Hepatitis C Virus Impairs p53 via Persistent Overexpression of 3��-Hydroxysterol ��24-Reductase

Persistent infection with hepatitis C virus (HCV) induces tumorigenicity in hepatocytes. To gain insight into the mechanisms underlying this process, we generated monoclonal antibodies on a genome-wide scale against an HCV-expressing human hepatoblastoma-derived cell line, RzM6-LC, showing augmented tumorigenicity. We identified 3β-hydroxysterol Δ24-reductase (DHCR24) from this screen and showed that its expression reflected tumorigenicity. HCV induced the DHCR24 overexpression in human hepatocytes. Ectopic or HCV-induced DHCR24 overexpression resulted in resistance to oxidative stress-induced apoptosis and suppressed p53 activity. DHCR24 overexpression in these cells paralleled the increased interaction between p53 and MDM2 (also known as HDM2), a p53-specific E3 ubiquitin ligase, in the cytoplasm. Persistent DHCR24 overexpression did not alter the phosphorylation status of p53 but resulted in decreased acetylation of p53 at lysine residues 373 and 382 in the nucleus after treatment with hydrogen peroxide. Taken together, these results suggest that DHCR24 is elevated in response to HCV infection and inhibits the p53 stress response by stimulating the accumulation of the MDM2-p53 complex in the cytoplasm and by inhibiting the acetylation of p53 in the nucleus.     

Isolation of a Leptothrix Strain,OUMS1, from Ocherous Deposits in Groundwater

Leptothrix species in aquatic environments produce uniquely shaped hollow microtubules composed of aquatic inorganic and bacterium-derived organic hybrids. Our group termed this biologically derived iron oxide as “biogenous iron oxide (BIOX)”. The artificial synthesis of most industrial iron oxides requires massive energy and is costly while BIOX from natural environments is energy and cost effective. The BIOX microtubules could potentially be used as novel industrial functional resources for catalysts, adsorbents and pigments, among others if effective and efficient applications are developed. For these purposes, a reproducible system to regulate bacteria and their BIOX productivity must be established to supply a sufficient amount of BIOX upon industrial demand. However, the bacterial species and the mechanism of BIOX microtubule formation are currently poorly understood. In this study, a novel Leptothrix sp. strain designated OUMS1 was successfully isolated from ocherous deposits in groundwater by testing various culture media and conditions. Morphological and physiological characters and elemental composition were compared with those of the known strain L. cholodnii SP-6 and the differences between these two strains were shown. The successful isolation of OUMS1 led us to establish a basic system to accumulate biological knowledge of Leptothrix and to promote the understanding of the mechanism of microtubule formation. Additional geochemical studies of the OUMS1-related microstructures are expected provide an attractive approach to study the broad industrial application of bacteria-derived iron oxides.     

Structural basis of cargo recognition by the myosin-X MyTH4-FERM domain

Myosin-X is an important unconventional myosin that is critical for cargo transportation to filopodia tips and is also utilized in spindle assembly by interacting with microtubules. We present a series of structural and biochemical studies of the myosin-X tail domain cassette, consisting of myosin tail homology 4 (MyTH4) and FERM domains in complex with its specific cargo, a netrin receptor DCC (deleted in colorectal cancer). The MyTH4 domain is folded into a helical VHS-like structure and is associated with the FERM domain. We found an unexpected binding mode of the DCC peptide to the subdomain C groove of the FERM domain, which is distinct from previously reported β-β associations found in radixin-adhesion molecule complexes. We also revealed direct interactions between the MyTH4-FERM cassette and tubulin C-terminal acidic tails, and identified a positively charged patch of the MyTH4 domain, which is involved in tubulin binding. We demonstrated that both DCC and integrin bindings interfere with microtubule binding and that DCC binding interferes with integrin binding. Our results provide the molecular basis by which myosin-X facilitates alternative dual binding to cargos and microtubules.     

Efflux of sphingomyelin, cholesterol, and phosphatidylcholine by ABCG1

Cholesterol and phospholipids are essential to the body, but an excess of cholesterol or lipids is toxic and a risk factor for arteriosclerosis. ABCG1, one of the half-type ABC proteins, is thought to be involved in cholesterol homeostasis. To explore the role of ABCG1 in cholesterol homeostasis, we examined its subcellular localization and function. ABCG1 and ABCG1-K120M, a WalkerA lysine mutant, were localized to the plasma membrane in HEK293 cells stably expressing ABCG1 and formed a homodimer. A stable transformant expressing ABCG1 exhibited efflux of cholesterol and choline phospholipids in the presence of BSA, and the cholesterol efflux was enhanced by the presence of HDL, whereas cells expressing ABCG1-K120M did not, suggesting that ATP binding and/or hydrolysis is required for the efflux. Mass and TLC analyses revealed that ABCG1 and ABCA1 secrete several species of sphingomyelin (SM) and phosphatidylcholine (PC), and SMs were preferentially secreted by ABCG1, whereas PCs were preferentially secreted by ABCA1. These results suggest that ABCA1 and ABCG1 mediate the lipid efflux in different mechanisms, in which different species of phospholipids are secreted, and function coordinately in the removal of cholesterol and phospholipids from peripheral cells.     

24(S)‐hydroxycholesterol is actively eliminated from neuronal cells by ABCA1

High cholesterol turnover catalyzed by cholesterol 24‐hydroxylase is essential for neural functions, especially learning. Because 24(S)‐hydroxycholesterol (24‐OHC), produced by 24‐hydroxylase, induces apoptosis of neuronal cells, it is vital to eliminate it rapidly from cells. Here, using differentiated SH‐SY5Y neuron‐like cells as a model, we examined whether 24‐OHC is actively eliminated via transporters induced by its accumulation. The expression of ABCA1 and ABCG1 was induced by 24‐OHC, as well as TO901317 and retinoic acid, which are ligands of the nuclear receptors liver X receptor/retinoid X receptor (LXR/RXR). When the expression of ABCA1 and ABCG1 was induced, 24‐OHC efflux was stimulated in the presence of high‐density lipoprotein (HDL), whereas apolipoprotein A‐I was not an efficient acceptor. The efflux was suppressed by the addition of siRNA against ABCA1, but not by ABCG1 siRNA. To confirm the role of each transporter, we analyzed human embryonic kidney 293 cells stably expressing human ABCA1 or ABCG1; we clearly observed 24‐OHC efflux in the presence of HDL, whereas efflux in the presence of apolipoprotein A‐I was marginal. Furthermore, the treatment of primary cerebral neurons with LXR/RXR ligands suppressed the toxicity of 24‐OHC. These results suggest that ABCA1 actively eliminates 24‐OHC in the presence of HDL as a lipid acceptor and protects neuronal cells.     

Assessment of sustainable development based on the capital approach

This study analyzes countries’ sustainability conditions using panel data of genuine savings (GS) by calculating the average, trend, and stability of the countries’ GS flow. We identify the pattern of GS changes in the past three decades on the basis of these three capital flow measures and the level of capital stock (wealth), and subsequently identify the factors that determine the GS change patterns. We find that the quality of institutional and population growth, along with natural resource abundance, substantially influences capital accumulation in the long term. In particular, our results indicate that only once does a good performance of GS not ensure sustainable development. For example, countries like Kenya performed well in terms of capital accumulation in the first period, but this performance was not maintained in the following period, as a result of the poor quality of institutions and governance. On the contrary, some countries, like Chile, where GS performance was poor in the beginning, eventually achieve a sustained pathway if they have good institutions and governance. In addition, we suggest that even countries in the best performing group for capital change are on an unsustainable path, which will eventually start decreasing in wealth. The exception is Norway, which is socially well developed, but has a lower population density and abundant natural resources. Our study proves that analyzing relevant indicators in the long term is crucial to understanding sustainability.     

A Docosahexaenoic Acid-Derived Pro-resolving Agent,Maresin 1, Protects Motor Neuron Cells Death

Maresin 1 is a novel pro-resolving mediator derived from docosahexaenoic acid (DHA), with potent anti-inflammation effects against several animal models, including brain ischemia, sepsis, and lung fibrosis. However, its effect against motor neuron cell death is still not investigated. Therefore, we investigated the effects of maresin 1 on several stress-induced motor neuron cell death. Maresin 1 suppressed combinatorial stress which was evoked by superoxide dismutase 1 (SOD1)^G93A and serum-free, -induced motor neuron cells death in a concentration-dependent manner, and had a stronger neuroprotective effective than DHA. Maresin 1 also had neuroprotective effects against transactivation response DNA-binding protein (TDP)-43^A315T and serum-free stress, H₂O₂, and tunicamycin-induced cell death. Maresin 1 reduced the reactive oxygen species (ROS) production caused by SOD1^G93A or TDP-43^A315T. Moreover, maresin 1 suppressed the NF-κB activation induced by SOD1^G93A and serum-free stress. These data indicate that maresin 1 has motor neuron protective effects against several stresses by reduction of ROS production or attenuation of the NF-κB activation. Maresin 1 also had neuroprotective effects against H₂O₂, and tunicamycin-induced cell death in a concentration-dependent manner. Finally, maresin 1 ameliorated the motor function deficits of spinal muscular atrophy model in which endoplasmic reticulum stress was upregulated. Thus, maresin 1 may be beneficial to protect against motor neuron diseases.     

Physiological Characterization of RNA-Binding Protein-Deficient Cells from Synechococcus sp. Strain PCC7942

The unicellular cyanobacteria, Synechococcus sp. strains PCC7942and PCC6301, have two small RNA-binding proteins, Rbp1 and Rbp2.In this study, native gel electrophoresis of the nuclease-treatedSynechococcus cell extracts showed that both Rbps are associatedin vivo with RNA but not with DNA. This indicates that theyare bona fide RNA-binding proteins. To address the functionof Rbps, we have characterized the mutants deficient in Rbp1or Rbp2. The Rbp1 deficient cells showed the same growth curve,cell color and cell viability as the wild-type strain at 30°C.The Rbp2-less mutant also grew well as wild-type but exhibiteda yellow-green color, and its cell viability was significantlyreduced. On exposure of the Rbp1-deficient mutant cells to atemperature of 10°C for one week, cell viability was completelylost. Western blot analysis showed that Rbp1 increases onlyin response to a temperature shift from 30 to 10°C, whereasRbp2 accumulates at a constant rate at cold temperature. Interestingly,translation elongation factor Tu was significantly decreasedin Rbp2-deficient cells but not in Rbp1-deficient cells. Thus,each Rbp appears to have a distinct role in cellular function. (Received June 28, 1999; Accepted September 24, 1999)