Radioimmune, radiobinding and HPLC analysis of 2-5A and related oligonucleotides from intact cells

The enzyme (2-5A synthetase) which synthesizes ppp(A2'p)nA where n=2 to 4 (collectively referred to as 2-5A) is widely distributed in a variety of cells and tissues in amounts which increase response to interferon and vary with growth and hormone status. 2-5A activates a nuclease which inhibits protein synthesis. The non-phosphorylated 'core' of 2-5A ((A2'p)nA, n=2 to 4) can inhibit DNA synthesis and cell growth. Here we describe convenient and sensitive radioimmune (RI) and radiobinding (RB) assays for core and 2-5A. In combination with more satisfactory high performance liquid chromatography (HPCL) methods using reverse-phase C18 columns, these assays have been used to detect core and 2-5A in crude extracts from interferon-treated cells. The novel 2-5A synthetase products NAD2'p5' A2'p5'A and A5'p45'A2'p5'A2'p5'A (ref. 13), which can also be detected using the RB assay, were not found in significant amounts. The natural occurrence of core has not been described previously.     

Evidence for fixed charge in the nexus

The nexus or gap junction has been characterized as a low-resistance junction as well as a highly permeable junctional membrane to many molecules. The transfer of electrical current from one cell interior to another, the aqueous solubility of dyes used to trace cell to cell communication and the fact that these molecules move across the nexus more rapidly than the plasma membrane have led to the hypothesis of an aqueous channel in the junction. Both Ca2+ (ref.11) and H+ (ref. 12) are thought to alter nexal membrane conductance, and a voltage-sensitive gate has been demonstrated within the junction. Recently, Flagg-Newton et al. have concluded that mammalian junctions may contain fixed charge or be of smaller diameter than arthropod junctions. Here we have investigated these alternatives by examining the permeability of nexuses of septa of the median giant axon of Lumbricus terrestris with various derivatives of fluorescein. Both carboxyfluorescein and aminofluorescein were found to have depressed permeabilities relative to their predicted permeabilities based on molecular size and weight (MW). Flourescein diffusion was significantly suppressed in axons pre-injected with aminofluorescein but carboxyfluorescein had no such effect (Table 1). These data suggest the existence of fixed anionic charge within the nexal channel which may have affinity for amino groups.     

Neuronal argyrophilia in early degenerative states: A light and electron-microscopic study of theGlees andNauta techniques

Zusammenfassung Die Grundlagen für die Silbermethoden nachNauta undGlees werden elektronen-mikroskopisch untersucht und die Verteilung der Silbergranula im Gewebe festgestellt.