The relationship of common saprophytic and pathogenic fungi to the Forssman and other antigens of the sheep erythrocyte

Summary 1) Culture filtrates and mats from 20 different species of fungi cultured on Sabouraud dextrose broth and asparagine synthetic medium were examined for the presence of Forssman activity utilizing a hemolysis inhibition test.2) None of the uninoculated media contained any Forssman activity.3) Seventeen of the fungi exhibited no such activity.4) Single preparations of mats ofCandida tropicalis, Fusarium moniliforme andHistoplasma capsulatum appeared to contain Forssman activity. Further studies, however, employing different preparations of these fungi in the hemolysis inhibition test and the initial preparations in an hemagglutination inhibition test did not corroborate the original findings.5) Preparations of three of seven different fungi, none of which possessed Forssman activity, were found to contain blood group A activity.6) Preparations of six different fungi without Forssman activity, including the three with the blood group A properties, were found not to inhibit the anti-sheep erythrocyte agglutinins in the serum from a patient with infectious mononucleosis.7) The importance of the sheep erythrocyte as a heterogenetic antigen is discussed.This study was supported in part by USPHS GRANTS A1-01478-07, 5 T1 A1 52-05 and 5T1-AM-5265-05 and the Dermatologic Research Foundation of California, Inc.     

Discovery of imidazo[1,2-b]pyridazines as IKKβ inhibitors. Part 3: exploration of effective compounds in arthritis models

We have discovered imidazo[1,2-b]pyridazine derivatives that show suppressive activity of inflammation in arthritis models. We optimized the substructures of imidazo[1,2-b]pyridazine derivatives to combine potent IKKβ inhibitory activity, TNFα inhibitory activity invivo and excellent pharmacokinetics. The compound we have acquired, which had both potent activities and good pharmacokinetic profiles based on improved physicochemical properties, demonstrated efficacy on collagen-induced arthritis models in mice and rats.     

Discovery of novel 7-membered cyclic amide derivatives that inhibit 11beta-hydroxysteroid dehydrogenase type 1

A series of novel 5-trans-hydroxyadamantan-2-yl-5,6,7,8-tetrahydropyrazolo[4,3-c]azepin-4(1H)-ones that inhibit 11beta-hydroxysteroid dehydrogenase type 1 are described. We discovered these 7-membered cyclic amide derivatives by introducing a distinctive linker through pharmacophore analysis of known ligands included in X-ray co-crystal structures. Further optimization using docking studies led to highly potent inhibitors 15b and 27, which furthermore showed the potent efficacy in in vivo studies.     

Selective localization of G protein γ5 subunit in the subventricular zone of the lateral ventricle and rostral migratory stream of the adult rat brain

G proteins play important roles in transmembrane signal transduction, and various isoforms of each subunit, alpha, beta and gamma, are highly expressed in the brain. The Ggamma5 subunit is a minor isoform in the adult brain, but we have previously shown it to be highly expressed in the proliferative region of the ventricular zone in the rat embryonic brain. We show here that Ggamma5 is also selectively localized in a proliferative region in the adult rat brain, including the subventricular zone of the lateral ventricle and rostral migratory stream. The Galphai2 subunit colocalized with Ggamma5 in these regions, the two subunits being present in neuronal precursors and ependymal cells but not in proliferating astrocytes. In addition, intense staining of Ggamma5 was seen in axons of the olfactory neurons, which are known to regenerate. These results suggest specific roles for Ggamma5 in precursor cells during neurogenesis so that this isoform might be a useful biological marker.     

Is intracytoplasmic membrane structure a generic criterion? It does not coincide with phylogenetic interrelationships among phototrophic purple nonsulfur bacteria

The 16S rRNA or rRNA gene sequences of the type strains of 5 species of Rhodobacter, Rhodopseudomonas blastica and Paracoccus denitrificans were determined. The sequence analysis revealed that Rhodobacter species, whose intracytoplasmic membrane systems were characteristically vesicular, composed a sole cluster. Rhodopseudomonas blastica, whose intracytoplasmic membrane system was lamellar, was included in the cluster of Rhodobacter. The phylogenetic co-clustering of these bacteria conformed to their possessing of the identical types of carotenoids. Paracoccus denitrificans, which is nonphototrophic, is a right member of the Rhodobacter cluster. Rhodobacter species, Rhodopseudomonas blastica and Paracoccus denitrificans are apart from the other phototrophic bacteria and have the common deletions of 21 bases at the positions 1258 to 1278 (Escherichia coli numbering system). It was demonstrated that the morphological character intracyto-plasmic membrane structure, that has been regarded as a generic criterion does not reflect the phylogeny in the phototrophic bacteria. The transfer of Rhodopseudomonas blastica to the genus Rhodobacter is proposed.     

The real factor for polypeptide elongation in Dictyostelium cells is EF-2B, not EF-2A

Polypeptide elongation factor 2 (EF-2) plays an essential role in protein synthesis and is believed to be indispensable for cell proliferation. Recently, it has been demonstrated that there are two kinds of EF-2 (EF-2A and EF-2B with 76.6% of sequence identity at the amino acid level) in Dictyostelium discoideum. Although the knockout of EF-2A slightly impaired cytokinesis, EF-2A null cells exhibited almost normal protein synthesis and cell growth, suggesting that there is another molecule capable of compensating for EF-2 function. Since EF-2B is the most likely candidate, we examined its function using ef-2b knockdown cells prepared by the RNAi method. Our results strongly suggest that EF-2B is required for protein synthesis and cell proliferation, functioning as the real EF-2. Interestingly, the expressions of ef-2a and ef-2b mRNAs during development are reversely regulated, and the ef-2b expression is greatly augmented in ef-2a null cells.     

Mammalian Bax initiates plant cell death through organelle destruction

Mammalian Bax is known to cause cell death when expressed in plants. We examined transgenic plants expressing both Bax and organelle-targeted green fluorescent protein to determine the cellular changes that occur during Bax-induced cell death. The mitochondria changed morphologically from being bacilli-shaped to being round, eventually becoming swollen. Mitochondria streaming also stopped. The chloroplasts lost membrane function and their contents leaked out, followed by the disruption of the vacuole. Light was not essential for Bax-induced ion leakage or organelle disruption. These results indicate that Bax induces temporal and spatial cell death events at the organelle level in the plant. A heterologous system, using Bax, would therefor be available to investigate cell death, which is commonly conserved in animals and plantsElectronic Supplementary Material Supplementary material is available for this article at     

Properties of a tobacco DNA methyltransferase, NtMET1 and its involvement in chromatin movement during cell division

BACKGROUND AND AIMS: Plants possess three types of DNA methyltransferase, among which methyltransferase type 1 (MET1) is considered to play a major role by maintaining the CpG methylation patterns. However, little information is available as to its enzymatic activity, interacting proteins and spatial and temporal behaviours during DNA replication. In the present study, one example, NtMET1 from tobacco plants, was selected and an analysis was made of its biochemical properties and cellular localization. METHODS: NtMET1 was expressed in Sf9 insect cells, and a purified sample was subjected to a standard in vitro methylation assay. Intramolecular interaction was examined by the yeast two-hybrid and pull-down assays. Transgenic tobacco plants (Nicotiana tabacum) over-expressing NtMET1 were constructed via Agrobacterium-mediated transformation. Cellular localization was examined by fluorescence protein fusion, which was expressed in tobacco bright yellow 2 cells. KEY RESULTS: In vitro assays showed no detectable methylation activity when both hemimethylated and unmethylated DNA samples were used as the substrate. In planta assays with over-expressing transgenic lines showed no hypermethylation but rather hypomethylation of genomc DNA. The inability of methylation was conceivably due to a tight intramolecular interaction between the N- and C-terminal regions with the catalytic domain residing on the C-terminus being completely masked. Cellular localization analyses indicated that NtMET1 localized to the nucleus in the resting stage and migrates to the cytoplasm during mitosis, particularly at metaphase. The pattern observed resembled that of Ran GTPase, and in vitro pull-down assays showed a clear interaction between NtMET1 and AtRAN3, an Arabidopsis orthologue of tobacco Ran GTPase, NtRan-A1. CONCLUSIONS: The results suggest that enzymatic activity of NtMET1 is well adjusted by its own intra/intermolecular interaction and perhaps by interactions with other proteins, one of which was found to be Ran GTPase. Results also revealed that NtMET1 becomes localized to the vicinity of chromatin with the aid of Ran GTPase during cell division, and may play an important role in progress through mitosis independently of methylation activity.     

Structural basis of digoxin that antagonizes RORgamma t receptor activity and suppresses Th17 cell differentiation and interleukin (IL)-17 production

The retinoic acid-related orphan nuclear receptor γt (RORγt)/RORγ2 is well known as a master regulator of interleukin 17 (IL-17)-producing helper T (Th17) cell development. To develop a therapeutic agent against Th17-mediated autoimmune diseases, we screened chemical compounds and successfully found that digoxin inhibited IL-17 production. Further studies revealed that digoxin bound to the ligand binding domain of RORγt and suppressed Th17 differentiation without affecting Th1 differentiation. To better understand the structural basis for the inhibitory activity of digoxin, we determined the crystal structure of the RORγt ligand-binding domain in complex with digoxin at 2.2 Å resolution. The structure reveals that digoxin binds to the ligand-binding pocket protruding between helices H3 and H11 from the pocket. In addition, digoxin disrupts the key interaction important for the agonistic activity, resulting in preventing the positioning of helix H12 in the active conformation, thus antagonizing coactivator interaction. Functional studies demonstrated that digoxin inhibited RORγt activity and decreased IL-17 production but not RORα activity. Digoxin inhibited IL-17 production in CD4⁺ T cells from experimental autoimmune encephalomyelitis mice. Our data indicates that RORγt is a promising therapeutic target for Th17-derived autoimmune diseases and our structural data will help to design novel RORγt antagonists.     

MULTIPLE FtsZ RING FORMATION AND REDUPLICATED CHLOROPLAST DNA IN NANNOCHLORIS BACILLARIS (CHLOROPHYTA,TREBOUXIOPHYCEAE) UNDER PHOSPHATE‐ENRICHED CULTURE1

We examined the effects of phosphate enrichment on chloroplasts of the unicellular green alga Nannochloris bacillaris Naumann. The doubling time of cells was similar in phosphate‐limited (no β‐glycerophosphate) and phosphate‐enriched (2 mM β‐glycerophosphate) media. The lengths of cells and chloroplasts were similar, regardless of phosphate concentration. The relationship between the ring formation of the prokaryote‐derived chloroplast division protein FtsZ and phosphate concentration was examined using indirect fluorescent antibody staining. The number of FtsZ rings increased as the phosphate concentration of the medium increased. Multiple FtsZ rings were formed in cells in phosphate‐enriched medium; up to six FtsZ rings per chloroplast were observed. The number of FtsZ rings increased as the chloroplast grew. The FtsZ ring located near the center of the chloroplast had the strongest fluorescence. The FtsZ ring at the relative center of all FtsZ rings was used for division. Plastid division rings did not multiply in phosphate‐enriched culture. The chloroplast DNA content was 2.3 times greater in phosphate‐enriched than in phosphate‐limited culture and decreased in cells cultured in phosphate‐enriched medium containing 5‐fluorodeoxyuridine (FdUr). In the presence of FdUr, only one FtsZ ring formed, even under phosphate enrichment. This finding suggests that excessive chloroplast DNA replication induces multiple FtsZ ring formation in phosphate‐enriched culture. We propose a multiple FtsZ ring formation model under phosphate enrichment.