The adipocyte-derived hormone adiponectin has been proposed to play important roles in the regulation of energy homeostasis and insulin sensitivity, and it has been reported to exhibit putative antiatherogenic properties in vitro. In this study we generated adiponectin-deficient mice to directly investigate whether adiponectin has a physiological protective role against diabetes and atherosclerosis in vivo. Heterozygous adiponectin-deficient (adipo ؉/؊ ) mice showed mild insulin resistance, while homozygous adiponectin-deficient (adipo ؊/؊ ) mice showed moderate insulin resistance with glucose intolerance despite body weight gain similar to that of wild-type mice. Moreover, adipo ؊/؊ mice showed 2-fold more neointimal formation in response to external vascular cuff injury than wild-type mice (p ‫؍‬ 0.01). This study provides the first direct evidence that adiponectin plays a protective role against insulin resistance and atherosclerosis in vivo.

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Mice with a targeted mutation of the gastric inhibitory polypeptide (GIP) receptor gene (GIPR) were generated to determine the role of GIP as a mediator of signals from the gut to pancreatic ␤ cells. GIPR؊͞؊ mice have higher blood glucose levels with impaired initial insulin response after oral glucose load. Although blood glucose levels after meal ingestion are not increased by high-fat diet in GIPR؉͞؉ mice because of compensatory higher insulin secretion, they are significantly increased in GIPR؊͞؊ mice because of the lack of such enhancement. Accordingly, early insulin secretion mediated by GIP determines glucose tolerance after oral glucose load in vivo, and because GIP plays an important role in the compensatory enhancement of insulin secretion produced by a high insulin demand, a defect in this entero-insular axis may contribute to the pathogenesis of diabetes.

Hepatitis E virus (HEV) is a causative agent of acute hepatitis. The crystal structure of HEV-like particles (HEV-LP) consisting of capsid protein was determined at 3.5-Å resolution. The capsid protein exhibited a quite different folding at the protruding and middle domains from the members of the families of Caliciviridae and Tombusviridae, while the shell domain shared the common folding. Tyr-288 at the 5-fold axis plays key roles in the assembly of HEV-LP, and aromatic amino acid residues are well conserved among the structurally related viruses. Mutational analyses indicated that the protruding domain is involved in the binding to the cells susceptive to HEV infection and has some neutralization epitopes. These structural and biological findings are important for understanding the molecular mechanisms of assembly and entry of HEV and also provide clues in the development of preventive and prophylactic measures for hepatitis E.capsid ͉ HEV ͉ VLP

The homeostasis of the plasma phosphate level is essential for many biological processes including skeletal mineralization. The reabsorption of phosphate in the kidney is a major determinant of the plasma levels of phosphate. Phosphatonin is a hormone-like factor that specifically inhibits phosphate uptake in renal proximal epithelial cells. Recent studies on tumor-induced osteomalacia suggested that phosphatonin was potentially identical to fibroblast growth factor (FGF)-23. However, as purified recombinant FGF-23 could not inhibit phosphate uptake in renal proximal epithelial cells, the mechanism of action of FGF-23 remains to be elucidated. Therefore, we examined the mechanism of action of FGF-23 in cultured renal proximal epithelial cells, opossum kidney cells. FGF-23 was found to require heparinlike molecules for its inhibitory activity on phosphate uptake. FGF-23 binds to the FGF receptor 3c, which is mainly expressed in opossum kidney cells, with high affinity. An inhibitor for tyrosine kinases of the FGF receptor, SU 5402, blocked the activity of FGF-23. FGF-23 activated the mitogen-activated protein kinase (MAPK) pathway, which is the major intracellular signaling pathway of FGF. Inhibitors of the MAPK pathway, PD98059 and SB203580, also blocked the activity of FGF-23.ThepresentfindingshaverevealedanovelMAPKdependent mechanism of the regulation of phosphate uptake by FGF signaling.Phosphate is a nutrient essential for many biological processes including skeletal mineralization and energy metabolism (1). The homeostasis of the plasma phosphate level is essential for these processes. The reabsorption of phosphate in the kidney is a major determinant of the plasma phosphate level. Reabsorption is largely regulated by the type II sodium-dependent phosphate (Na/P i ) cotransporter that is expressed in renal proximal epithelial cells (1). The activity of the type-II Na/P i cotransporter is regulated by hormones, such as parathyroid hormone (PTH) 1 and 1,25-dihydroxyvitamin D (1,25(OH) 2 D), which have opposite effects. PTH and 1,25(OH) 2 D decrease and increase the reabsorption of phosphate in renal proximal tubules, respectively (1). Tumor-induced osteomalacia is a renal phosphate-wasting disorder resulting in low serum phosphorus concentration and osteomalacia. Removal of the tumors responsible for tumorinduced osteomalacia normalizes phosphate metabolism. The responsible tumors secrete a heat-sensitive molecule of ϳ25 kDa designated as "phosphatonin" that specifically inhibits sodium-dependent phosphate transport in cultured renal proximal epithelial cells. Recent studies on tumor-induced osteomalacia revealed that phosphatonin was potentially identical to fibroblast growth factor (FGF)-23, which is a new member of the FGF family (2, 3, 5). Autosomal dominant hypophosphataemic rickets is also a renal phosphate-wasting disorder resulting in low serum phosphorus concentration, rickets, and osteomalacia. The ADHR gene was also potentially identified to be FGF-23 with missense mutations (4). However, as puri...

Japanese encephalitis virus (JEV) belongs to the genus Flavivirus within the family Flaviviridae. Members of the genus Flavivirus are predominantly arthropodborne viruses and frequently cause significant morbidity and mortality in mammals and birds (6). JEV is distributed in the south and southeast regions of Asia and kept in a zoonotic transmission cycle between pigs or birds and mosquitoes (6,50,57). JEV spreads to dead-end hosts, including humans, through the bite of JEVinfected mosquitoes and causes infection of the central nervous system, with a high mortality rate (6, 57). JEV has a single-stranded positive-strand RNA genome approximately 11 kb in length, which is capped at the 5Ј end but lacks modification of the 3Ј terminus by polyadenylation (34). The genomic RNA encodes a single large open reading frame, and a polyprotein translated from the genome is cleaved co-and posttranslationally by host and viral proteases to yield three structural proteins, the core, precursor membrane (prM), and envelope (E) proteins, and seven nonstructural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 (53). Although the core protein has very little amino acid homology to other flaviviruses-for example, the core protein of JEV has only 25% homology to that of tick-borne encephalitis virus (TBEV)-the structural properties, such as the hydrophobicity profile, abundances of basic amino acid residues, and secondary structures, are very similar (11,20,36). The flavivirus core proteins commonly contain two hydrophobic sequences in the center and a carboxyl-terminal end, and the carboxyl-terminal hydrophobic region serves as a signal sequence of prM. The signal-anchor sequence is cleaved off by the viral protease NS2B-3, and this cleavage is required for the subsequent liberation of the amino terminus of prM by the host signal peptidase (35,52,63). The mature core protein, released from the endoplasmic reticulum (ER) membrane, is believed to bind to the genomic RNA via the basic amino acid clusters at the amino and carboxyl termini and forms nucleocapsids (23). The central hydrophobic region of the core protein may be associated with the ER membrane, and this interaction is believed to facilitate the assembly of nucleocapsid and two membrane proteins, prM and E, and to bud into the ER lumen as virions (39). The removal of the central hydrophobic region of the TBEV core protein increased the production of the subviral particles that consist of (pr)M and E proteins but that lack a core protein and genomic RNA (26,27).In addition to their role as structural proteins, core proteins of dengue virus (DEN) and Kunjin virus (KUN) are localized not only in the cytoplasm but also in the nucleus, especially in the nucleoli of several infected cell lines (4,38,55,59,61). Transport from the cytoplasm to the nucleus occurs through nuclear pore complexes that penetrate the double lipid layers of the nuclear envelope. Small molecules up to 9 nm in diameter (Ͻ50 kDa) can freely diffuse through the nuclear pore complexes, while most macromolecu...

Bioenergy is efficiently produced in the mitochondria by the respiratory system consisting of complexes I-V. In various organisms, complex I can be replaced by the alternative NADH-quinone oxidoreductase (NDH-2), which catalyzes the transfer of an electron from NADH via FAD to quinone, without proton pumping. The Ndi1 protein from Saccharomyces cerevisiae is a monotopic membrane protein, directed to the matrix. A number of studies have investigated the potential use of Ndi1 as a therapeutic agent against complex I disorders, and the NDH-2 enzymes have emerged as potential therapeutic targets for treatments against the causative agents of malaria and tuberculosis. Here we present the crystal structures of Ndi1 in its substrate-free, NAD + -and ubiquinone-(UQ2) complexed states. The structures reveal that Ndi1 is a peripheral membrane protein forming an intimate dimer, in which packing of the monomeric units within the dimer creates an amphiphilic membrane-anchor domain structure. Crucially, the structures of the Ndi1-NAD + and Ndi1-UQ2 complexes show overlapping binding sites for the NAD + and quinone substrates.alternative complex I | structural biology B ioenergy is efficiently produced in the mitochondria by the respiratory system consisting of complexes I-V. The first of these complexes, NDH-1 (complex I, proton pumping NADH-Q oxidoreductase) serves as the major entry point into the respiratory chain, for electrons derived from metabolic processes. In various organisms, complex I can be replaced by the alternative NADH-quinone oxidoreductase (NDH-2), which catalyzes the transfer of an electron from NADH via FAD to quinone, without proton pumping (1). The NDH-2 enzymes are found in bacteria and the mitochondria of plants and fungi, but crucially, not in mammalian mitochondria. Plant and fungal mitochondria possess two types of NDH-2: one is directed to the matrix and catalyzes NADH oxidation in the matrix (designated the internal NADH dehydrogenase or Ndi), and the other faces the intermembrane space and oxidizes NADH in the cytoplasmic space (designated the external NADH dehydrogenase or Nde).

The NS3 protein of Japanese encephalitis virus (JEV) is a large multifunctional protein possessing protease, helicase, and nucleoside 5'-triphosphatase (NTPase) activities, and plays important roles in the processing of a viral polyprotein and replication. To clarify the enzymatic properties of NS3 protein from a structural point of view, an enzymatically active fragment of the JEV NTPase/helicase catalytic domain was expressed in bacteria and the crystal structure was determined at 1.8 A resolution. JEV helicase is composed of three domains, displays an asymmetric distribution of charges on its surface, and contains a tunnel large enough to accommodate single-stranded RNA. Each of the motifs I (Walker A motif), II (Walker B motif) and VI was composed of an NTP-binding pocket. Mutation analyses revealed that all of the residues in the Walker A motif (Gly(199), Lys(200) and Thr(201)), in addition to the polar residues within the NTP-binding pocket (Gln(457), Arg(461) and Arg(464)), and also Arg(458) in the outside of the pocket in the motif IV were crucial for ATPase and helicase activities and virus replication. Lys(200) was particularly indispensable, and could not be exchanged for other amino acid residues without sacrificing these activities. The structure of the NTP-binding pocket of JEV is well conserved in dengue virus and yellow fever virus, while different from that of hepatitis C virus. The detailed structural comparison among the viruses of the family Flaviviridae should help in clarifying the molecular mechanism of viral replication and in providing rationale for the development of appropriate therapeutics.