Maintenance of Bone Homeostasis by DLL1‐Mediated Notch Signaling

Adult bone mass is maintained through a balance of the activities of osteoblasts and osteoclasts. Although Notch signaling has been shown to maintain bone homeostasis by controlling the commitment, differentiation, and function of cells in both the osteoblast and osteoclast lineages, the precise mechanisms by which Notch performs such diverse and complex roles in bone physiology remain unclear. By using a transgenic approach that modified the expression of delta‐like 1 (DLL1) or Jagged1 (JAG1) in an osteoblast‐specific manner, we investigated the ligand‐specific effects of Notch signaling in bone homeostasis. This study demonstrated for the first time that the proper regulation of DLL1 expression, but not JAG1 expression, in osteoblasts is essential for the maintenance of bone remodeling. DLL1‐induced Notch signaling was responsible for the expansion of the bone‐forming cell pool by promoting the proliferation of committed but immature osteoblasts. However, DLL1‐Notch signaling inhibited further differentiation of the expanded osteoblasts to become fully matured functional osteoblasts, thereby substantially decreasing bone formation. Osteoblast‐specific expression of DLL1 did not alter the intrinsic differentiation ability of cells of the osteoclast lineage. However, maturational arrest of osteoblasts caused by the DLL1 transgene impaired the maturation and function of osteoclasts due to a failed osteoblast‐osteoclast coupling, resulting in severe suppression of bone metabolic turnover. Taken together, DLL1‐mediated Notch signaling is critical for proper bone remodeling as it regulates the differentiation and function of both osteoblasts and osteoclasts. Our study elucidates the importance of ligand‐specific activation of Notch signaling in the maintenance of bone homeostasis. J. Cell. Physiol. 232: 2569–2580, 2017. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals Inc.     

Genome 3'-end repair in dengue virus type 2

Genomes of RNA viruses encounter a continual threat from host cellular ribonucleases. Therefore, viruses have evolved mechanisms to protect the integrity of their genomes. To study the mechanism of 3′-end repair in dengue virus-2 in mammalian cells, a series of 3′-end deletions in the genome were evaluated for virus replication by detection of viral antigen NS1 and by sequence analysis. Limited deletions did not cause any delay in the detection of NS1 within 5 d. However, deletions of 7–10 nucleotides caused a delay of 9 d in the detection of NS1. Sequence analysis of RNAs from recovered viruses showed that at early times, virus progenies evolved through RNA molecules of heterogeneous lengths and nucleotide sequences at the 3′ end, suggesting a possible role for terminal nucleotidyl transferase activity of the viral polymerase (NS5). However, this diversity gradually diminished and consensus sequences emerged. Template activities of 3′-end mutants in the synthesis of negative-strand RNA in vitro by purified NS5 correlate well with the abilities of mutant RNAs to repair and produce virus progenies. Using the Mfold program for RNA structure prediction, we show that if the 3′ stem–loop (3′ SL) structure was abrogated by mutations, viruses eventually restored the 3′ SL structure. Taken together, these results favor a two-step repair process: non-template-based nucleotide addition followed by evolutionary selection of 3′-end sequences based on the best-fit RNA structure that can support viral replication.     

Fungal colonization and decomposition of Castanopsis sieboldii leaves in a subtropical forest

Fungi play a crucial role in the decomposition of lignin in fallen leaves but few studies have examined the functional roles of ligninolytic fungi associated with the decomposition of fallen leaves on tropical forest soils. This study examined fungal populations responsible for lignin decomposition in Castanopsis sieboldii leaves in a subtropical evergreen broad-leaved forest in southern Japan. Fallen leaves of C. sieboldii are characterized by the occurrence of bleached portions attributable to fungal colonization of leaf tissues and decomposition of lignin. The bleached area accounted for 29.7%, on average, of the total area of C. sieboldii fallen leaves in the study site. Leaf mass per unit area (LMA) and lignin content were lower in the bleached area than in the surrounding nonbleached area of the same leaves, indicating that removal of lignin enhanced mass loss from leaf tissues and created small-scale heterogeneity of decomposition within single leaves. An unidentified species of Lachnocladiaceae (Basidiomycetes) was isolated frequently from the bleached area and caused selective decomposition of lignin in leaves under pure culture conditions, indicating that this fungus was responsible for the bleaching. The greater hyphal length of basidiomycetes in the bleached area than in the nonbleached area supported the finding that this Lachnocladiaceae sp. was associated with the bleaching. The relatively rapid decomposition of C. sieboldii leaves on the subtropical forest soil is partly attributable to colonization of the litter by this Lachnocladiaceae sp.     

ATP-Dependent but Proton Gradient-Independent Polyphosphate-Synthesizing Activity in Extraradical Hyphae of an Arbuscular Mycorrhizal Fungus

Arbuscular mycorrhizal (AM) fungi benefit their host plants by supplying phosphate obtained from the soil. Polyphosphate is thought to act as the key intermediate in this process, but little is currently understood about how polyphosphate is synthesized or translocated within arbuscular mycorrhizas. Glomus sp. strain HR1 was grown with marigold in a mesh bag compartment system, and extraradical hyphae were harvested and fractionated by density gradient centrifugation. Using this approach, three distinct layers were obtained: layers 1 and 2 were composed of amorphous and membranous materials, together with mitochondria, lipid bodies, and electron-opaque bodies, and layer 3 was composed mainly of partially broken hyphae and fragmented cell walls. The polyphosphate kinase/luciferase system, a highly sensitive polyphosphate detection method, enabled the detection of polyphosphate-synthesizing activity in layer 2 in the presence of ATP. This activity was inhibited by vanadate but not by bafilomycin A₁ or a protonophore, suggesting that ATP may not energize the reaction through H⁺-ATPase but may act as a direct substrate in the reaction. This report represents the first demonstration that AM fungi possess polyphosphate-synthesizing activity that is localized in the organelle fraction and not in the cytosol or at the plasma membrane.Arbuscular mycorrhizal (AM) fungi are obligate biotrophs that form symbiotic associations with most land plants (29). These fungi promote the growth of host plants via enhanced uptake of phosphate (P_i) and thus play important roles in the terrestrial phosphorus cycle. In the symbiotic phase, AM fungi take up P_i from soil through an extensive network of extraradical hyphae and rapidly accumulate inorganic polyphosphate (polyP). This accumulation was as rapid as that for a polyP-hyperaccumulating bacterium found in activated sludge (6). PolyP is a linear polymer of three to hundreds of molecules of P_i linked by high-energy phosphoanhydride bonds and has been found across all classes of organisms (19). Although polyP is considered to play a central role in long-distance translocation of P_i in AM fungal associations (4, 10, 30, 31), the translocation mechanism, metabolism, and dynamics in the fungi have not been elucidated due to the difficulty in obtaining sufficient fungal material for analysis.Many enzymes/genes involved in polyP synthesis/metabolism have been identified and characterized in prokaryotes (19). For instance, exopolyphosphatase hydrolyzes the terminal high-energy bonds of polyP, and polyphosphate glucokinase (PPGK) transfers the terminal P_i residue to glucose. Polyphosphate kinase 1 (PPK1) is responsible both for polyP synthesis, using ATP as a phosphoryl donor, and for the reverse ATP-generating reaction. This enzyme is bound to the plasma membrane (18) and has been found in a wide range of bacteria (17). Unlike the case for prokaryotes, knowledge of polyP synthesis/metabolism in eukaryotes remains limited. The first eukaryotic PPK genes, DdPPK1 (32) and DdPPK2 (14), were identified from the social slime mold Dictyostelium discoideum. The products of these genes, as known for bacterial PPK1s, are responsible both for polyP synthesis and for the ATP-generating reaction and have been suggested to be associated with vacuoles or small vesicles (14, 32). Although several homologues of bacterial PPK1 genes have now been found in the genomes of eukaryotic microorganisms (17), yeast Candida humicola is the only organism apart from D. discoideum for which PPK-like activity has been confirmed (22). The model organism Saccharomyces cerevisiae is known to accumulate polyP, to up to 10% of its dry weight (19). A unique polyP synthetic pathway different from those of PPK1 has been proposed for S. cerevisiae based on the observation that vacuolar-type H⁺-ATPase (V-ATPase)-defective mutants could not accumulate polyP (23). In this hypothetical pathway, P_i would be polymerized by an analogous system (enzyme) of mitochondrial F₁-ATPase on the vacuolar membrane, using the proton motive force created by V-ATPase (23). On the other hand, Hothorn et al. (16) demonstrated very recently that vacuolar transporter chaperone 4 (VTC4), a small transmembrane protein associated with the membrane, polymerizes P_i by using the γ-P_i residue of ATP as a phosphoryl donor in S. cerevisiae.More than 2 decades ago, Capaccio and Callow (3) reported the presence of polyP-hydrolyzing, -metabolizing (PPGK), and -synthesizing (PPK-like) activities in the soluble (cytosolic) fractions of the hyphae of the AM fungus Glomus mosseae. Recently, polyP-hydrolyzing activity was found in both the cytosolic and insoluble (membrane) fractions and then characterized (8). PPGK activity has also been confirmed in the cytosolic fraction, although the activity was quite low and hexokinase (ATP-hexose phosphotransferase) activity appeared to dominate in the glucose phosphorylation process (9). PPK-like activity, however, could not be detected in the same fraction (10), and this seems likely because all other prokaryotic (reviewed in reference 17) and eukaryotic (14, 16, 22, 32) polyP-synthesizing enzymes, so far, are associated with membranes. These observations suggest that AM fungi possess a polyP-synthesizing enzyme that is probably associated with membranes and that ATP may be essential in the synthesis as a phosphoryl donor or via H⁺-ATPase, as suggested by Ogawa et al. (23). In this study, a cell fractionation technique was applied to demonstrate polyP-synthesizing activity in an AM fungus, and then the role of ATP in the synthesis was investigated.     

Dynamics of cell wall components of Magnaporthe grisea during infectious structure development

Oligosaccharides derived from cell wall of fungal pathogens induce host primary immune responses. To understand fungal strategies circumventing the host plant immune responses, cell wall polysaccharide localization was investigated using fluorescent labels during infectious structure differentiation in the rice blast fungus Magnaporthe grisea . α-1,3-glucan was labelled only on appressoria developing on plastic surfaces, whereas it was detected on both germ tubes and appressoria on plant surfaces. Chitin, chitosan and β-1,3-glucan were detected on germ tubes and appressoria regardless of the substrate. Major polysaccharides labelled at accessible surface of infectious hyphae were α-1,3-glucan and chitosan, but after enzymatic digestion of α-1,3-glucan, β-1,3-glucan and chitin became detectable. Immunoelectron microscopic analysis showed α-1,3-glucan and β-1,3-glucan intermixed in the cell wall of infectious hyphae; however, α-1,3-glucan tended to be distributed farther from the fungal cell membrane. The fungal cell wall became more tolerant to chitinase digestion upon accumulation of α-1,3-glucan. Accumulation of α-1,3-glucan was dependent on the Mps1 MAP kinase pathway, which was activated by a plant wax derivative, 1,16-hexadecanediol. Taken together, α-1,3-glucan spatially and functionally masks β-1,3-glucan and chitin in the cell wall of infectious hyphae. Thus, a dynamic change of composition of cell wall polysaccharides occurs during plant infection in M. grisea .     

Tonometric biosensor with a differential pressure sensor for chemo-mechanical measurement of glucose

A tonometric biosensor for glucose was constructed using a chemo-mechanical reaction unit and a differential pressure sensor. The reaction unit was fabricated by using both liquid and gas cells separated by an enzyme diaphragm membrane, in which glucose oxidase was immobilized onto the single (gas cell) side of the dialysis membrane. By applying glucose solution (0, 25.0, 50.0, 100, 150 and 200 mmol/l) into the liquid cell of the chemo-mechanical reaction unit, the pressure in the gas cell decreased continuously with a steady de-pressure slope because the oxygen consumption in the gas cell was induced by the glucose oxidase (GOD) enzyme reaction at the enzyme side of the porous diaphragm membrane. The steady de-pressure slope in the gas cell showed the linear relationship with the glucose concentration in the liquid cell between 25.0 and 200.0 mmol/l (correlation coefficient of 0.998). A substrate regeneration cycle coupling GOD with l-ascorbic acid (AsA: 0, 1.0, 3.0, 10.0 and 50.0 mmol/l; as reducing reagent system) was applied to the chemo-mechanical reaction unit in order to amplify the output signal of the tonometric biosensor. 3.0 mmol/l concentration of AsA could optimally amplify the sensor signal more than 2.5 times in comparison with that of non-AsA reagent.     

Discovery and biological profile of isoindolinone derivatives as novel metabotropic glutamate receptor 1 antagonists: A potential treatment for psychotic disorders

We describe here the discovery and biological profile of a series of isoindolinone derivatives as developed mGluR1 antagonists. Our combined strategy of rapid parallel synthesis and conventional medicinal optimization successfully led to N-cyclopropyl 22 and N-isopropyl isoindolinone analogs 21 and 23 with improved in vivo DMPK profiles. Moreover the most advanced analog 23 showed an oral antipsychotic-like effect at a dose of 1 mg/kg in an animal model.