Expression, Purification and Characterization of Ricin vectors used for exogenous antigen delivery into the MHC Class I presentation pathway

Disarmed versions of the cytotoxin ricin can deliver fused peptides into target cells leading to MHC class I-restricted antigen presentation [Smith et al. J Immunol 2002; 169:99-107]. The ricin delivery vector must contain an attenuated catalytic domain to prevent target cell death, and the fused peptide epitope must remain intact for delivery and functional loading to MHC class I molecules. Expression in E. coli and purification by cation exchange chromatography of the fusion protein is described. Before used for delivery, the activity of the vector must be characterized in vitro, via an N-glycosidase assay, and in vivo, by a cytotoxicity assay. The presence of an intact epitope must be confirmed using mass spectrometry by comparing the actual mass with the predicted mass

Far Infrared and Submillimeter Emission from Galactic and Extragalactic Photo-Dissociation Regions

Photodissociation Region (PDR) models are computed over a wide range of physical conditions, from those appropriate to giant molecular clouds illuminated by the interstellar radiation field to the conditions experienced by circumstellar disks very close to hot massive stars. These models use the most up-to-date values of atomic and molecular data, the most current chemical rate coefficients, and the newest grain photoelectric heating rates which include treatments of small grains and large molecules. In addition, we examine the effects of metallicity and cloud extinction on the predicted line intensities. Results are presented for PDR models with densities over the range n=10^1-10^7 cm^-3 and for incident far-ultraviolet radiation fields over the range G_0=10^-0.5-10^6.5, for metallicities Z=1 and 0.1 times the local Galactic value, and for a range of PDR cloud sizes. We present line strength and/or line ratio plots for a variety of useful PDR diagnostics: [C II] 158 micron, [O I] 63 and 145 micron, [C I] 370 and 609 micron, CO J=1-0, J=2-1, J=3-2, J=6-5 and J=15-14, as well as the strength of the far-infrared continuum. These plots will be useful for the interpretation of Galactic and extragalactic far infrared and submillimeter spectra observable with ISO, SOFIA, SWAS, FIRST and other orbital and suborbital platforms. As examples, we apply our results to ISO and ground based observations of M82, NGC 278, and the Large Magellenic Cloud.Comment: 54 pages, 20 figures, accepted for publication in The Astrophysical Journa

Free ricin A chain, proricin, and native toxin have different cellular fates when expressed in tobacco protoplasts.

The catalytic A subunit of ricin can inactivate eukaryotic ribosomes, including those of Ricinus communis where the toxin is naturally produced. How such plant cells avoid intoxication has remained an open question. Here we report the transient expression of a number of ricin A chain-encoding cDNA constructs in tobacco protoplasts. Ricin A chain entered the endoplasmic reticulum lumen, where it was efficiently glycosylated, but it was toxic to the cells and disappeared with time in a brefeldin A-insensitive manner, suggesting reverse translocation to the cytosol and eventual degradation. Proricin (the natural precursor form containing A and B chains joined together by a linker sequence) was glycosylated, transported to the vacuole, and processed to its mature form, but was not toxic. Free ricin A chain and proricin were not secreted, whereas free ricin B chain was found entirely in the extracellular medium. The coexpression of ricin A and B chains resulted in the formation of disulfide-linked, transport-competent heterodimers, which were secreted, with a concomitant reduction in the observed cytotoxicity. These results suggest that the production of ricin as a precursor is essential for its routing to the vacuole and for protection of ricin-producing cells

The FeSi phase diagram to 150 GPa

The melting curve of FeSi has been determined to 150 GPa in the laser-heated diamond anvil cell (LH-DAC) on the basis of discontinuities in the power versus temperature function. A multianvil experimental cross-check at 12 GPa using textural criteria as a proxy for melting is in good agreement with our LH-DAC results. The melting point of FeSi reaches ∼4000 K at the core mantle boundary and an extrapolated value of 4900 K at the inner-core boundary (ICB). We also present the melting curve as determined by the Lindemann melting law; this agrees well with our experimental curve to 70 GPa and then diverges to higher temperatures, reaching 6200 K at the ICB. These temperatures are substantially higher than previous LH-DAC determinations. The boundary of the ε-FeSi → CsCl-FeSi subsolidus transition has also been determined by synchrotron-based X-ray diffraction at high pressures, and the results confirm a negative Clapeyron slope for the transition. We conclude that if present, FeSi is likely to be solid within the D″ layer and is unlikely to be present within the inner core for any plausible bulk core silicon content.9 page(s