Association of insomnia symptoms and non-restorative sleep with Typus melancholicus: a Japanese general population survey

Sleep and Biological Rhythms - This study aimed to investigate the association between insomnia symptoms and non-restorative sleep (NRS) in individuals with Typus melancholicus, a personality trait...     

Restoration of genetic diversity from soil seed banks in a threatened aquatic plant, <Emphasis Type="Italic">Nymphoides peltata</Emphasis>

Populations of a threatened aquatic plant, Nymphoides peltata, have rapidly degenerated under the influence of recent artificial changes in Lake Kasumigaura of Japan. To estimate the potential of soil seed banks for genetic restoration of the species, we used 10 microsatellite markers to analyze the genetic variation in adults and in seedlings that emerged from soil seed banks. About 187 leaf samples from the cultured stocks that were collected in 17 adult subpopulations in 1995 and 2000 and from three subpopulations that were newly discovered in 2002 were analyzed. As a result, only 18 genets could be identified, suggesting that clonal diversity of the adult population had already become extremely low. Genetic tests were performed on 430 seedlings from seed banks at six locations of natural lakeshores and three of the restoration sites that were artificially constructed in an attempt to assign them to the remnant adult population; many of the seedlings showed genetic variation different from the adults. Furthermore, the seedlings preserved seven alleles that had been lost from remnant adults. However, they had lower average numbers of alleles and heterozygosity levels (NA = 1.5–3.1, H _E = 0.146–0.487) than the remnant adults (NA = 3.5, H _E = 0.539) and showed high inbreeding coefficients, suggesting that the seed banks were produced by inbreeding. Thus, although the seed banks had a certain potential to restore genetic diversity, the fitness reduction in seed banks caused by inbreeding could affect the success of restoration based on seed banks.     

Liquid chromatography-electrospray ionization-tandem mass spectrometry for simultaneous analysis of chlorogenic acids and their metabolites in human plasma

A method using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) was developed for the simultaneous analysis of nine chlorogenic acids (CGAs), three isomers each of caffeoylquinic acids (CQAs), feruloylquinic acids (FQAs) and dicaffeoylquinic acids (dCQAs), and their two metabolites, caffeic acid (CA) and ferulic acid (FA), in human plasma. In simultaneous multiple reaction monitoring (MRM) measurements using ESI-MS/MS with a negative ion mode, a deprotonated molecular ion derived from each of the 11 molecules was used as a precursor ion while three diagnostic product ions characteristic for each were selected for the qualitative analysis. To obtain maximal intensities for all diagnostic product ions, the collision energy was optimized for each one. LC separation was achieved under conditions of a reversed-phase Inertsil ODS-2 column combined with a gradient elution system using 50mM acetic acid with 3% acetonitrile aqueous solution and 50 mM acetic acid with 100% acetonitrile. In the quantitative analysis, one of the three diagnostic product ions for each of the 11 molecules was selected. Application of simultaneous LC-ESI-MS/MS MRM measurements to analyze the 11 standards spiked into blank human plasma indicated that all diagnostic product ions were detected without any interference, and that the sensitivity, linearity and recovery of this method were acceptable. When using this method to analyze those 11 molecules in the plasma after oral ingestion of 250 ml of a drink containing a green coffee bean extract (300 mg CGAs), all 11 molecules were identified and CQAs, FQAs and FA were quantified. CQAs, FQAs and dCQAs in human plasma were detected for the first time. This method should be useful to understand the biological and pharmacological effects of CGAs, such as improvement of human hypertension.     

Trophic polymorphism in bluegill sunfish (<Emphasis Type="Italic">Lepomis macrochirus</Emphasis>) introduced into Lake Biwa: evidence from stable isotope analysis

Trophic polymorphism was recently reported in introduced bluegill (Lepomis macrochirus) in Lake Biwa, Japan, where three morphs are specialized in benthic invertebrates (benthivorous type), submerged aquatic plants (herbivorous type), and zooplankton (planktivorous type). We evaluated the long-term effects of food resource utilization by these trophic morphs using stable isotope ratios, δ¹⁵N and δ¹³C. A significant difference in δ¹⁵N was found between the benthivorous and planktivorous types. The planktivorous type had the higher δ¹⁵N value, which corresponded with the value expected from its prey, zooplankton. The lower δ¹⁵N value of the benthivorous type would be derived from the lower δ¹⁵N values of benthic prey organisms compared to zooplankton. These results support previous findings that the benthivorous and planktivorous types have different food resource utilization. In contrast, the δ¹⁵N and δ¹³C values of the herbivorous type were distinctly different from the expected values, indicating that this type was unlikely to utilize aquatic plants substantially, contradicting the results of the dietary analysis.     

Ionizing radiation suppresses FAP-1 mRNA level in A549 cells via p53 activation

Ionizing radiation (IR) is known to upregulate cell surface Fas through p53 activation in various cells. However, the signaling pathway intermediating between p53 activation and cell surface Fas upregulation remains to be elucidated. Recently, Fas-associated phosphatase-1 (FAP-1) has been reported to associate with Fas and inhibit cell surface Fas expression. We evaluated the expression of FAP-1 mRNA following IR in A549 cells. Ionizing radiation inhibited the expression of FAP-1 mRNA. Pretreatment with p53 inhibitor pifithrin alpha cancelled the IR-induced downregulation of FAP-1 mRNA. These results suggest that IR-induced p53 activation may upregulate cell surface Fas via the down-modulation of FAP-1.     

2-Deoxy-D-ribose inhibits hypoxia-induced apoptosis by suppressing the phosphorylation of p38 MAPK

An angiogenic factor, platelet-derived endothelial cell growth factor/thymidine phosphorylase (TP), stimulates the chemotaxis of endothelial cells and confers resistance to apoptosis induced by hypoxia. 2-Deoxy-d-ribose, a degradation product of thymidine generated by TP enzymatic activity, partially prevented hypoxia-induced apoptosis. 2-Deoxy-d-ribose inhibited hypoxia-induced phosphorylation of p38 mitogen-activated protein kinase (MAPK) but not c-jun NH(2)-terminal kinase/stress-activated protein kinase in human leukemia HL-60 cells. 2-Deoxy-d-ribose also suppressed the levels of Bax attached to mitochondria under hypoxic conditions. SB203580, a specific inhibitor of the p38 MAPK, suppressed the hypoxia-induced apoptosis of HL-60 cells. These findings suggest that one of the molecular bases for resistance to hypoxia-induced apoptosis conferred by 2-deoxy-d-ribose is the inhibition of the p38 signaling pathway. The expression levels of TP are elevated in many malignant solid tumors and thus the 2-deoxy-d-ribose generated by TP in these tumors may play an important role in tumor progression by preventing hypoxia-induced apoptosis.     

Spatial localizations of Mam22 and Mam12 in the magnetosomes of Magnetospirillum magnetotacticum

Magnetospirillum magnetotacticum possesses intracellular magnetite particles with a chain-like structure, termed magnetosomes. The bacterium expresses 22-kDa and 12-kDa magnetosome-associated proteins, termed Mam22 (MamA) and Mam12 (MamC), respectively. In this study, we investigated the structure of the purified magnetosomes with transmission electron microscopic techniques and found that the magnetosomes consisted of four compartments, i.e., magnetite crystal, magnetosomal membrane, interparticle connection, and magnetosomal matrix. Furthermore, we determined the precise localizations of Mam22 and Mam12 using immunogold staining of the purified magnetosomes and ultrathin sections of the bacterial cells. Interestingly, most Mam22 existed in the magnetosomal matrix, whereas Mam12 was strictly localized in the magnetosomal membrane. Moreover, the recombinant Mam22 was attached to the magnetosomal matrix of the Mam22-deficient magnetosomes prepared by alkaline treatment, such as 0.1 M Caps-NaOH buffer (pH 11.0). The spatial localization of the magnetosome-associated proteins in the magnetosomal chain provides useful information to elucidate the functional roles of these proteins.     

Subtilisin-like proprotein convertase activity is necessary for left–right axis determination in Xenopus neurula embryos

Signaling by members of TGF-β superfamily requires the activity of a family of site-specific endopeptidases, known as Subtilisin-like proprotein convertases (SPCs), which cleave these ligands into mature, active forms. To explore the role of SPCs in lateral plate mesoderm (LPM) differentiation in Xenopus, two SPC inhibitors, decanoyl-Arg-Val-Lys-Arg-chloromethylketone (Dec-RVKR-CMK) and hexa-arginine, were injected into the left and right LPM of Xenopus neurulae. Left-side injection caused heart-specific left–right reversal, and this phenotype was rescued by co-injection of mature Nodal protein. In contrast, right-side injection caused left–right reversal of both the heart and gut. Tailbud embryos were less sensitive to SPC inhibitors than neurula embryos. Injection of inhibitors into either side of neurula embryos completely abolished expression of the left-LPM-specific genes, Xnr-1, antivin, and pitx2. SPC1 enzyme (Furin) was injected into the left or right LPM of mid-neurula embryos to determine the effect of enhancing SPC activity. Left-side injection of SPC1 did not cause a significant left–right reversal of the internal organs. However, right-side injection of SPC1 strongly induced the expression of Xnr-1 and pitx2 in the right LPM, and caused 100% left–right reversal of both the heart and gut. These results suggest that moderate level of SPC activity in the right LPM of the neurulae is necessary for proper left–right specification. Taken together, SPC enzymatic activity must be present in both LPMs for expression of the left-handed genes and left–right axis determination of the heart and gut in Xenopus embryos.     

14-3-3 zeta protein secreted by tumor associated monocytes/macrophages from ascites of epithelial ovarian cancer patients

Tumor associated monocytes/macrophages (MO/MA) are known contributors to the immune-inflammatory cell environment of advanced epithelial ovarian carcinoma (EOC). The secreted proteome of ascitic MO/MA was examined as an aid to the discovery of novel proteins in EOC that are likely to have biological relevance in the inflammatory pathways of EOC. Ascitic fluid MO/MA were isolated from EOC patients, grown short-term in serum-free media. MO/MA supernatants were analyzed for secreted proteins by HPLC fractionation followed by LC-tandem mass spectrometric analysis. The 14-3-3 zeta adaptor protein was identified in supernatants of three of three EOC patients but not in supernatants of buffy coat monocytes isolated from normal donors or the established monocyte cell line THP1. Moreover, 14-3-3 zeta was identified in ascitic fluids in eight of eight chemotherapy-naïve patients by both immunoblot and mass spectrometric analysis. Immunofluorescent staining for 14-3-3 zeta demonstrated expression of the protein on ascitic and peritumoral macrophages in EOC patients. 14-3-3 zeta was also expressed on endothelial cells in the peritumoral stroma and partially on tumor cells. Uptake of 14-3-3 zeta was observed in EOC cell lines co-cultured with the recombinant protein expressed in E. coli. It is demonstrated for the first time that the important adaptor protein 14-3-3 zeta is common to the secretome of ascitic MO/MA and the ascites of advanced EOC patients.