Messenger RNA orientation on the ribosome. Placement by electron microscopy of antibody-complementary oligodeoxynucleotide complexes

Messenger RNA orients on the small ribosomal subunit by base pairing with a complementary sequence in ribosomal RNA. We have positioned this ribosomal RNA segment and thus oriented the mRNA using a new technique--localization of an antibody-recognizable modified complementary oligodeoxynucleotide by electron microscopy. A synthetic oligodeoxynucleotide complementary to the message-positioning ribosomal RNA sequence was modified at either or both ends with different antigenic markers. Electron microscopy of subunit-oligodeoxynucleotide-antibody complexes allowed separate placement of each terminal marker of the oligodeoxynucleotide probe. The 5'-end of the complementary sequence contacts the subunit at the platform tip (rRNA nucleotide 1542). The message then extends along the interior side of the platform to the level of the fork of the cleft separating the platform from the subunit body, and displaced slightly to the convex side of the platform (rRNA nucleotide 1531). Based on our results and data from other laboratories, we propose a model for the positioning of messenger RNA on the 30 S subunit.     

Diethyldithiocarbamate and Nitric Oxide Synergize with Oxidants and with Membrane-Damaging Agents to Injure Mammalian Cells

The effect of diethyldithiocarbamate (DDC) and sodium nitroprusside (SNP) on the killing of endothe-lial cells and on the release of arachidonate by mixtures of oxidants and membrane-damaging agents was studied in a tissue culture model employing bovine aortic endothelial cells labeled either with ⁵¹Chromium or ³arachidonic acid. While exposure to low, subtoxic concentrations of oxidants (reagent H₂O₂, glucose-oxidase generated peroxide, xanthine xanthine oxidase, AAPH-generated peroxyl radical, menadione-generated oxidants) did not result either in cell death or in the loss of membrane-associated arachidonic acid, the addition of subtoxic amounts of a variety of membrane-damaging agents (streptolysin S, PLA₂, histone, taurocholate, wheatgerm agglutinin) resulted in a synergistic cell death. However, no significant amounts of arachidonate were released unless proteinases were also present. The addition to these reaction mixtures of subtoxic amounts of DDC (an SOD inhibitor and a copper chelator) not only very markedly enhanced cell death but also resulted in the release of large amounts of arachidonate (in the complete absence of added proteinases). Furthermore, the inclusion in DDC-containing reaction mixtures of subtoxic amounts of SNP, a generator of NO, further enhanced, in a synergistic manner, both cell killing and the release of arachidonate. Cell killing and the release of arachidonate induced by the DDC and SNP- containing mixtures of agonists were strongly inhibited by catalase, glutathione, N-acetyl cysteine, vitamin A, and by a nonpenetrating PLA_z inhibitor as well as by tetracyclines. A partial inhibition of cell killing was also obtained by 1,10-phenanthroline and by antimycin. It is suggested that DDC might amplify cell damage by forming intracellular, loosely-bound complexes with copper and probably also by depleting antioxidant thiols. It is also suggested that “cocktails” containing oxidants, membrane-damaging agents, DDC, and SNP might be beneficial for killing of tumor cells in vivo and for the assessment of the toxicity of xenobiotics in vitro.     

Multiple bands produced by interaction of a single macromolecule with carrier ampholytes during isoelectric focusing

Equilibrium isoelectric focusing patterns have been computed for reversible, carrier ampholyte-induced macromolecular isomerization reactions. The calculations predict that an amphoteric macromolecule, interacting with n species of ampholyte located at different positions along the isoelectric focusing column, can give a pattern showing n + 1 well-resolved peaks under appropriate conditions.