Suppressive effects of coumarins from Mammea siamensis on inducible nitric oxide synthase expression in RAW264.7 cells

A methanol extract of the flowers of Mammea siamensis (Calophyllaceae) was found to inhibit nitric oxide (NO) production in lipopolysaccharide-activated RAW264.7 cells. From the extract, two new geranylated coumarins, mammeasins A (1) and B (2), were isolated together with 17 known compounds including 15 coumarins. The structures of 1 and 2 were determined on the basis of their spectroscopic properties as well as of their chemical evidence. Among the isolates, 1 (IC(50)=1.8μM), 2 (6.4μM), surangins B (3, 5.0μM), C (4, 6.8μM), and D (5, 6.2μM), kayeassamins E (7, 6.1μM), F (8, 6.0μM), and G (9, 0.8μM), mammea A/AD (11, 1.3μM), and mammea E/BB (16, 7.9μM) showed NO production inhibitory activity. Compounds 1, 9, and 11 were found to inhibit induction of inducible nitric oxide synthase (iNOS). With regard to mechanism of action of these active constituents (1, 9, and 11), suppression of STAT1 activation is suggested to be mainly involved in their suppression of iNOS induction.     

Comprehensive application of an mtDsRed2-Tg mouse strain for mitochondrial imaging

Mitochondria are essential for many cellular functions such as oxidative phosphorylation and calcium homeostasis; consequently, mitochondrial dysfunction could cause many diseases, including neurological disorders. Recently, mitochondrial dynamics, such as fusion, fission, and transportation, have been visualized in living cells by using time-lapse imaging systems. The changes in mitochondrial morphology could be an indicator for estimating the activity of mitochondrial biological function. Here, we report a transgenic mouse strain, mtDsRed2-Tg, which expresses a red fluorescent protein, DsRed2, exclusively in mitochondria. Mitochondrial morphology could be clearly observed in various tissues of this strain under confocal microscope. Recently, many transgenic mouse strains in which enhanced green fluorescent protein (EGFP)-tagged proteins of interest are expressed have been established for physiological analysis in vivo. After mating these strains with mtDsRed2-Tg mice, red-colored mitochondria and green-colored proteins were detected simultaneously using fluorescent imaging systems, and the interactions between mitochondria and those proteins could be morphologically analyzed in cells and tissues of the F₁ hybrids. Thus, mtDsRed2-Tg mice can be a powerful tool for bioimaging studies on mitochondrial functions.     

Iron-dependent binding of bovine milk α-casein with holo-lactoferrin, but not holo-transferrin

Bovine milk α-casein has been identified as an iron- and heme-binding protein. However, the physiological role of its iron-binding remains to be elucidated in more detail. α-Casein was immobilized on CNBr-activated Sepharose 4B beads, and the α-casein agarose beads efficiently bound hemin as well as ferrous ammonium sulfate (Fe(2+)) as compared with control beads. Additionally, α-casein-beads bound bovine holo-lactoferrin (Lf), but not holo-transferrin. Lf caused the release of Fe(2+) which had bound to the α-casein-agarose beads beforehand. These results suggest that bovine α-casein iron-dependently binds holo-bovine Lf more strongly than Fe(2+), and that strong binding between them may play a physiological role in regulating iron homeostasis in the bovine mammary gland.     

Antidiabetogenic oligostilbenoids and 3-ethyl-4-phenyl-3,4-dihydroisocoumarins from the bark of Shorea roxburghii

A methanol extract of the bark of Shorea roxburghii (Dipterocarpaceae) was found to inhibit plasma glucose elevation in sucrose-loaded mice. From the extract, three new 3-ethyl-4-phenyl-3,4-dihydroisocoumarins, 1'S-dihydrophayomphenol A(2) (1) and phayomphenols B(1) (2) and B(2) (3), were isolated together with 24 known compounds including 20 stilbenoids and oligostilbenoids. The structures of 1-3 were determined on the basis of their spectroscopic properties as well as of chemical evidences. Among the isolates, (-)-hopeaphenol (6), hemsleyanol D (8), (+)-α-viniferin (15), and (-)-balanocarpol (18) showed inhibitory activity against plasma glucose elevation in sucrose-loaded rats at doses of 100-200mg/kg, p.o. To clarify the mode of action of the antihyperglycemic property, effects of these oligostilbenoids on gastric emptying in mice, those on glucose uptake in isolated intestinal tissues as well as inhibitory activities against rat intestinal α-glucosidase and rat lens aldose reductase were examined.     

Replication and infectivity of a novel genotype 1b hepatitis C virus clone

Hepatitis C virus infection is a major public health problem because of an estimated 170 million carriers worldwide. Genotype 1b is the major subtype of HCV in many countries and is resistant to interferon therapy. Study of the viral life cycle is important for understanding the mechanisms of interferon resistance of genotype 1b HCV strains. For such studies, genotype 1b HCV strains that can replicate and produce infectious virus particles in cultured cells are required. In the present study, we isolated HCV cDNA, which we named the NC1 strain, from a patient with acute severe hepatitis. Subgenomic replicon experiments revealed that several mutations enhanced the colony-formation efficiency of the NC1 replicon. The full-length NC1 genome with these adaptive mutations could replicate in cultured cells and produce infectious virus particles. The density gradient profile and morphology of the secreted virus particles were similar to those reported for the JFH-1 virus. Further introduction of a combination of mutations of the NS3 and NS5a regions into the NC1 mutants further enhanced secreted core protein levels and infectious virus titers in the culture medium of HCV-RNA-transfected cells. However, the virus infection efficiency was not sufficient for autonomous virus propagation in cultured cells. In conclusion, we established a novel cell culture-adapted genotype 1b HCV strain, termed NC1, which can produce infectious virus when the viral RNA is transfected into cells. This system provides an important opportunity for studying the life cycle of the genotype 1b HCV.     

Onset of hepatic erythropoiesis after malarial infection in mice

Plasmodium yoelii-infected erythrocytes were injected into mice with or without 6.5 Gy irradiation. This irradiation suppressed erythropoiesis and induced severe immunosuppression. However, these mice showed a rather delayed infection, suggesting that fresh erythrocytes may become malarial targets. In other words, malarial infection did not persist without newly generated erythrocytes in mice. We then examined erythropoiesis in the liver and bone marrow of mice with malaria. Surprisingly, erythropoiesis began in the liver. At this time, the serum level of erythropoietin (EPO) was prominently elevated and the EPO mRNA also became detectable in the kidney. Many clusters of red blood cells appeared de novo in the parenchymal space of the liver. These results revealed that malarial infection had a potential to induce the onset of hepatic erythropoiesis in mice.     

Structure of the metabotropic glutamate receptor

In the twelve years since the molecular elucidation of the metabotropic glutamate receptor subtype 1, a class III family of G-protein-coupled receptors has emerged; members of this family include the calcium-sensing receptor, the GABA(B) receptor, some odorant receptors and some taste receptors. Atomic structures of the ligand-binding core of the original metabotropic glutamate receptor 1 obtained using X-ray crystallography provide a foundation for determining the initial receptor activation of this important family of G-protein-coupled receptors.     

X-ray and biochemical anatomy of an archaeal XPF/Rad1/Mus81 family nuclease: similarity between its endonuclease domain and restriction enzymes

The XPF/Rad1/Mus81-dependent nuclease family specifically cleaves branched structures generated during DNA repair, replication, and recombination, and is essential for maintaining genome stability. Here, we report the domain organization of an archaeal homolog (Hef) of this family and the X-ray crystal structure of the middle domain, with the nuclease activity. The nuclease domain architecture exhibits remarkable similarity to those of restriction endonucleases, including the correspondence of the GDX(n)ERKX(3)D signature motif in Hef to the PDX(n)(E/D)XK motif in restriction enzymes. This structural study also suggests that the XPF/Rad1/Mus81/ERCC1 proteins form a dimer through each interface of the nuclease domain and the helix-hairpin-helix domain. Simultaneous disruptions of both interfaces result in their dissociation into separate monomers, with strikingly reduced endonuclease activities.