Cell surface-expressed moesin-like HDL/apoA-I binding protein promotes cholesterol efflux from human macrophages

HDL and its major component, apolipoprotein A-I (apoA-I), play a central role in reverse cholesterol transport. We recently reported the involvement of a glycosylphosphatidylinositol anchor (GPI anchor) in the binding of HDL and apoA-I on human macrophages, and purified an 80 kDa HDL/apoA-I binding protein. In the present study, we characterized the GPI-anchored HDL/apoA-I binding protein from macrophages. The HDL/apoA-I binding protein was purified from macrophages and digested with endopeptidase, and the resultant fragments were sequenced. Cholesterol efflux, flow cytometry, immunoblotting, and immunohistochemical analyses were performed to characterize the HDL/apoA-I binding protein. Two parts of seven amino acid sequences completely matched those of moesin. Flow cytometry, immunoblotting, and immunohistochemistry using anti-moesin antibody showed that the HDL/apoA-I binding protein was N-glycosylated and expressed on the cell surface. It was termed moesin-like protein. Treatment of macrophages with anti-moesin antibody blocked the binding of HDL/apoA-I and suppressed cholesterol efflux. The moesin-like protein was exclusively expressed on macrophages and was upregulated by cholesterol loading and cell differentiation. Our results indicate that the moesin-like HDL/apoA-I binding protein is specifically expressed on the surface of human macrophages and promotes cholesterol efflux from macrophages.-Matsuyama, A, N. Sakai, H. Hiraoka, K-i. Hirano, and S. Yamashita. Cell surface-expressed moesin-like HDL/apoA-I binding protein promotes cholesterol efflux from human macrophages.     

Expansion of trait of gibberellin-induced dormancy in the Tertiary relict species of the genus Dioscorea (Dioscoreaceae)

Effects of the application of gibberellin A₃ (GA₃) on sprouting of rhizomes and germination of seeds were tested in five species of the genus Dioscorea which are considered to be Tertiary relicts growing disjunctively in the Appalachians, the Balkans, and the Caucasus. Applied GA₃ at higher than 1 micro;M markedly inhibited sprouting of rhizomes of all species. However, inhibition of seed germination of these species by GA₃ application was not observed. It is hypothesized that before disjunction in the Tertiary period GA-induced dormancy probably prevailed in rhizomes of all members of the genus Dioscorea.     

The Effects of Various Ions on Resting and Spike Potentials of Barnacle Muscle Fibers

Effects of monovalent cations and some anions on the electrical properties of the barnacle muscle fiber membrane were studied when the intra- or extracellular concentrations of those ions were altered by longitudinal intra-cellular injection. The resting potential of the normal fiber decreases linearly with increase of logarithm of [K⁺]_out and the decrement for a tenfold increase in [K⁺]_out is 58 mv when the product, [K⁺]_out ·[Cl^-]_out, is kept constant. It also decreases with decreasing [K⁺]_in but is always less than expected theoretically. The deviation becomes larger as [K⁺]_in increases and the resting potential finally starts to decrease with increasing [K⁺]_in for [K⁺]_in > 250 mM. When the internal K⁺ concentration is decreased the overshoot of the spike potential increases and the time course of the spike potential becomes more prolonged. In substituting for the internal K⁺, Na⁺ and sucrose affect the resting and spike potentials similarly. Some organic cations (guanidine, choline, tris, and TMA) behave like sucrose while some other organic cations (TEA, TPA, and TBA) have a specific effect and prolong the spike potential if they are applied intracellularly or extracellularly. In all cases the active membrane potential increases linearly with the logarithm of [Ca⁺⁺]_out/[K⁺]_in and the increment is about 29 mv for tenfold increase in this ratio. The fiber membrane is permeable to Cl^- and other smaller anions (Br^- and I^-) but not to acetate^- and larger anions (citrate^-, sulfate^-, and methanesulfonate^-).     

Receptive field mechanisms of ganglion cells in the cat retina

On the basis of anatomical and physiological results of the vertebrate retina, a method is proposed for analysing the respective fields of ganglion cells in the cat retina. In the model, we assume the following: (a) Ganglion cells receive their input from bipolar and/or amacrine cells. (b) The nonlinearity of ganglion cell responses is due to the activities of transient type amacrine cells. The method has been proved to be effective. According to the results of this investigation, the receptive field properties of X type and Y type ganglion cells are heterogeneous. Thus, it may be considered that their receptive fields consist of center and surround mechanisms. The receptive field properties of X-cells are almost linear and the X-cells seem to receive most of their input from bipolar cells. On the other hand, the ones of Y-cells are highly nonlinear. Consequently, it is conceivable that the Y-cells receive their input mainly from transient type amacrine cells.     

Changes in expression of inhibin subunits in the cyclic golden hamster (Mesocricetus auratus) and the regulation of inhibin alpha subunit production by luteinizing hormone

In the present study, changes in localization of each inhibin subunit in the ovary were investigated during the estrous cycle of the golden hamster. The effect of LH surge on changes in localization in inhibin alpha subunit in the ovary was also investigated. Inhibin alpha subunit was localized in granulosa cells of various stages of follicles throughout the estrous cycle. Inhibin alpha subunit was also present in numerous interstitial cells on days 1 and 2 (day 1 = day of ovulation), but the number of positive interstitial cells was fewer on days 3 and almost disappeared on day 4 of the estrous cycle. Newly formed luteal cells were also positive for inhibin alpha subunit on days 1 and 2. On the other hand, positive reactions for inhibin beta A and beta B subunits were only present in the granulosa cells of healthy antral follicles. However, a positive reaction for inhibin beta B subunit in peripheral mural granulosa cells disappeared on days 3 and 4 of the estrous cycle. Treatment with LHRH-AS at 1100 h on day 4 completely blocked the luteinizing hormone (LH) surge and ovulation, although relatively high concentrations of plasma follicle-stimulating hormone (FSH) were maintained throughout the experiment. There were few positive reactions for inhibin alpha subunit in theca and interstitial cells 24 hr after LHRH-AS injection. The effect of LHRH-AS treatment was blocked by a single injection of 10 IU human chorionic gonadotropin. These results suggest that the major source of dimeric inhibin in the cyclic hamster was granulosa cells of healthy antral follicles. Different distribution pattern of inhibin beta A from beta B subunits in large antral follicles on days 3 and 4 of the estrous cycle suggests different secretion patterns of inhibin A from B on these days. Furthermore, the LH surge may be an important factor to induce production of inhibin alpha subunit in interstitial cells of the cyclic hamster.     

Binding of AlF-C, an Orc1-binding transcriptional regulator, enhances replicator activity of the rat aldolase B origin

A region encompassing the rat aldolase B gene (aldB) promoter acts as a chromosomal origin of DNA replication (origin) in rat aldolase B-nonexpressing hepatoma cells. To examine replicator function of the aldB origin, we constructed recombinant mouse cell lines in which the rat aldB origin and the mutant derivatives were inserted into the same position at the mouse chromosome 8 by cre-mediated recombination. Nascent strand abundance assays revealed that the rat origin acts as a replicator at the ectopic mouse locus. Mutation of site C in the rat origin, which binds an Orc1-binding protein AlF-C in vitro, resulted in a significant reduction of the replicator activity in the mouse cells. Chromatin immunoprecipitation (ChIP) assays indicated that the reduction of replicator activity was paralleled with the reduced binding of AlF-C and Orc1, suggesting that sequence-specific binding of AlF-C to the ectopic rat origin leads to enhanced replicator activity in cooperation with Orc1. Involvement of AlF-C in replication in vivo was further examined for the aldB origin at its original rat locus and for a different rat origin identified in the present study, which contained an AlF-C-binding site. ChIP assays revealed that both replication origins bind AlF-C and Orc1. We think that the results presented here may represent one mode of origin recognition in mammalian cells.     

Characterization of Tudor-sn-containing granules in the silkworm,Bombyx mori

The Tudor-sn protein, which contains four staphylococcal nuclease domains and a Tudor domain, is a ubiquitous protein found in almost all organisms. It has been reported that Tudor-sn in mammals participates in various cellular pathways involved in gene regulation, cell growth, and development. In insects, we have previously identified a Tudor-sn ortholog in the silkworm, Bombyx mori, and detected its interactions between with Argonaute proteins. The role of Tudor-sn in silkworm, however, still remains largely unknown. In this study, we demonstrated that silkworm Tudor-sn is a stress granule (SG) protein, and determined its interactions with other SG proteins using Bimolecular Fluorescence Complementation assay and Insect Two-Hybrid method. Depletions of Argonaute proteins and SG-marker protein Tia1 by RNAi impaired the involvement of Tudor-sn in the SG formation. Protein domain deletion analysis of Tudor-sn demonstrated that SN2 is the key domain required for the aggregation of Tudor-sn in SGs.     

Naked gene therapy of hepatocyte growth factor for dextran sulfate sodium-induced colitis in mice

Ulcerative colitis (UC) is progressive and relapsing disease. To explore the therapeutic effects of naked gene therapy of hepatocyte growth factor (HGF) on UC, the SRalpha promoter driving HGF gene was intrarectally administered to the mice in which colitis was induced by dextran sulfate sodium (DSS). Expression of the transgene was seen in surface epithelium, lamina propria, and muscularis mucosae. The HGF-treated mice showed reduced colonic mucosal damage and increased body weights, compared with control mice (P < 0.01 and P < 0.05, respectively). The HGF-treated mice displayed increased number of PCNA-positive cells and decreased number of apoptotic cells than in control mice (P < 0.01, each). Phosphorylated AKT was dramatically increased after HGF gene administration, however, phosphorylated ERK1/2 was not altered. Microarray analysis revealed that HGF induced expression of proliferation- and apoptosis-associated genes. These data suggest that naked HGF gene delivery causes therapeutic effects through regulation of many downstream genes.     

Molecular Conformation of 2′-Deoxy-2′-methylidene-cytidine: A Potent Antineoplastic Nucleoside

Abstract

2′-Deoxy-2′-methylidenecytidine (DMDC), a potent inhibitor of the growth of tumor cells, was crystallized with two different forms. One is dihydrated (DMDC·2H₂O) and the other is its hydrochloride salt (DMDC·HCLl). Both crystal and molecular structures have been determined by the X-ray diffraction method. In both forms the glycosidic and sugar conformations are anti and C(4′)-exo, respectively, whereas the conformation about the exocyclic bond is trans for DMDC·2H₂O and gauche ⁺ for DMDC·HCl. Proton nuclear magnetic resonance data of DMDC indicate a preference for the anti C(4′)-exo conformation found in the solid state. These molecular conformations were compared with the related pyrimidine nucleosides. When the cytosine bases are brought into coincidence, DMDC displays the exocyclic C(4′)-C(5′) bond located on the very close position to those of pyrimidine nucleosides with typical overall conformations. On the other hand, the hydroxyl O(3′)-H groups are separated by ca. 3 Å in the cases of DMDC and other pyrimidine nucleosides which have the C(2′)-endo sugar conformation. This result may be useful for the implication about the mechanism of the biological activity of DMDC.