Structure of Carbohydrate Moiety of Asterosaponin A

Partial acid hydrolysis of asterosaponin A, a steroidal saponin, afforded two new disaccharides in addition to O-(6-deoxy-α-d-glucopyranosyl)-(l→4)-6-deoxy-d-glucose which has been characterized in the preceding paper. The formers were demonstrated as O-(6-deoxy-α-d-galactopyranosyl)-(1→4)-6-deoxy-d-glucose and O-(6-deoxy-α-d-galactopyranosyl)-(l→4)-6-deoxy-d-galactose, respectively.

Accordingly, the structure of carbohydrate moiety being composed of two moles each of 6-deoxy-d-galactose and 6-deoxy-d-glucose, was established as O-(6-deoxy-α-d-galactopyranosyl)-(l→4)-O-(6-deoxy-α-d-galactopyranosyl)-(l→4)-O-(6-deoxy-α-d-glucopyranosyl)-(l→4)-6-deoxy-d-glucose, which is attached to the steroidal aglycone through an O-acetal glycosidic linkage.     

Isolation of Extracellular Cobalt-free Corrinoid from Methanosarcina barkeri

Cobalt-free corrinoids (CFCs) were isolated from Methanosarcina barkeri Fusaro cells growing on a methanol minimum medium. The methanogen cells excreted a trace of CFCs (9.1 μg/I) into the culture medium when cobalt-deficient methanol medium was used. Several CFCs were separated by column chromatographies on ion exchangers and paper electrophoresis, where a major CFC showed a similar characteristic to that of nucleotide-free corrinoid, Factor B (cobinamide), suggesting to be hydrogenobinamide. By chemical insertion of Co^{2 +}, Cu^{2 +}, and Zn²⁺ into CFCs, the corresponding corrinoid and its metal analogues were observed. Bioassay using Escherichia coli 215 revealed that the major CFC (a yellow product obtained after alkaline treatment) and its copper and zinc analogues were inactive as cobalamin but were active as antimetabolites of cobalamin. However, the CFC greatly stimulated the cell growth of M. barkeri grown under cobalt-deficient conditions.     

Demonstration of Antigenic and Genotypic Variation in Orientia tsutsugamushi Which Were Isolated in Japan,and Their Classification into Type and Subtype

A total of 40 strains of Orientia tsutsugamushi (34 isolates from patients and trombiculid mites in Japan, and 6 prototype strains of antigenic variants) were examined for classification based on the reactivities with type-specific monoclonal antibodies in indirect immunofluorescence tests, and on the restriction fragment length polymorphism of a polymerase chain reaction (PCR)-amplified 56-kilodalton type-specific antigenic protein gene. By these methods, several antigenic and genotypic variants were found among the strains, and these variants were classified into types and further into subtypes. These results suggest that there are many variants in O. tsutsugamushi, and the methods used here seem to be useful for the systematic classification of the numerous variants. A strain which may be a new type distinguishable from those identified previously was also found in this study. Furthermore, variety in the degree of pathogenicity in mice related to type and/or subtype classification were observed.     

Cutting edge: Deficiency of macrophage migration inhibitory factor impairs murine airway allergic responses

Increased levels of macrophage migration inhibitory factor (MIF) in serum, sputum, and bronchioalveolar lavage fluid (BALF) from asthmatic patients and time/dose-dependent expression of MIF in eosinophils in response to phorbol myristate acetate suggest the participation of MIF in airway inflammation. In this study, we examined inflammation in OVA-sensitized mouse lungs in wild-type and MIF-deficient mice (MIF(-/-)). We report increased MIF in the lung and BALF of sensitized wild-type mice. MIF(-/-) mice demonstrated significant reductions in serum IgE and alveolar inflammatory cell recruitment. Reduced Th1/Th2 cytokines and chemokines also were detected in serum or BALF from MIF(-/-) mice. Importantly, alveolar macrophages and mast cells, but not dendritic cells or splenocytes, from MIF(-/-) mice demonstrated impaired CD4+ T cell activation, and the reconstitution of wild-type mast cells in MIF(-/-) mice restored the phenotype of OVA-induced airway inflammation, revealing a novel and essential role of mast cell-derived MIF in experimentally induced airway allergic diseases.     

Characterization of a multidomain adaptor protein, p140Cap, as part of a pre-synaptic complex

p140Cap (Cas-associated protein) is an adaptor protein considered to play pivotal roles in cell adhesion, growth and Src tyrosine kinase-related signaling in non-neuronal cells. It is also reported to interact with a pre-synaptic membrane protein, synaptosome-associated protein of 25 kDa, and may participate in neuronal secretion. However, properties and precise functions of p140Cap in neuronal cells are almost unknown. Here we show, using biochemical analyses, that p140Cap is expressed in rat brain in a developmental stage-dependent manner, and is relatively abundant in the synaptic plasma membrane fraction in adults. Immunohistochemistry showed localization of p140Cap in the neuropil in rat brain and immunofluorescent analyses detected p140Cap at synapses of primary cultured rat hippocampal neurons. Electron microscopy further revealed localization at pre- and post-synapses. Screening of p140Cap-binding proteins identified a multidomain adaptor protein, vinexin, whose third Src-homology 3 domain interacts with the C-terminal Pro-rich motif of p140Cap. Immunocomplexes between the two proteins were confirmed in COS7 and rat brain. We also clarified that a pre-synaptic protein, synaptophysin, interacts with p140Cap. These results suggest that p140Cap is involved in neurotransmitter release, synapse formation/maintenance, and signaling.     

Synergistic biofilm formation by Treponema denticola and Porphyromonas gingivalis

Biofilm formation is an important step in the etiology of periodontal diseases. In this study, in vitro biofilm formation by Treponema denticola and Porphyromonas gingivalis 381 displayed synergistic effects. Confocal microscopy demonstrated that P. gingivalis attaches to the substratum first as a primary colonizer followed by coaggregation with T. denticola to form a mixed biofilm. The T. denticola flagella mutant as well as the cytoplasmic filament mutant were shown to be essential for biofilm formation as well as coaggregation with P. gingivalis. The major fimbriae and Arg-gingipain B of P. gingivalis also play important roles in biofilm formation with T. denticola.     

Effect of dietary fiber, glucomannan, on absorption of sulfonylurea in man

In order to clarify whether a dietary fiber has any effect upon the intestinal absorption of sulfonylurea, changes in plasma concentration of glibenclamide were determined during a six-hour period in nine healthy volunteers who took 2.5 mg of glibenclamide together with a breakfast and 3.9 g of glucomannan in a form of konjac powder and were compared with those of the control experiment in which the same amount of the hypoglycemic agent was given without the dietary fiber. In the control, mean plasma glibenclamide level increased rapidly, reaching a peak at 60 min and decreased gradually thereafter, whereas an increase in plasma glibenclamide level was blunted in the test experiment, thus plasma concentration of glibenclamide being lower at 30, 60, 90 and 150 min compared with the corresponding value of the control (31.7 +/- 24.5 ng/ml vs 76.4 +/- 25.0 ng/ml at 30 min; 51.3 +/- 35.5 ng/ml vs 120.9 +/- 56.0 ng/ml at 60 min; 60.0 +/- 38.8 ng/ml vs 117.4 +/- 53.1 ng/ml at 90 min; 54.0 +/- 31.5 ng/ml vs 100.7 +/- 46.5 ng/ml at 150 min). Mean plasma glucose concentration was significantly lower at 30 min in the test experiment than in the control despite the lower level of plasma glibenclamide in the former. The results suggest that glucomannan may influence the intestinal absorption of glibenclamide. A dietary fiber must be prescribed in due consideration of these facts.     

Structural basis for the unique multifaceted interaction of DPPA3 with the UHRF1 PHD finger

Ubiquitin-like with PHD and RING finger domain-containing protein 1 (UHRF1)-dependent DNA methylation is essential for maintaining cell fate during cell proliferation. Developmental pluripotency-associated 3 (DPPA3) is an intrinsically disordered protein that specifically interacts with UHRF1 and promotes passive DNA demethylation by inhibiting UHRF1 chromatin localization. However, the molecular basis of how DPPA3 interacts with and inhibits UHRF1 remains unclear. We aimed to determine the structure of the mouse UHRF1 plant homeodomain (PHD) complexed with DPPA3 using nuclear magnetic resonance. Induced α-helices in DPPA3 upon binding of UHRF1 PHD contribute to stable complex formation with multifaceted interactions, unlike canonical ligand proteins of the PHD domain. Mutations in the binding interface and unfolding of the DPPA3 helical structure inhibited binding to UHRF1 and its chromatin localization. Our results provide structural insights into the mechanism and specificity underlying the inhibition of UHRF1 by DPPA3.