Mapping of a Region of the PA-X Protein of Influenza A Virus That Is Important for Its Shutoff Activity

Influenza A virus PA-X comprises an N-terminal PA endonuclease domain and a C-terminal PA-X-specific domain. PA-X reduces host and viral mRNA accumulation via its endonuclease function. Here, we found that the N-terminal 15 amino acids, particularly six basic amino acids, in the C-terminal PA-X-specific region are important for PA-X shutoff activity. These six basic amino acids enabled a PA deletion mutant to suppress protein expression at a level comparable to that of wild-type PA-X.     

CARTS biogenesis requires VAP–lipid transfer protein complexes functioning at the endoplasmic reticulum–Golgi interface

Vesicle-associated membrane protein–associated protein (VAP) is an endoplasmic reticulum (ER)-resident integral membrane protein that controls a nonvesicular mode of ceramide and cholesterol transfer from the ER to the Golgi complex by interacting with ceramide transfer protein and oxysterol-binding protein (OSBP), respectively. We report that VAP and its interacting proteins are required for the processing and secretion of pancreatic adenocarcinoma up-regulated factor, whose transport from the trans-Golgi network (TGN) to the cell surface is mediated by transport carriers called “carriers of the trans-Golgi network to the cell surface” (CARTS). In VAP-depleted cells, diacylglycerol level at the TGN was decreased and CARTS formation was impaired. We found that VAP forms a complex with not only OSBP but also Sac1 phosphoinositide phosphatase at specialized ER subdomains that are closely apposed to the trans-Golgi/TGN, most likely reflecting membrane contact sites. Immobilization of ER–Golgi contacts dramatically reduced CARTS production, indicating that association–dissociation dynamics of the two membranes are important. On the basis of these findings, we propose that the ER–Golgi contacts play a pivotal role in lipid metabolism to control the biogenesis of transport carriers from the TGN.     

Cascading effects of larval Crucian carp introduction on phytoplankton and microbial communities in a paddy field: top-down and bottom-up controls

Effects of fish predation propagate through aquatic food webs, where the classical grazing food chain and microbial loop are interwoven by trophic interactions. The overall impact on aquatic food webs is further complicated because fish may also exert bottom-up controls through nutrient regeneration. Yet, we still have limited information about cascading effects among fish, zooplankton, phytoplankton, and microbes. In this study, we performed a mesocosm experiment to evaluate effects of fish introduction on plankton communities. Six plots were set in factorial combination with fish introduction and rice straw plowing in a paddy field, and the experiment was continued for 4 weeks. Introduction of fish significantly increased chlorophyll a concentrations in smaller size fractions (<15 μm) and abundances of filamentous bacteria (>5 μm in length) and heterotrophic nanoflagellates in 3–15 μm fraction. Microbes in 0.8–3 μm fraction showed increasing but not significant trends in response to fish introduction. These results indicate cascading effects of fish predation operating via two pathways, one through grazing food chain and the other through microbial food web. Phytoplankton community compositions shifted in similar fashion in all plots until 1 week after fish introduction, and then diverged between plots with and without fish thereafter. Bottom-up effects of fish introduction were suggested by increases of total chlorophyll a and inedible phytoplankton species in response to fish introduction. This study provides an example of how fish predation regulates biomass and structure of phytoplankton and microbial communities.     

Novel and potent calcium-sensing receptor antagonists: discovery of (5R)-N-[1-ethyl-1-(4-ethylphenyl)propyl]-2,7,7-trimethyl-5-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide monotosylate (TAK-075) as an orally active bone anabolic agent

The calcium-sensing receptor antagonist (CaSR) has been recognized as a promising target of anabolic agents for treating osteoporosis. In the course of developing a new drug candidate for osteoporosis, we found tetrahydropyrazolopyrimidine derivative 1 to be an orally active CaSR antagonist that stimulated transient PTH secretion in rats. However, compound 1 showed poor physical and chemical stability. In order to work out this compound's chemical stability and further understand its in vivo efficacy, we focused on modifying the 2-position of the tetrahydropyrazolopyrimidine. As a result of chemical modification, we discovered (5R)-N-[1-ethyl-1-(4-ethylphenyl)propyl]-2,7,7-trimethyl-5-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide monotosylate 10m (TAK-075), which showed improved solubility, chemical stability, and in vivo efficacy. Furthermore, we describe that evaluating the active metabolite is important during repeated treatment with short-acting CaSR antagonists.     

ARHGAP18, a GTPase-activating protein for RhoA, controls cell shape, spreading, and motility

Rho GTPases are molecular switches that transmit biochemical signals in response to extracellular stimuli to elicit changes in the actin cytoskeleton. Rho GTPases cycle between an active, GTP-bound state and an inactive, GDP-bound state. These states are regulated by two distinct families of proteins-guanine nucleotide exchange factors and GTPase-activating proteins (GAPs). We studied the role of a previously uncharacterized GAP, ARHGAP18 (MacGAP). Overexpression of ARHGAP18 suppressed the activity of RhoA and disrupted stress fiber formation. Conversely, silencing of ARHGAP18 by small interfering RNA transfection-enhanced stress fiber formation and induced rounding of cells. We examined the role of ARHGAP18 in cell spreading and migration. Immunofluorescence analysis revealed that ARHGAP18 was localized to the leading edge during cell spreading and migration. ARHGAP18-knockdown cells showed impaired spreading, premature formation of stress fibers, and sustained activation of RhoA upon cell attachment. In addition, knockdown and overexpression of ARHGAP18 resulted in the inhibition and promotion of cell migration, respectively. Furthermore, ARHGAP18 was required for the polarization of cells for migration. Our results define ARHGAP18 as one of the crucial factors for the regulation of RhoA for the control of cell shape, spreading, and migration.     

The habitat disruption induces immune-suppression and oxidative stress in honey bees

The honey bee is a major insect used for pollination of many commercial crops worldwide. Although the use of honey bees for pollination can disrupt the habitat, the effects on their physiology have never been determined. Recently, honey bee colonies have often collapsed when introduced in greenhouses for pollination in Japan. Thus, suppressing colony collapses and maintaining the number of worker bees in the colonies is essential for successful long-term pollination in greenhouses and recycling of honey bee colonies. To understand the physiological states of honey bees used for long-term pollination in greenhouses, we characterized their gene expression profiles by microarray. We found that the greenhouse environment changes the gene expression profiles and induces immune-suppression and oxidative stress in honey bees. In fact, the increase of the number of Nosema microsporidia and protein carbonyl content was observed in honey bees during pollination in greenhouses. Thus, honey bee colonies are likely to collapse during pollination in greenhouses when heavily infested with pathogens. Degradation of honey bee habitat by changing the outside environment of the colony, during pollination services for example, imposes negative impacts on honey bees. Thus, worldwide use of honey bees for crop pollination in general could be one of reasons for the decline of managed honey bee colonies.     

Comparative proteomic analysis of Methanothermobacter themautotrophicus ΔH in pure culture and in co-culture with a butyrate-oxidizing bacterium

To understand the physiological basis of methanogenic archaea living on interspecies H(2) transfer, the protein expression of a hydrogenotrophic methanogen, Methanothermobacter thermautotrophicus strain ΔH, was investigated in both pure culture and syntrophic coculture with an anaerobic butyrate oxidizer Syntrophothermus lipocalidus strain TGB-C1 as an H(2) supplier. Comparative proteomic analysis showed that global protein expression of methanogen cells in the model coculture was substantially different from that of pure cultured cells. In brief, in syntrophic coculture, although methanogenesis-driven energy generation appeared to be maintained by shifting the pathway to the alternative methyl coenzyme M reductase isozyme I and cofactor F(420)-dependent process, the machinery proteins involved in carbon fixation, amino acid synthesis, and RNA/DNA metabolisms tended to be down-regulated, indicating restrained cell growth rather than vigorous proliferation. In addition, our proteome analysis revealed that α subunits of proteasome were differentially acetylated between the two culture conditions. Since the relevant modification has been suspected to regulate proteolytic activity of the proteasome, the global protein turnover rate could be controlled under syntrophic growth conditions. To our knowledge, the present study is the first report on N-acetylation of proteasome subunits in methanogenic archaea. These results clearly indicated that physiological adaptation of hydrogenotrophic methanogens to syntrophic growth is more complicated than that of hitherto proposed.     

Combination of hTERT and bmi-1, E6, or E7 induces prolongation of the life span of bone marrow stromal cells from an elderly donor without affecting their neurogenic potential

Murine bone marrow stromal cells differentiate not only into mesodermal derivatives, such as osteocytes, chondrocytes, adipocytes, skeletal myocytes, and cardiomyocytes, but also into neuroectodermal cells in vitro. Human bone marrow stromal cells are easy to isolate but difficult to study because of their limited life span. To overcome this problem, we attempted to prolong the life span of bone marrow stromal cells and investigated whether bone marrow stromal cells modified with bmi-1, hTERT, E6, and E7 retained their differentiated capability, or multipotency. In this study, we demonstrated that the life span of bone marrow stromal cells derived from a 91-year-old donor could be extended and that the stromal cells with an extended life span differentiated into neuronal cells in vitro. We examined the neuronally differentiated cells morphologically, physiologically, and biologically and compared the gene profiles of undifferentiated and differentiated cells. The neuronally differentiated cells exhibited characteristics similar to those of midbrain neuronal progenitors. Thus, the results of this study support the possible use of autologous-cell graft systems to treat central nervous system diseases in geriatric patients.