AKs are the only AKs with three catalytic sites. Further study of the SpAKr will aid in understanding the active site of this interesting and important ATP synthase.     

Characterization of a major secretory protein in the cane toad (Bufo marinus) choroid plexus as an amphibian lipocalin-type prostaglandin D synthase

Here we report the enzymatic and ligand-binding properties of a major secretory protein in the choroid plexus of cane toad, Bufo marinus, whose protein is homologous with lipocalin-type prostaglandin (PG) D synthase (L-PGDS) and is recombinantly expressed in Xenopus A6 cells and Escherichia coli. The toad protein bound all-trans retinal, bile pigment, and thyroid hormones with high affinities (K(d)=0.17 to 2.00 microM). The toad protein also catalysed the L-PGDS activity, which was accelerated in the presence of GSH or DTT, similar to the mammalian enzyme. The K(m) value for PGH(2) (17 microM) of the toad protein was almost the same as that of rat L-PGDS (14 microM), whereas the turnover number (6 min(-1)) was approximately 28 fold lower than that of rat L-PGDS. Site-directed mutagenesis based on a modeled structure of the toad protein revealed that Cys(59) and Thr(61) residues were crucial for the PGDS activity. The quadruple Gly(39)Ser/Ala(75)Ser/Ser(140)Thr/Phe(142)Tyr mutant of the toad protein, resembling mouse L-PGDS, showed a 1.6 fold increase in the turnover number and a shift in the optimum pH for the PGDS activity from 9.0 to 8.5. Our results suggest that the toad protein is a prototype of L-PGDS with a highly functional ligand-binding pocket and yet with a primitive catalytic pocket.     

The genetic structure of endangered indigenous populations of the amago salmon, <Emphasis Type="Italic">Oncorhynchus</Emphasis><Emphasis Type="Italic">masou</Emphasis><Emphasis Type="Italic">ishikawae</Emphasis>, in Japan

The amago salmon, Oncorhynchus masou ishikawae, is an endemic subspecies of O. masou in Japan. Owing to the extensive stocking of hatchery fish throughout Japan, indigenous populations of O. m. ishikawae are now on the verge of extinction. We examined the genetic effects of stocking hatchery fish on wild populations in the River Koza, Japan, using microsatellite and mitochondrial DNA (mtDNA) markers. For mtDNA, haplotype mt1, which is common in wild populations, was present exclusively in isolated wild populations assumed to be unaffected by previous stocking, while it was never observed in hatchery fish. Genetic diversity was much higher in wild populations in the stocked area, which shared many mtDNA haplotypes with hatchery fish, than in isolated wild populations with haplotype mt1. Pairwise F _ST estimates based on microsatellites showed significant differentiation among the isolated populations with many microsatellite loci monomorphic. Significant deviation from Hardy–Weinberg equilibrium was observed in wild populations in the area subject to stocking, where a Bayesian-based assignment test showed a high level of introgression with hatchery fish. These results suggest that wild populations with haplotype mt1, which became isolated through anthropogenic environmental change in the 1950–1960s, represent indigenous populations of O. m. ishikawae in the River Koza. They have low genetic diversity, most likely caused by genetic bottlenecks following damming and environmental deterioration, while stocking of hatchery fish over the past 30 years apparently had a large impact on the genetic structure of wild populations in the main channel of the River Koza.     

Norlichexanthone produced by cultured endolichenic fungus induced from Pertusaria laeviganda and its antioxidant activity

Endolichenic fungi, nonobligate microfungi that live in lichen, are promising as new bioresources of pharmacological compounds. We found that norlichexanthone isolated from the endolichenic fungus in Pertusaria laeviganda exhibited high antioxidant activity. Norlichexanthone produced by endolichenic fungus had the antioxidant activity with same level of ascorbic acid. This is the first report of high antioxidant activity of norlichexanthone.

Abbreviations: AAPH: 2,2?-azobis (2-methylpropionamidine) dihydrochloride; DPPH: 2,2-diphenyl-1-picrylhydrazyl; FL: fluorescein sodium salt; HPLC-PDA: high-performance liquid chromatography with photodiode array; LC-ESI-MS: liquid chromatography with electrospray ionization mass spectrometry; ORAC: oxygen radical absorbance capacity; PB: phosphate buffer; ROS: reactive oxygen species; TLC: thin-layer chromatography     

Hsp105α suppresses Adriamycin-induced cell death via nuclear localization signal-dependent nuclear accumulation

Heat shock protein 105 (Hsp105) is a molecular chaperone, and the isoforms Hsp105α and Hsp105β exhibit distinct functions with different subcellular localizations. Hsp105β localizes in the nucleus and induces the expression of the major heat shock protein Hsp70, whereas cytoplasmic Hsp105α is less effective in inducing Hsp70 expression. Hsp105 shuttles between the cytoplasm and the nucleus; the subcellular localization is governed by the relative activities of the nuclear localization signal (NLS) and nuclear export signal (NES). Here, we show that nuclear accumulation of Hsp105α but not Hsp105β is involved in Adriamycin (ADR) sensitivity. Knockdown of Hsp105α induces cell death at low ADR concentration, at which ADR is less effective in inducing cell death in the presence of Hsp105α. Of note, Hsp105 is localized in the nucleus under these conditions, even though Hsp105β is not expressed, indicating that Hsp105α accumulates in the nucleus in response to ADR treatment. The exogenously expressed Hsp105α but not its NLS mutant localizes in the nucleus of ADR-treated cells. In addition, the expression level of the nuclear export protein chromosomal maintenance 1 (CRM1) was decreased by ADR treatment of cells, and CRM1 knockdown caused nuclear accumulation of Hsp105α both in the presence and absence of ADR. These results indicating that Hsp105α accumulates in the nucleus in a manner dependent on the NLS activity via the suppression of nuclear export. Our findings suggest a role of nuclear Hsp105α in the sensitivity against DNA-damaging agents in tumor cells.