Myelin Basic Protein-Specific TCR/HLA-DRB5*01:01 Transgenic Mice Support the Etiologic Role of DRB5*01:01 in Multiple Sclerosis

Genetic susceptibility to multiple sclerosis (MS) has been linked to the HLA-DR15 haplotype consisting of DRB1*15:01(DR2b) and DRB5*01:01(DR2a) alleles. Given almost complete linkage disequilibrium of the two alleles, recent studies suggested differential roles in susceptibility (DR2b) or protection from MS (DR2a). Our objective was to assess the potential contribution of DR2a to disease etiology in MS using a humanized model of autoimmunity. To assess the potential contribution of DR2a to disease etiology, we created DR2a humanized transgenic (Tg) mice and subsequently crossed them to Tg mice expressing TL3A6, an MS patient-derived myelin basic protein 83-99-specific TCR. In TL3A6/DR2a Tg mice, CD4 Tg T cells escape thymic and peripheral deletion and initiate spontaneous experimental autoimmune encephalomyelitis (EAE) at low rates, depending on the level of DR2a expression. The ability to induce active EAE was also increased in animals expressing higher levels of DR2a. Inflammatory infiltrates and neuronal damage were present throughout the spinal cord, consistent with a classical ascending EAE phenotype with minor involvement of the cerebellum, brainstem, and peripheral nerve roots in spontaneous, as well as actively induced, disease. These studies emphasize the pathologic contribution of the DR2a allele to the development of autoimmunity when expressed as the sole MHC class II molecule, as well as strongly argue for DR2a as a contributor to the CNS autoimmunity in MS.     

Reactive oxygen species production and activation mechanism of the rice NADPH oxidase OsRbohB

Reactive oxygen species (ROS) produced by plant NADPH oxidases (NOXes) are important in plant innate immunity. The Oryza sativa respiratory burst oxidase homologue B (OsRbohB) gene encodes a NOX the regulatory mechanisms of which are largely unknown. Here, we used a heterologous expression system to demonstrate that OsRbohB shows ROS-producing activity. Treatment with ionomycin, a Ca(2+) ionophore, and calyculin A, a protein phosphatase inhibitor, activated ROS-producing activity; it was thus OsRbohB activated by both Ca(2+) and protein phosphorylation. Mutation analyses revealed that not only the first EF-hand motif but also the upstream amino-terminal region were necessary for Ca(2+)-dependent activation, while these regions are not required for phosphorylation-induced ROS production.     

Direct evidence of generation and accumulation of β-sheet-rich prion protein in scrapie-infected neuroblastoma cells with human IgG1 antibody specific for β-form prion protein

We prepared β-sheet-rich recombinant full-length prion protein (β-form PrP) (Jackson, G. S., Hosszu, L. L., Power, A., Hill, A. F., Kenney, J., Saibil, H., Craven, C. J., Waltho, J. P., Clarke, A. R., and Collinge, J. (1999) Science 283, 1935-1937). Using this β-form PrP and a human single chain Fv-displaying phage library, we have established a human IgG1 antibody specific to β-form but not α-form PrP, PRB7 IgG. When prion-infected ScN2a cells were cultured with PRB7 IgG, they generated and accumulated PRB7-binding granules in the cytoplasm with time, consequently becoming apoptotic cells bearing very large PRB7-bound aggregates. The SAF32 antibody recognizing the N-terminal octarepeat region of full-length PrP stained distinct granules in these cells as determined by confocal laser microscopy observation. When the accumulation of proteinase K-resistant PrP was examined in prion-infected ScN2a cells cultured in the presence of PRB7 IgG or SAF32, it was strongly inhibited by SAF32 but not at all by PRB7 IgG. Thus, we demonstrated direct evidence of the generation and accumulation of β-sheet-rich PrP in ScN2a cells de novo. These results suggest first that PRB7-bound PrP is not responsible for the accumulation of β-form PrP aggregates, which are rather an end product resulting in the triggering of apoptotic cell death, and second that SAF32-bound PrP lacking the PRB7-recognizing β-form may represent so-called PrP(Sc) with prion propagation activity. PRB7 is the first human antibody specific to β-form PrP and has become a powerful tool for the characterization of the biochemical nature of prion and its pathology.     

Limonoid compounds inhibit sphingomyelin biosynthesis by preventing CERT protein-dependent extraction of ceramides from the endoplasmic reticulum

To identify novel inhibitors of sphingomyelin (SM) metabolism, a new and selective high throughput microscopy-based screening based on the toxicity of the SM-specific toxin, lysenin, was developed. Out of a library of 2011 natural compounds, the limonoid, 3-chloro-8β-hydroxycarapin-3,8-hemiacetal (CHC), rendered cells resistant to lysenin by decreasing cell surface SM. CHC treatment selectively inhibited the de novo biosynthesis of SM without affecting glycolipid and glycerophospholipid biosynthesis. Pretreatment with brefeldin A abolished the limonoid-induced inhibition of SM synthesis suggesting that the transport of ceramide (Cer) from the endoplasmic reticulum to the Golgi apparatus is affected. Unlike the Cer transporter (CERT) inhibitor HPA-12, CHC did not change the transport of a fluorescent short chain Cer analog to the Golgi apparatus or the formation of fluorescent and short chain SM from the corresponding Cer. Nevertheless, CHC inhibited the conversion of de novo synthesized Cer to SM. We show that CHC specifically inhibited the CERT-mediated extraction of Cer from the endoplasmic reticulum membranes in vitro. Subsequent biochemical screening of 21 limonoids revealed that some of them, such as 8β-hydroxycarapin-3,8-hemiacetal and gedunin, which exhibits anti-cancer activity, inhibited SM biosynthesis and CERT-mediated extraction of Cer from membranes. Model membrane studies suggest that 8β-hydroxycarapin-3,8-hemiacetal reduced the miscibility of Cer with membrane lipids and thus induced the formation of Cer-rich membrane domains. Our study shows that certain limonoids are novel inhibitors of SM biosynthesis and suggests that some biological activities of these limonoids are related to their effect on the ceramide metabolism.     

Synthesis and biological evaluation of deoxy-hematoxylin derivatives as a novel class of anti-HIV-1 agents

SAR studies for the exploration a novel class of anti-human immunodeficiency virus type 1 (HIV-1) agents based on the hematoxylin structure (1) are described. The systematic deoxygenations of 1 including asymmetric synthesis were conducted to obtain a compound showing high potencies for inhibiting the nuclear import and viral replication as anti-HIV-1 agent. Among all, C-3-deoxygenated analog 16 exhibited most promising biological activities as anti-HIV-1 agent such as lower cytotoxicity (16:1; >80:40 μM), stronger inhibition of nuclear import (0.5:1.3 μM), and viral replication in HIV-1-infected TZM-bl cells (24.6:100 μM), human peripheral blood mononuclear cells (PMBCs) (30.1 μM: toxic). Different spectra of inhibitory activities against infected three healthy humans macrophages with high (donor A) and low (donor B and C) amounts of virus were also observed. Thus 16 showed 10-times stronger activity than 1 (16:1; 0.1:<1.0 μM) in the case of A, while 16 and 1 showed comparable activities in the cases of B and C (>0.01 and >0.00 1μM). The comparison of the inhibition of viral p24 antigen production was clearly indicated that compound 16 is at least twofold more potent anti-viral activity than 1. Thus, structures and actions of deoxy analogs particularly 16 could provide valuable information for the development of a novel class of anti-HIV-1 agents.     

Inflorescence architecture affects pollinator behaviour and mating success in Spiranthes sinensis (Orchidaceae)

? Despite the wide inflorescence diversity among angiosperms, the effects of inflorescence architecture (three-dimensional flower arrangement) on pollinator behaviour and mating success have not been sufficiently studied in natural plant populations. ? Here, we investigated how inflorescence architecture affected inter- and intra-plant pollinator movements and consequent mating success in a field population of Spiranthes sinensis var. amoena (S. sinensis). In this species, the flowers are helically arranged around the stem, and the degree of twisting varies greatly among individuals. The large variation in inflorescence architecture in S. sinensis results from variation in a single structural parameter, the helical angle (the angular distance between neighbour-flower directions). ? The numbers of visits per inflorescence and successive probes per visit by leaf-cutting bees decreased with helical angle, indicating that individual flowers of tightly twisted inflorescences received less visitations. As expected from pollinator behaviour, pollinia removal and fruit set of individual flowers decreased with helical angle. Meanwhile, geitonogamy decreased in tightly twisted inflorescences. ? Our novel findings demonstrate that natural variation in inflorescence architecture significantly affects pollinator behaviour and reproductive success, suggesting that inflorescence architecture can evolve under pollinator-mediated natural selection in plant populations. We also discuss how diverse inflorescence architectures may have been maintained in S. sinensis populations.     

Dephosphorylation of Cdc20 is required for its C-box-dependent activation of the APC/C

The anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase is tightly regulated to ensure programmed proteolysis in cells. The activity of the APC/C is positively controlled by cyclin-dependent kinase (CDK), but a second level of control must also exist because phosphorylation inactivates Cdc20, a mitotic APC/C co-activator. How Cdc20 is dephosphorylated specifically, when CDK is high, has remained unexplained. Here, we show that phosphatases are crucial to activate the APC/C. Cdc20 is phosphorylated at six conserved residues (S50/T64/T68/T79/S114/S165) by CDK in Xenopus egg extracts. When all the threonine residues are phosphorylated, Cdc20 binding to and activation of the APC/C are inhibited. Their dephosphorylation is regulated depending on the sites and protein phosphatase 2A, active in mitosis, is essential to dephosphorylate the threonine residues and activate the APC/C. Consistently, most of the Cdc20 bound to the APC/C in anaphase evades phosphorylation at T79. Furthermore, we show that the 'activation domain' of Cdc20 associates with the Apc6 and Apc8 core subunits. Our data suggest that dephosphorylation of Cdc20 is required for its loading and activation of the APC/C ubiquitin ligase.     

The postbranchial digestive tract of the ascidian, Polyandrocarpa misakiensis (Tunicata: Ascidiacea). 2. Stomach

The organization of the stomach in the compound styelid ascidian, Polyandrocarpa misakiensis, is described, and the morphology and cell types of the stomach is discussed from the phylogenetic viewpoint. The stomach is a sac-like organ whose wall is formed into longitudinal folds. The stomach consists of external and internal epithelium. The internal epithelium is simple columnar, except for the bottom of the folds. There are five cell types: absorptive cells, zymogenic cells, endocrine cells, ciliated mucous cells, and undifferentiated cells. The absorptive cells have numerous microvilli. The apical region of these cells is occupied by coated vesicles. The zymogenic cells have a conical outline and a few microvilli on their apical surfaces. There are secretory granules in the apical region of zymogenic cells. The endocrine cells have low cell height and electron-dense granules around the nucleus. Endocrine cells have one or two cilia and a few microvilli on the apical surfaces. The basolateral part of these cells often bulges into the adjoining cells. Immunoelectron microscopy revealed that some endocrine cells have serotonin-like immunoreactivity. The ciliated mucous cells are restricted to a single ventral groove. They have numerous microvilli and a few cilia on their apical surfaces. Moderately electron-dense granules are accumulated in the apical part of the ciliated mucous cells. Undifferentiated cells, filled with free ribosomes, form a pseudostratified epithelium in the base of each fold. The nucleus of undifferentiated cells has a prominent nucleolus. The pseudostratified epithelium of the pyloric caecum consists of electron-dense and electron-light cells.