The Regulation of Rat Liver Xanthine Oxidase CONVERSION IN VITRO OF THE ENZYME ACTIVITY FROM DEHYDROGENASE (TYPE D) TO OXIDASE (TYPE O)

Abstract The aerobic oxidation of xanthine by rat liver supernatant was greatly stimulated by the addition of methylene blue or of NAD+: the latter was reduced during the reaction. Storage of the supernatant at -20° brought about an enhancement of the xanthine oxidation rate measured without addition of cofactors. A similar "activation" was caused by prior incubation at 37° of the unfractionated liver homogenate, or of the supernatant separated after sonic disruption of the homogenate. The same effect was obtained by treatment with solvents, or by prior incubation at 37° of the supernatant in the presence of proteolytic enzymes or under anaerobic conditions. The presence of xanthine accelerated the effect of proteolytic enzymes and of anaerobiosis. Only the changes caused by anaerobiosis could be reversed by incubating the supernatant in air before the assay. The reaction rate was apparently unaffected by these treatments if activity of the enzyme was measured in the presence of methylene blue or of NAD+. The latter, however, was not reduced during the oxidation of xanthine by "activated" supernatants stored at -20° if the reaction was run in the presence of oxygen. If the reaction was in anaerobiosis, uric acid and a corresponding amount of NADH were formed by fresh, supernatant, and by supernatants activated at -20° or by prior incubation in anaerobiosis, but not by supernatant activated by trypsin. The hypothesis is formulated that most of the xanthine oxidase of rat liver supernatant is a dehydrogenase (Type D), and may be converted (activated) into an oxidase (Type O)

Intrinsic Absorption in the Spectrum of Mrk 279: Simultaneous Chandra, FUSE, and STIS Observations

We present a study of the intrinsic X-ray and far-ultraviolet absorption in the Seyfert 1.5 galaxy Markarian 279 using simultaneous observations from the Chandra X-ray Observatory, the Space Telescope Imaging Spectrograph aboard the Hubble Space Telescope, and the Far Ultraviolet Spectroscopic Explorer (FUSE). We also present FUSE observations made at three additional epochs. We detect the Fe K-alpha emission line in the Chandra spectrum, and its flux is consistent with the low X-ray continuum flux level of Mrk 279 at the time of the observation. Due to low signal-to-noise ratios in the Chandra spectrum, no O VII or O VIII absorption features are observable in the Chandra data, but the UV spectra reveal strong and complex absorption from HI and high-ionization species such as O VI, N V, and C IV, as well as from low-ionization species such as C III, N III, C II, and N II in some velocity components. The far-UV spectral coverage of the FUSE data provides information on high-order Lyman series absorption, which we use to calculate the optical depths and line and continuum covering fractions in the intrinsic HI absorbing gas in a self-consistent fashion. The UV continuum flux of Mrk 279 decreases by a factor of ~7.5 over the time spanning these observations and we discuss the implications of the response of the absorption features to this change. From arguments based on the velocities, profile shapes, covering fractions and variability of the UV absorption, we conclude that some of the absorption components, particularly those showing prominent low-ionization lines, are likely associated with the host galaxy of Mrk 279, and possibly with its interaction with a close companion galaxy, while the remainder arises in a nuclear outflow.Comment: To appear in 2004 May ApJS; double-column format; 58 pages, incl. 29 figures, 9 tables; minor changes to tex

Delivery of the ibosome-inactivating protein, gelonin, to Iymphoma cells via CD22 and CD38 using bispecilic antibodies

It is well established that bispecific antibodies (BsAbs) can be used effectively in targeting the ribosome-inactivating protein (RIP), saporin, against neoplastic B cells. We have now extended this delivery system for use with gelonin. By measuring antigen-binding characteristics and epitope mapping a panel of anti-gelonin MAbs using the IAsys resonant mirror bisensor, we were able to rapidly select the most suitable for making BaAbs. The Fab’ fragments from these MAbs were chemically conjugated with Fab’ from either anti-CD22 or anti-CD38. Cytotoxicity assays showed that BsAbs were highly efficient at delivering gelonin to cultured Daudi cells and achieved levels of toxicity which correlated closely with the affinity of the BsAbs. Using pairs of anti-CD22 BsAbs we were able to generate bivalent BsAb-gelonin complexes which achieved IC50 values of 2 x 10 -11 M gelonin, a potency which is equivalent to that reached by saporin in this targeting system. However, because gelonin is 5-10 times less toxic than saporin, the therapeutic ratio for gelonin is superior, making it potentially a more useful agent for human treatment. Cytotoxicity assays and kinetic analysis showed that targeting gelonin via CD38 was 2-5 times less effective than delivery through CD22. However, with a pair of BsAbs designed to co-target gelonin via CD22 and CD38, the cytotoxicity achieved equalled that obtained with a pair of anti-CD22 BsAbs (IC50 = 1 x 10 -11 M). This important result suggests that the anti-CD38 helps bind the gelonin to the cell and is then ‘dragged’ or ‘piggy-backed’ into the cell by the anti-CD22 BsAb. The implication of these findings for cancer therapy is discussed. © 1995 Stockton Press. All rights reserved

The Host Galaxies of Narrow-Line Seyfert 1s: Evidence for Bar-Driven Fueling

We present a study of the host-galaxy morphologies of narrow- and broad-line Seyfert 1 galaxies (NLS1s and BLS1s) based on broad-band optical images from the Hubble Space Telescope archives. We find that large-scale stellar bars, starting at ~1 kpc from the nucleus, are much more common in NLS1s than BLS1s. Furthermore, the fraction of NLS1 spirals that have bars increases with decreasing full-width at half-maximum (FWHM) of the broad component of H-beta. These results suggest a link between the large-scale bars, which can support high fueling rates to the inner kpc, and the high mass-accretion rates associated with the supermassive black holes in NLS1s.Comment: 19 pages, 4 figures (1a, 1b, 2, and 3), Accepted for publication in the Astronomical Journa

BeppoSAX observations of Narrow-Line Seyfert 1 galaxies: II. Ionized iron features in Arakelian 564

The BeppoSAX observations of the bright Narrow-Line Seyfert 1 galaxy Ark 564 are presented along with a high quality optical spectrum taken at the 1.5m telescope at La Silla. The 0.1-10 keV X-ray spectrum is characterized by a strong soft component which is best described by blackbody-like emission with a temperature of about 160 eV. At higher energies a steep (Gamma = 2.4) power-law tail is present. There is evidence of an ionized reflector in the form of an iron line and edge. We do not find significant evidence of soft X-ray features if the spectrum is modelled with a two component continuum. The optical and X-ray spectral properties support the hypothesis of a high accretion rate onto a low mass black hole.Comment: 9 pages, 7 figures, accepted by Astronomy and Astrophysic

Increased levels of RNA oxidation enhance the reversion frequency in aging pro-apoptotic yeast mutants

Despite recent advances in understanding the complexity of RNA processes, regulation of the metabolism of oxidized cellular RNAs and the mechanisms through which oxidized ribonucleotides affect mRNA translation, and consequently cell viability, are not well characterized. We show here that the level of oxidized RNAs is markedly increased in a yeast decapping Kllsm4Δ1 mutant, which accumulates mRNAs, ages much faster that the wild type strain and undergoes regulated-cell-death. We also found that in Kllsm4Δ1 cells the mutation rate increases during chronological life span indicating that the capacity to han- dle oxidized RNAs in yeast declines with aging. Lowering intracellular ROS levels by antioxidants recovers the wild- type phenotype of mutant cells, including reduced amount of oxidized RNAs and lower mutation rate. Since mRNA oxidation was reported to occur in different neurodegen- erative diseases, decapping-deficient cells may represent a useful tool for deciphering molecular mechanisms of cell response to such conditions, providing new insights into RNA modification-based pathogenesis