High-throughput scintillation proximity assay for stearoyl-CoA desaturase-1

Stearoyl-CoA desaturase (SCD) catalyzes the synthesis of monounsaturated fatty acids and has been implicated in a number of disease states, including obesity and diabetes. To find small-molecule inhibitor leads, a high-throughput scintillation proximity assay (SPA) was developed using the hydrophobic binding characteristics of a glass microsphere scintillant bead to capture SCD1 from a crude lysate of recombinant SCD1 in Sf9 lysate coupled with the strong binding characteristics of an azetidine compound ([(3)H]AZE). The SPA assay was stable over 24 h and could detect compounds with micromolar to nanomolar potencies. A robust 1536-well high-throughput screening assay was developed with good signal-to-noise ratio (10:1) and excellent Z' factor (0.8). A screening collection of 1.6 million compounds was screened at 11 μM, and approximately 7700 compounds were identified as initial hits, exhibiting at least 35% inhibition of [(3)H]AZE binding. Further screening and confirmation with an SCD enzyme activity assay led to a number of new structural leads for inhibition of the enzyme. The SPA assay complements the enzyme activity assay for SCD1 as a tool for the discovery of novel leads in drug discovery.     

Bioactive ellagitannins from Cunonia macrophylla, an endemic Cunoniaceae from New Caledonia

Chemical study of Cunonia macrophylla, a New Caledonian Cunoniaceae, based on bioactive effects of a crude methanol extract of the leaves, detected bioactive tannins for the first time in this plant family. These ellagitannins have been identified as ellagic acid-4-O-beta-D-xylopyranoside (6), mallorepanin (3), mallotinic acid (1) along with corilagin (2), chebulagic acid (4), ellagic acid (5) and gallic acid (7) and have been shown to possess antimicrobial activity and to inhibit xanthine oxidase. Antimicrobial effects on bacterial human pathogens (Staphylococcus aureus, Corynebacterium accolans) and on a plant pathogen (Erwinia carotovora) as well as on a human pathogenic yeast (Candida albicans) were investigated. Activity is reported here for the first time for compounds 1, 3, 4 and 6. The inhibitory effects of all molecules against xanthine oxidase in relation to their structure was evaluated and compared. Compound 6 presented the best activity and seems to be of considerable interest for further studies.     

Glutamate synthase in rice roots. Studies on the electron donor specificity

Rice root glutamate synthase activity was assayed with various reducing systems. Ferredoxin-dependent glutamate synthase (EC 1.4.7.1) and pyridine nucleotide-dependent glutamate synthase (NADH, EC 1.4.1.14; or NADPH, EC 1.4.1.13) exhibited a strict specificity for the electron donor. The ferredoxin-dependent glutamate synthase from rice roots could accept electrons from photoreduced ferredoxin in an illuminated reconstituted spinach chloroplast system. Thioredoxin, a potent electron carrier, was not able to provide either ferredoxin-dependent or pyridine nucleotide-dependent glutamate synthase with electrons as no glutamate formation was detected in the presence of reduced thioredoxin f or m.     

Cross-Presentation of Human Cytomegalovirus pp65 (UL83) to CD8+ T Cells Is Regulated by Virus-Induced, Soluble-Mediator-Dependent Maturation of Dendritic Cells

Cytotoxic CD8+ T lymphocytes (CTL) directed against the matrix protein pp65 are major effectors in controlling infection against human cytomegalovirus (HCMV), a persistent virus of the Betaherpesvirus family. We previously suggested that cross-presentation of pp65 by nonpermissive dendritic cells (DCs) could overcome viral strategies that interfere with activation of CTL (G. Arrode, C. Boccaccio, J. Lule, S. Allart, N. Moinard, J. Abastado, A. Alam, and C. Davrinche, J. Virol. 74:10018-10024, 2000). It is well established that mature DCs are very potent in initiating T-cell-mediated immunity. Consequently, the DC maturation process is a key step targeted by viruses in order to avoid an immune response. Here, we report that immature DCs maintained in coculture with infected human (MRC5) fibroblasts acquired pp65 from early-infected cells for cross-presentation to specific HLA-A2-restricted CTL. In contrast, coculture of DCs in the presence of late-infected cells decreased their capacity to stimulate CTL. Analyses of DC maturation after either coculture with infected MRC5 cells or incubation with infected-cell-conditioned medium revealed that acquisition of a mature phenotype was a prerequisite for efficient stimulation of CTL and that soluble factors secreted by infected cells were responsible for both up and down regulation of CD83 expression on DCs. We identified transforming growth factor beta1 secreted by late HCMV-infected cells as one of these down regulating mediators. These findings suggest that HCMV has devised another means to compromise immune surveillance mechanisms. Together, our data indicate that recognition of HCMV-infected cells by DCs has to occur early after infection to avoid immune evasion and to allow generation of anti-HCMV CTL.     

Myosin Va binding to neurofilaments is essential for correct myosin Va distribution and transport and neurofilament density

The identification of molecular motors that modulate the neuronal cytoskeleton has been elusive. Here, we show that a molecular motor protein, myosin Va, is present in high proportions in the cytoskeleton of mouse CNS and peripheral nerves. Immunoelectron microscopy, coimmunoprecipitation, and blot overlay analyses demonstrate that myosin Va in axons associates with neurofilaments, and that the NF-L subunit is its major ligand. A physiological association is indicated by observations that the level of myosin Va is reduced in axons of NF-L-null mice lacking neurofilaments and increased in mice overexpressing NF-L, but unchanged in NF-H-null mice. In vivo pulse-labeled myosin Va advances along axons at slow transport rates overlapping with those of neurofilament proteins and actin, both of which coimmunoprecipitate with myosin Va. Eliminating neurofilaments from mice selectively accelerates myosin Va translocation and redistributes myosin Va to the actin-rich subaxolemma and membranous organelles. Finally, peripheral axons of dilute-lethal mice, lacking functional myosin Va, display selectively increased neurofilament number and levels of neurofilament proteins without altering axon caliber. These results identify myosin Va as a neurofilament-associated protein, and show that this association is essential to establish the normal distribution, axonal transport, and content of myosin Va, and the proper numbers of neurofilaments in axons.     

Plant snoRNA database

The Plant snoRNA database (http://www.scri.sari.ac.uk/plant_snoRNA/) provides information on small nucleolar RNAs from Arabidopsis and eighteen other plant species. Information includes sequences, expression data, methylation and pseudouridylation target modification sites, initial gene organization (polycistronic, single gene and intronic) and the number of gene variants. The Arabidopsis information is divided into box C/D and box H/ACA snoRNAs, and within each of these groups, by target sites in rRNA, snRNA or unknown. Alignments of orthologous genes and gene variants from different plant species are available for many snoRNA genes. Plant snoRNA genes have been given a standard nomenclature, designed wherever possible, to provide a consistent identity with yeast and human orthologues.     

Comparison of Gold and Silver/Gold Bimetallic Surface for Highly Sensitive Near-infrared SPR Sensor at 1550 nm

A large majority of surface plasmon resonance (SPR) sensors reported in the literature are designed to operate in the visible electromagnetic spectrum. However, the near-infrared, particularly at the telecommunications wavelength of 1550 nm, is also especially attractive for SPR sensing applications. In fact, SPR sensors operating in this region benefit from narrower resonance and deeper field penetration. In this paper, we report a theoretical and experimental study of an SPR sensor operating at a fixed wavelength of 1550 nm. The influence of the choice of metals and the interrogation methods on the sensitivity of the resulting SPR sensor is investigated. Two types of sensor chips (simple gold (Au) and bimetallic silver/Au structure) and three interrogation methods (monitoring of the position of the reflectivity minimum, the position of the centroid, and the intensity evolution of the reflectivity) are examined. We show that a refractive index resolution of 2.7?×?10^?6 refractive index unit can be easily obtained, and with further optimization of the measurement system, the ultimate limit of detection is expected to be even lowered. Therefore, the approach discussed here already shows a promising potential for highly sensitive SPR sensors.     

Tum—mutation P35B generates the MHC-binding site of a new antigenic peptide

Immunogenic tumor cell variant P35 was obtained by mutagen treatment of mouse mastocytoma P815. It express a potent new antigen recognized by syngeneic cytolytic T lymphocytes (CTL). This antigen is the result of a point mutation in a gene that is expressed by most healthy cells. A decapeptide encoded by the region spanning the mutation sensitized P815 cells to the relevant CTL, whereas the homologous decapeptide corresponding to the normal sequence did not. Only the mutant decapeptide was capable of enhancing the expression of the D^d-presenting molecule at the cell surface, indicating that the mutation generates a motif which enables the antigenic peptide to bind to D^d. Correspondence to: T. Boon.