Purification and characterization of UDP-glucose: hydroxycoumarin 7-O-glucosyltransferase, with broad substrate specificity from tobacco cultured cells

The enzyme UDP-glucose: hydroxycoumarin 7-O-glucosyltransferase (CGTase), which catalyzes the formation of scopolin from scopoletin, was purified approximately 1200-fold from a culture of 2,4-D-treated tobacco cells (Nicotiana tabacum L. cv. Bright Yellow T-13) with a yield of 7%. Purification to apparent homogeneity, as judged by SDS-PAGE, was achieved by sequential anion-exchange chromatography, hydroxyapatite chromatography, gel filtration, a second round of anion-exchange chromatography, and affinity chromatography on UDP-glucuronic acid agarose. The purified enzyme had a pH optimum of 7.5, an isoelectric point (pI) of 5.0, and a molecular mass of 49 kDa. The enzyme did not require metal cofactors for activity. Its activity was inhibited by Zn2+, Co2+ and Cu2+ ions, as well as by SH-blocking reagents. The K-m values for UDP-glucose, scopoletin and esculetin were 43, 150 and 25 mu M. respectively. A study of the initial rate of the reaction suggested that the reaction proceeded via a sequential mechanism. The purified enzyme preferred hydroxycoumarins as substrates but also exhibited significant activity with flavonoids. A database search using the amino terminus amino acid sequence of CGTase revealed strong homology to the amino acid sequences of other glucosyltransferases in plants.ArticlePlant Science. 157(1):105-112 (2000)journal articl

CCQM-K90, formaldehyde in nitrogen, 2 μmol mol− 1 Final report

The CCQM-K90 comparison is designed to evaluate the level of comparability of national metrology institutes (NMI) or designated institutes (DI) measurement capabilities for formaldehyde in nitrogen at a nominal mole fraction of 2 μmol mol−1. The comparison was organised by the BIPM using a suite of gas mixtures prepared by a producer of specialty calibration gases. The BIPM assigned the formaldehyde mole fraction in the mixtures by comparison with primary mixtures generated dynamically by permeation coupled with continuous weighing in a magnetic suspension balance. The BIPM developed two dynamic sources of formaldehyde in nitrogen that provide two independent values of the formaldehyde mole fraction: the first one based on diffusion of trioxane followed by thermal conversion to formaldehyde, the second one based on permeation of formaldehyde from paraformaldehyde contained in a permeation tube. Two independent analytical methods, based on cavity ring down spectroscopy (CRDS) and Fourier transform infrared spectroscopy (FTIR) were used for the assignment procedure. Each participating institute was provided with one transfer standard and value assigned the formaldehyde mole fraction in the standard based on its own measurement capabilities. The stability of the formaldehyde mole fraction in transfer standards was deduced from repeated measurements performed at the BIPM before and after measurements performed at participating institutes. In addition, 5 control standards were kept at the BIPM for regular measurements during the course of the comparison. Temporal trends that approximately describe the linear decrease of the amount-of-substance fraction of formaldehyde in nitrogen in the transfer standards over time were estimated by two different mathematical treatments, the outcomes of which were proposed to participants. The two treatments also differed in the way measurement uncertainties arising from measurements performed at the BIPM were propagated to the uncertainty of the trend parameters, as well as how the dispersion of the dates when measurements were made by the participants was taken into account. Upon decision of the participants, the Key Comparison Reference Values were assigned by the BIPM using the largest uncertainty for measurements performed at the BIPM, linear regression without weight to calculate the trend parameters, and not taking into account the dispersion of dates for measurements made by the participant. Each transfer standard was assigned its own reference value and associated expanded uncertainty. An expression for the degree of equivalence between each participating institute and the KCRV was calculated from the comparison results and measurement uncertainties submitted by participating laboratories. Results of the alternative mathematical treatment are presented in annex of this report

NK Cell–Like Behavior of Vα14i NK T Cells during MCMV Infection

Immunity to the murine cytomegalovirus (MCMV) is critically dependent on the innate response for initial containment of viral replication, resolution of active infection, and proper induction of the adaptive phase of the anti-viral response. In contrast to NK cells, the Vα14 invariant natural killer T cell response to MCMV has not been examined. We found that Vα14i NK T cells become activated and produce significant levels of IFN-γ, but do not proliferate or produce IL-4 following MCMV infection. In vivo treatment with an anti-CD1d mAb and adoptive transfer of Vα14i NK T cells into MCMV-infected CD1d−/− mice demonstrate that CD1d is dispensable for Vα14i NK T cell activation. In contrast, both IFN-α/β and IL-12 are required for optimal activation. Vα14i NK T cell–derived IFN-γ is partially dependent on IFN-α/β but highly dependent on IL-12. Vα14i NK T cells contribute to the immune response to MCMV and amplify NK cell–derived IFN-γ. Importantly, mortality is increased in CD1d−/− mice in response to high dose MCMV infection when compared to heterozygote littermate controls. Collectively, these findings illustrate the plasticity of Vα14i NK T cells that act as effector T cells during bacterial infection, but have NK cell–like behavior during the innate immune response to MCMV infection

Trends in life science grid: from computing grid to knowledge grid

BACKGROUND: Grid computing has great potential to become a standard cyberinfrastructure for life sciences which often require high-performance computing and large data handling which exceeds the computing capacity of a single institution. RESULTS: This survey reviews the latest grid technologies from the viewpoints of computing grid, data grid and knowledge grid. Computing grid technologies have been matured enough to solve high-throughput real-world life scientific problems. Data grid technologies are strong candidates for realizing "resourceome" for bioinformatics. Knowledge grids should be designed not only from sharing explicit knowledge on computers but also from community formulation for sharing tacit knowledge among a community. CONCLUSION: Extending the concept of grid from computing grid to knowledge grid, it is possible to make use of a grid as not only sharable computing resources, but also as time and place in which people work together, create knowledge, and share knowledge and experiences in a community

Purification and Characterization of a Novel Bacteriocin Produced by Enterococcus faecalis Strain RJ-11

APPEAL FROM THE THIRD JUDICIAL DISTRICT COURT, SALT LAKE COUNTY, JUDGE LESLIE A. LEWIS PRESIDING, DENYING DEFENDANT\u27S MOTION TO SUPPRESS EVIDENC