C(8)-substituted 1-azabicyclo[3.3.1]non-3-enes: a novel scaffold for muscarinic receptor ligands

The [3.3.1]-bicyclic amine, exo-8-benzyloxymethyl-3-ethoxycarbonyl-4-hydroxy-1-azabicyclo[3.3.1]non-3-ene (1), has been shown to be a potent competitive antagonist against the hM(1)-hM(5) muscarinic receptors. This heterocyclic system has not been extensively evaluated despite the notable activities reported for other bicyclic amines. Synthetic strategies permitted the selective alteration of five structural sites in 1. Pharmacological evaluation demonstrated that modification of either the C(3) alkoxycarbonyl or the C(4) enol units in 1 gave compounds with high affinity for the hM(1)-hM(5) muscarinic receptors with selectivity for the hM(2) receptor.     

Analysis of plastid DNA-like sequences within the nuclear genomes of higher plants

A wide-ranging examination of plastid (pt)DNA sequence homologies within higher plant nuclear genomes (promiscuous DNA) was undertaken. Digestion with methylation-sensitive restriction enzymes and Southern analysis was used to distinguish plastid and nuclear DNA in order to assess the extent of variability of promiscuous sequences within and between plant species. Some species, such as Gossypium hirsutum (cotton), Nicotiana tabacum (tobacco), and Chenopodium quinoa, showed homogenity of these sequences, while intraspecific sequence variation was observed among different cultivars of Pisum sativum (pea), Hordeum vulgare (barley), and Triticum aestivum (wheat). Hypervariability of plastid sequence homologies was identified in the nuclear genomes of Spinacea oleracea (spinach) and Beta vulgaris (beet), in which individual plants were shown to possess a unique spectrum of nuclear sequences with ptDNA homology. This hypervariability apparently extended to somatic variation in B. vulgaris. No sequences with ptDNA homology were identified by this method in the nuclear genome of Arabidopsis thaliana.      

Indoleamine 2,3-dioxygenase-expressing dendritic cells are involved in the generation of CD4+CD25+ regulatory T cells in Peyer's patches in an orally tolerized, collagen-induced arthritis mouse model

Introduction

The present study was devised to understand the role of systemic indoleamine 2,3-dioxygenase (IDO) in the tolerance induction for orally tolerized mice in collagen-induced arthritis (CIA). We examined whether IDO-expressing dendritic cells (DCs) are involved in the generation of CD4⁺CD25⁺ regulatory T cells during the induction of oral tolerance in a murine CIA model.

Methods

Type II collagen was fed six times to DBA/1 mice beginning 2 weeks before immunization, and the effect on arthritis was assessed. To examine the IDO expression, the DCs of messenger RNA and protein were analyzed by RT-PCR and Flow cytometry. In addition, a proliferative response assay was also carried out to determine the suppressive effects of DCs through IDO. The ability of DCs expressing IDO to induce CD4⁺CD25⁺ T regulatory cells was examined.

Results

CD11c⁺ DCs in Peyer's patches from orally tolerized mice expressed a higher level of IDO than DCs from nontolerized CIA mice. IDO-expressing CD11c⁺ DCs were involved in the suppression of type II collagen-specific T-cell proliferation and in the downregulation of proinflammatory T helper 1 cytokine production. The suppressive effect of IDO-expressing CD11c⁺ DCs was mediated by Foxp3⁺CD4⁺CD25⁺ regulatory T cells.

Conclusion

Our data suggest that tolerogenic CD11c⁺ DCs are closely linked with the induction of oral tolerance through an IDO-dependent mechanism and that this pathway may provide a new therapeutic modality to treat autoimmune arthritis.     

Activity assay of mammalian 2-cys peroxiredoxins using yeast thioredoxin reductase system

2-Cys peroxiredoxin (Prx) is a novel cellular peroxidase that reduces peroxides in the presence of thioredoxin, thioredoxin reductase, and nicotinamide adenine dinucleotide phosphate (NADPH) and that functions in H(2)O(2)-mediated signal transduction. Recent studies have shown that 2-cys Prx can be inactivated by cysteine overoxidation in conditions of oxidative stress. Therefore, peroxidase activity, rather than the protein level, of 2-cys Prx is the more important measure to predict its cellular function. Here, we introduce a modified activity assay method for mammalian 2-cys Prx based on yeast nonselenium thioredoxin reductase. Yeast thioredoxin reductase is expressed in Escherichia coli cells and purified at high yield (40 mg/L of culture broth) as an active flavoprotein by combined diethyl aminoethyl (DEAE) and phenyl hydrophobic chromatography. The optimal concentrations of yeast thioredoxin and thioredoxin reductase required to achieve maximum mammalian 2-cys Prx activity are 3.0 and 1.5 microM, respectively. This modified assay method is useful for measuring 2-cys Prx activity in cell lysates and can also be adapted for a 96-well plate reader for high-throughput screening of chemical compounds that target 2-cys Prx.     

Identification of Staphylococcus xylosus isolated from C57BL/6J-Nos2(tm1Lau) mice with dermatitis

Coagulase negative staphylococci (CNS) are significant pathogens, particularly in medical device related infections and in immunocompromised patients. Five CNS strains were isolated from 5 NOS2 knockout mice with dermatitis. Histologically, granulomatous dermatitis was found in the skin around the ear with epidermal ulceration. Dermal lesions included pustules, necrosis, and accumulations of neutrophils and macrophages. Isolates of the bacterial strains were identified to be Staphylococcus xylosus by the API STAPH kit and 16S-23S intergenic transcribed spacer-PCR. These results demonstrate the potential of this organism to be an opportunistic pathogen in immunocompromised mice.     

The Role of Elytra in Beetle Flight: I. Generation of Quasi-Static Aerodynamic Forces

We conducted a comprehensive study to investigate the aerodynamic characteristics and force generation of the elytra of abeetle,Allomyrina dichotoma.Our analysis included wind tunnel experiments and three-dimensional computational fluiddynamics simulations using ANSYS-CFX software.Our first approach was a quasi-static study that considered the effect ofinduced flapping flow due to the flapping motion of the fore-wings (elytra) at a frequency of around 30 Hz to 40 Hz.The dihedralangle was varied to represent flapping motion during the upstroke and downstroke.We found that an elytron producespositive lift at 0° geometric angle of attack,negative lift during the upstroke,and always produces drag during both the upstrokeand downstroke.We also found that the lift coefficient of an elytron does not drop even at a very high geometric angle of attack.For a beetle with a body weight of 5 g,based on the quasi-static method,the fore-wings (elytra) can produce lift of less than 1%of its body weight.     

Feedback control of adrenal steroidogenesis via H2O2-dependent, reversible inactivation of peroxiredoxin III in mitochondria

Certain members of the peroxiredoxin (Prx) family undergo inactivation through hyperoxidation of the catalytic cysteine to sulfinic acid during catalysis and are reactivated by sulfiredoxin; however, the physiological significance of this reversible regulatory process is unclear. We now show that PrxIII in mouse adrenal cortex is inactivated by H(2)O(2) produced by cytochrome P450 enzymes during corticosterone production stimulated by adrenocorticotropic hormone. Inactivation of PrxIII triggers a sequence of events including accumulation of H(2)O(2), activation of p38 mitogen-activated protein kinase, suppression of steroidogenic acute regulatory protein synthesis, and inhibition of steroidogenesis. Interestingly, levels of inactivated PrxIII, activated p38, and sulfiredoxin display circadian oscillations. Steroidogenic tissue-specific ablation of sulfiredoxin in mice resulted in the persistent accumulation of inactive PrxIII and suppression of the adrenal circadian rhythm of corticosterone production. The coupling of CYP11B1 activity to PrxIII inactivation provides a feedback regulatory mechanism for steroidogenesis that functions independently of the hypothalamic-pituitary-adrenal axis.     

Neuronal Synapse Formation Induced by Microglia and Interleukin 10

Recently, it was found that microglia regulated synaptic remodeling of the developing brain, but their mechanisms have not been well understood. In this study, the action of microglia on neuronal synapse formation was investigated, and the primary target of microglial processes was discovered. When the developing microglia were applied to cultured hippocampal neurons without direct contact, the numbers of dendritic spines and excitatory and inhibitory synapses significantly increased. In order to find out the main factor for synaptic formation, the effects of cytokines released from microglia were examined. When recombinant proteins of cytokines were applied to neuronal culture media, interleukin 10 increased the numbers of dendritic spines in addition to excitatory and inhibitory synapses. Interestingly, without external stimuli, the amount of interleukin 10 released from the intact microglia appeared to be sufficient for the induction of synaptic formation. The neutralizing antibodies of interleukin 10 receptors attenuated the induction of the synaptic formation by microglia. The expression of interleukin 10 receptor was newly found in the hippocampal neurons of early developmental stage. When interleukin 10 receptors on the hippocampal neurons were knocked down with specific shRNA, the induction of synaptic formation by microglia and interleukin 10 disappeared. Pretreatment with lipopolysaccharide inhibited microglia from inducing synaptic formation, and interleukin 1β antagonized the induction of synaptic formation by interleukin 10. In conclusion, the developing microglia regulated synaptic functions and neuronal development through the interactions of the interleukin 10 released from the microglia with interleukin 10 receptors expressed on the hippocampal neurons.