Interferon Treatment of Ehrlich Ascites Tumor Cells: Effects on Exogenous mRNA Translation and tRNA Inactivation in the Cell Extract

We reported earlier that in cell extracts that were prepared from interferon-treated Ehrlich ascites tumor cells and preincubated and passed through Sephadex G-25 (S30_INT), the translation of exogenous mRNA (viral and host) was impaired and the impairment could be overcome to a large extent by adding a crude tRNA preparation from Ehrlich ascites tumor cells but not from Escherichia coli. We find now that the rate of inactivation of some tRNA's (especially those specific for leucine, lysine, and serine) but not those of many others is faster in S30_INT than in corresponding extracts from control cells. This increased rate of tRNA inactivation may perhaps account for the need for added RNA to overcome at least partially the impairment of translation in S30_INT. The relationship of the increased rate of tRNA inactivation to the antiviral effect of interferon is unclear. So far no significant difference has been detected in the amount of tRNA needed to overcome the impairment of encephalomyocarditis virus RNA translation in S30_INT between tRNA from interferon-treated cells and tRNA from control cells. Furthermore, no difference was found in the rate of inactivation in S30_INT between leucine-specific tRNA's from interferon-treated and from control cells. tRNA's specific for leucine and lysine were not inactivated (unless very slowly) during incubation under our conditions in an extract from interferon-treated (or from control) cells unless the extract had been passed through Sephadex G-25 or dialyzed. The translation of exogenous mRNA was, however, impaired in an extract from interferon-treated cells that had not been passed through Sephadex G-25. This impairment was apparently not overcome by added tRNA.     

Fine structure of encystment of the quadriflagellate alga,Polytomella agilis

Summary The differentiation of resting cysts of the algaPolytomella agilis was examined by electron microscopy. During encystment the free-swimming, quadriflagellate unicells lose their flagella, sink to the bottom of the culture, and form a thick cell wall. Populations of cells at various stages of encystment were collected on microscope slides placed at the bottom of the culture flasks. The mature cyst wall consists of four layers which are laid down sequentially next to the plasma membrane. Freeze-etching has shown that the first layer of wall deposited consists of fibrils which are formed partly embedded within the plasma membrane. A proliferation of rough endoplasmic reticulum and Golgi bodies is seen in early stages of encystment followed by a reduction in size or number of these organelles and of plastids in the maturing cyst. Microtubular structures, including the basal bodies, dedifferentiate and are not observed in the later stages of encystment. The redifferentiation of the swimming cell during excystment is described in the companion paper.This work was supported by grant A6353 from the National Research Council of Canada to D. L.Brown and by the Inland Waters Directorate of Environment Canada.     

Release of low density lipoprotein from its cell surface receptor by sulfated glycosaminoglycans.

The sulfated glycosaminoglycan, heparin, was found to release 125I-labeled low density lipoprotein (125I-LDL) from its receptor site on the surface of normal human fibroblasts. Measurement of the amount of 125I-LDL released by heparin permitted the resolution of the total cellular uptake of 125I-LDL at 37 degrees C into two components: first, an initial rapid, high affinity binding of the lipoprotein to the surface receptor, from which the 125I-LDL could be released by heparin, and second, a slower process attributable to an endocytosis of the receptor-bound lipoprotein, which rendered it resistant to heparin release. At 4 degrees C the amount of heparin-releasable 125I-LDL was similar to that at 37 degrees C, but interiorization of the lipoprotein did not occur at the lower temperature. The physiologic importance of the cell surface LDL receptor was emphasized by the finding that mutant fibroblasts from a subject with homozygous Familial Hypercholesterolemia, which lack the ability to take up 125I-LDL at 37 degrees C, did not show cell surface binding of 125I-LDL, as measured by heparin release, at either 4 degrees C or 37 degrees C. Although heparin released 125I-LDL from its binding site, it did not release 3H-concanavalin A from its surface receptor, and conversely, alpha-methyl-D-mannopyranoside, which released 3H-concanavalin A, did not release surface-bound 125I-LDL. When added to the culture medium simultaneously with LDL, heparin prevented the binding of LDL to its receptor and hence prevented the LDL-mediated suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity. The uptake of LDL by fibroblasts is proposed as a model of receptor-mediated adsorptive endocytosis of macromolecules in human cells.     

Adjacent repeating units of xenopus laevis 5S DNA can be heterogeneous in length

The distribution of length heterogeneity in adjacent repeating units of X. laevis 5S DNA has been examined by “cloning” 5S DNA in bacteria. Fragments of 5S DNA produced by partial digestion with Hind III and containing 1, 4, and 5 repeating units have been inserted at the single Hind III site of the tetracycline-resistance plasmid, pSC101, and the hybrid plasmids cloned in E. coli. Adjacent 5S DNA repeats in the cloned multi-repeat fragments can differ in length. This finding rules out some mechanisms which have been proposed to account for the parallel evolution of tandem repeated DNAs. The results are consistent with an unequal crossing-over mechanism and place some constraints on the molecular processes in this recombinatory event.     

The acid-catalyzed decompostion of phenacylcobalamin: evidence for the formation of an enol-Co(III) pi-complex intermediate.

Phenacylcobalamin has been synthesized and characterized by thin-layer chromatography and uv-visible spectroscopy, as well as identification of the cobalt-containing and organic products of its cleavage in acid and base and by aerobic photolysis. The major organic product from all three cleavage reactions is acetophenone and the cobalt-containing product is aquacobalamin (or hydroxocobalamin, its conjugate base). In aqueous acidic solution (pH 0 to 7.3, ionic strength 1.0 M, and 25.0 degrees C), the kinetics of the formation of aquacobalamin are biphasic representing the linear sum of two exponential terms. The pH dependence of the first-order rate constant of both phases shows a first-order dependence on proton concentration but with an inflection point ot pH 3.55 for the faster phase and at pH 4.03 for the slower phase. This behavior is interpreted in terms of the specific acid catalyzed formation of an intermediate from both "base on" and "base off" phenacylcobalamin with different second-order rate constants for each form, followed by an intermediate decompotion step with a similar formal mechanism. The nature of the intermediate is discussed and it is concluded to be a pi-complex between cob(III)alamin and the enol of acetophenone.     

Cryocrystalglobulinemia.

A 40-year-old woman had cryocrystalglobulinemia with IgG2(K1). To date, 27 other cases of spontaneous crystallization of a plasma protein have been reported. In all, the protein, a cryoglobulin, has been found to be an IgG molecule. The disease most commonly associated with this phenomenon has been multiple myeloma. None of the patients have had Raynaud''s phenomenon, but many have had purpuric skin lesions made worse by exposure to cold. In the two cases of essential cryocrystalglobulinemia, crystals were found in the peripheral blood film. Immunologic, biochemical and ultrastructural studies have so far not demonstrated any property common to all cryocrystalglobulins.