Genome-wide association study of idiopathic hypersomnia in a Japanese population

Sleep and Biological Rhythms - Idiopathic hypersomnia (IH) is a rare sleep disorder characterized by excessive daytime sleepiness, great difficulty upon awakening, and prolonged sleep time. In...     

Normal human osteoclasts formed from peripheral blood monocytes express PTH type 1 receptors and are stimulated by PTH in the absence of osteoblasts

The prevailing view for many years has been that osteoclasts do not express parathyroid hormone (PTH) receptors and that PTH's effects on osteoclasts are mediated indirectly via osteoblasts. However, several recent reports suggest that osteoclasts express PTH receptors. In this study, we tested the hypothesis that human osteoclasts formed in vitro express functional PTH type 1 receptors (PTH1R). Peripheral blood monocytes (PBMC) were cultured on bone slices or plastic culture dishes with human recombinant RANK ligand (RANKL) and recombinant human macrophage colony-stimulating factor (M-CSF) for 16-21 days. This resulted in a mixed population of mono- and multi-nucleated cells, all of which stained positively for the human calcitonin receptor. The cells actively resorbed bone, as assessed by release of C-terminal telopeptide of type I collagen and the formation of abundant resorption pits. We obtained evidence for the presence of PTH1R in these cells by four independent techniques. First, using immunocytochemistry, positive staining for PTH1R was observed in both mono- and multi-nucleated cells intimately associated with resorption cavities. Second, PTH1R protein expression was demonstrated by Western blot analysis. Third, the cells expressed PTH1R mRNA at 21 days and treatment with 10(-7) M hPTH (1-34) reduced PTH1R mRNA expression by 35%. Finally, bone resorption was reproducibly increased by two to threefold when PTH (1-34) was added to the cultures. These findings provide strong support for a direct stimulatory action of PTH on human osteoclasts mediated by PTH1R. This suggests a dual regulatory mechanism, whereby PTH acts both directly on osteoclasts and also, indirectly, via osteoblasts.     

Paxillin mediates sensing of physical cues and regulates directional cell motility by controlling lamellipodia positioning

Physical interactions between cells and the extracellular matrix (ECM) guide directional migration by spatially controlling where cells form focal adhesions (FAs), which in turn regulate the extension of motile processes. Here we show that physical control of directional migration requires the FA scaffold protein paxillin. Using single-cell sized ECM islands to constrain cell shape, we found that fibroblasts cultured on square islands preferentially activated Rac and extended lamellipodia from corner, rather than side regions after 30 min stimulation with PDGF, but that cells lacking paxillin failed to restrict Rac activity to corners and formed small lamellipodia along their entire peripheries. This spatial preference was preceded by non-spatially constrained formation of both dorsal and lateral membrane ruffles from 5-10 min. Expression of paxillin N-terminal (paxN) or C-terminal (paxC) truncation mutants produced opposite, but complementary, effects on lamellipodia formation. Surprisingly, pax-/- and paxN cells also formed more circular dorsal ruffles (CDRs) than pax+ cells, while paxC cells formed fewer CDRs and extended larger lamellipodia even in the absence of PDGF. In a two-dimensional (2D) wound assay, pax-/- cells migrated at similar speeds to controls but lost directional persistence. Directional motility was rescued by expressing full-length paxillin or the N-terminus alone, but paxN cells migrated more slowly. In contrast, pax-/- and paxN cells exhibited increased migration in a three-dimensional (3D) invasion assay, with paxN cells invading Matrigel even in the absence of PDGF. These studies indicate that paxillin integrates physical and chemical motility signals by spatially constraining where cells will form motile processes, and thereby regulates directional migration both in 2D and 3D. These findings also suggest that CDRs may correspond to invasive protrusions that drive cell migration through 3D extracellular matrices.     

Identification and characterization of GCP16, a novel acylated Golgi protein that interacts with GCP170

GCP170, a member of the golgin family associated with the cytoplasmic face of the Golgi membrane, was found to have a Golgi localization signal at the NH2-terminal region (positions 137-237). Using this domain as bait in the yeast two-hybrid screening system, we identified a novel protein that interacted with GCP170. The 2.0-kilobase mRNA encoding a 137-amino acid protein of 16 kDa designated GCP16 was ubiquitously expressed. Immunofluorescence microscopy showed that GCP16 was co-localized with GCP170 and giantin in the Golgi region. Despite the absence of a hydrophobic domain sufficient for participating in membrane localization, GCP16 was found to be tightly associated with membranes like an integral membrane protein. Labeling experiments with [3H]palmitic acid and mutational analysis demonstrated that GCP16 was acylated at Cys69 and Cys72, accounting for its tight association with the membrane. A mutant without potential acylation sites (C69A/C72A) was no longer localized to the Golgi, indicating that the acylation is prerequisite for the Golgi localization of GCP16. Although the mutant GCP16, even when overexpressed, had no effect on protein transport, overexpression of the wild type GCP16 caused an inhibitory effect on protein transport from the Golgi to the cell surface. Taken together, these results indicate that GCP16 is the acylated membrane protein, associated with GCP170, and possibly involved in vesicular transport from the Golgi to the cell surface.     

Structural determination of five novel tetrasaccharides containing 3-O-sulfated D-glucuronic acid and two rare oligosaccharides containing a beta-D-glucose branch isolated from squid cartilage chondroitin sulfate E

Oversulfated chondroitin sulfate E (CS-E) derived from squid cartilage exhibits intriguing biological activities, which appear to reflect the biological activities of mammalian CS chains containing the so-called E disaccharide unit [GlcAbeta1-3GalNAc(4,6-O-disulfate)]. Previously, we isolated novel tetra- and hexasaccharides containing a rare GlcA(3-O-sulfate) at the nonreducing end after digestion of squid cartilage CS-E with testicular hyaluronidase. In this study, squid cartilage CS-E was extensively digested with chondroitinase AC-II, which yielded five highly sulfated novel tetrasaccharides and two odd-numbered oligosaccharides (tri- and pentasaccharides) containing D-Glc. Their structures were determined by fast atom bombardment mass spectrometry and (1)H NMR spectroscopy. The results revealed an internal GlcA(3-O-sulfate) residue for all the novel tetrasaccharide sequences, which rendered the oligosaccharides resistant to the enzyme. The results suggest that GlcA(3-O-sulfate) units are not clustered but rather interspersed in the CS-E polysaccahride chains, being preferentially located in the highly sulfated sequences. The predominant structure on the nearest nonreducing side of a GlcA(3-O-sulfate) residue was GalNAc(4-O-sulfate) (80%), whereas that on the reducing side was GalNAc(4,6-O-disulfate) (59%). The structural variety in the vicinity of the GlcA(3-O-sulfate) residue might represent the substrate specificity of the unidentified chondroitin GlcA 3-O-sulfotransferase. The results also revealed a trisaccharide and a pentasaccahride sequence, both of which contained a beta-d-Glc branch at the C6 position of the constituent GalNAc residue. Approximately 5 mol % of all disaccharide units were substituted by Glc in the CS-E preparation used.     

Foraging behavior of Adélie penguins during incubation period in Lützow-Holm Bay

The diving behavior of Adélie penguins Pygoscelis adeliae was investigated using time–depth recorders during the incubation period in the fast sea-ice area of Lützow-Holm Bay, Antarctica. Dive profiles and activity/time allocation suggested that penguins were obligated to walk on the fast-ice for 90–100 km until a polynya, which they used as an access to the pack-ice zone. Dive depth did not differ between males and females, though males’ dive duration was longer than that of females. Dive depth was slightly shallower and dive duration was shorter during the incubation than during the chick-rearing phase. Birds dove throughout the day, although less frequently around midnight, and there was no clear diel change in dive depth. This daily dive pattern during incubation period was similar to that previously observed during the chick-rearing period in a fast sea-ice area, but differed from that observed in sea-ice-free area. Variations in diving behavior resulted from different environmental conditions, such as foraging area with different sea-ice condition, as well as from different life history strategies.     

<Emphasis Type="Italic">N</Emphasis>-acetylneuraminic acid coupled human recombinant TNFα exhibits enhanced anti-tumor activity against Meth-A fibrosarcoma and reduced toxicity

In order to study the effect of glycosylation on its biological activities and to develop tumor necrosis factor α (TNFα) with less deleterious effects, N-acetylneuraminic acid (NeuAc) with a C9 spacer was chemically coupled to human recombinant TNFα. NeuAc-coupled TNFα (NeuAc-TNFα) exhibited reduced activities in vitro by about threefold compared to native TNFα. In this study, we examined a variety of TNFα activities in vivo. NeuAc-TNFα reduced activities in the up-regulation of serum levels of IL-6 and NOx, but comparable activity as native TNFα in the down-regulation of the serum level of glucose. However, NeuAc-TNFα was more potent than TNFα in the up-regulation of the serum level of serum amyloid A (SAA). NeuAc-TNFα was less toxic to mice. In addition, NeuAc-TNFα exhibited an augmented anti-tumor activity against Meth-A fibrosarcoma without hemorrhagic necrosis. These results indicate that coupling with NeuAc enabled us to develop neoglycoTNFα with selective activities in vivo, including enhanced anti-tumor activity but reduced toxicity.     

Roles of RecA protein in spontaneous mutagenesis in Escherichia coli

To verify the extent of contribution of spontaneous DNA lesions to spontaneous mutagenesis, we have developed a new genetic system to examine simultaneously both forward mutations and recombination events occurring within about 600 base pairs of a transgenic rpsL target sequence located on Escherichia coli chromosome. In a wild-type strain, the recombination events were occurring at a frequency comparable to that of point mutations within the rpsL sequence. When the cells were UV-irradiated, the recombination events were induced much more sharply than point mutations. In a recA null mutant, no recombination event was observed. These data suggest that the blockage of DNA replication, probably caused by spontaneous DNA lesions, occurs often in normally growing E. coli cells and is mainly processed by cellular functions requiring the RecA protein. However, the recA mutant strain showed elevated frequencies of single-base frameshifts and large deletions, implying a novel mutator action of this strain. A similar mutator action of the recA mutant was also observed with a plasmid-based rpsL mutation assay. Therefore, if the recombinogenic problems in DNA replication are not properly processed by the RecA function, these would be a potential source for mutagenesis leading to single-base frameshift and large deletion in E. coli. Furthermore, the single-base frameshifts induced in the recA-deficient cells appeared to be efficiently suppressed by the mutS-dependent mismatch repair system. Thus, it seems likely that the single-base frameshifts are derived from slippage errors that are not directly caused by DNA lesions but made indirectly during some kind of error-prone DNA synthesis in the recA mutant cells.     

In vitro development of one-cell embryos from outbred mice: Influence of culture medium composition

Summary One-cell embryos from outbred mice (CF1, CD-1, and Dub:ICR) were cultured in various modifications of egg culture medium (ECM). The best development was observed in medium in which inorganic salts of modified T6 medium (mT6) replaced those of ECM. In this modification (TE), 66% of one-cell CF1 embryos developed into blastocysts, comared to 46 and 43% for ECM and mT6, respectively. Moreover, the cell numbers of blastocysts developing in TE (74.9±3.3) were higher than the cell numbers of those developing in ECM (55.1±2.4). The culture requirements of embryos varied between different stocks of mice: Fewer CF1 embryos developed to the blastocyst stage than either Dub:ICR embryos (90%) or CD-1 embryos (84%). Lowering the osmolarity of the medium from 300 to 280 mOsm, increasing the concentration of KC1 from 1.42 to 25 mM, or omitting lactate from the medium during Day 1 of culture did not further improve development of embryos, in contrast to previous reports. However, the time at which embryos were transferred to outgrowth medium influenced their postblastocyst development. The best development was observed when embryos were transferred on Day 4 of culture at the late morula-early blastocyst stage. This work was supported by the Office of Health and Environmental Research, U.S. Department of Energy, Washington, DC, contract DE-AC03-76-SF01012.