The effect of poly-L-lysine on the uptake of reovirus double-stranded RNA in macrophages in vitro

The effect of polycations on cultured mouse peitoneal macrophages has been examined. Polycations, at concentrations greater than 5 µg/ml, are toxic for macrophages) as measured by failure of the cells to exclude vital dyes. At toxic concentrations polycations bind in large amounts to nuclei and endoplasmic reticulum, while at nontoxic levels polycations bind selectively to the cell surface. Nontoxic concentrations of polycations stimulate binding of reovirus double-stranded (ds) RNA to the macrophages by forming polycation-dsRNA complexes either in the medium or at the cell surface. These complexes enter the cell in endocytic vacuoles and are concentrated in secondary lysosomes. Despite exposure to the acid hydrolases within this cell compartment, the dsRNA and the polycation (poly-L-lysine) are conserved in a macromolecular form within the vacuolar system. The mechanism(s) by which the uptake of infectious nucleic acids and the induction of interferon by dsRNA are stimulated by polycations are discussed.     

THE INTERACTION OF PARTICULATE HORSERADISH PEROXIDASE (HRP)-ANTI HRP IMMUNE COMPLEXES WITH MOUSE PERITONEAL MACROPHAGES IN VITRO

The uptake, distribution, and fate of particulate horseradish peroxidase (HRP)-anti HRP aggregates has been studied in homogeneous monolayers of mouse macrophages in vitro. Macrophages rapidly interiorize the immune complexes after binding to the cell surface. The rate of interiorization is maximal for complexes formed in a broad zone of 4-fold antibody excess to equivalence and corresponds to a rate of 10% of the administered load/10⁶ cells per hour. This rate is 4000-fold greater than the uptake of soluble HRP. The binding and endocytosis of HRP-anti HRP by macrophages is mediated by the trypsin insensitive F_c receptor. Cytochemically, intracellular HRP is localized within membrane bound vacuoles. After uptake of HRP, the enzymatic activity is degraded exponentially with a half-life of 14–18 hr until enzyme is no longer detectable. This half-life is twice as long as that previously observed for soluble uncomplexed HRP and is related to the combination of HRP with anti-HRP rather than the absolute amounts of enzyme or antibody ingested. The half-life of HRP-¹²⁵I was 30 hr. Exocytosis of cell associated enzyme or TCA precipitable counts was not detected, nor were persistent surface complexes demonstrable. The extensive capacity of macrophages to interiorize and destroy large amounts of antigen after the formation of antibody illustrates a role of this cell in the efferent limb of the immune response.     

THE ENZYMATIC IODINATION OF THE RED CELL MEMBRANE

An enzymatic iodination procedure utilizing lactoperoxidase (LPO), radioactive iodide, and hydrogen peroxide generated by a glucose oxidase-glucose system has been described and utilized for a study of the red cell membrane. 97% of the incorporated isotope is in the erythrocyte ghost and 3% is associated with hemoglobin. No significant labeling of the red cell membrane occurs in the absence of LPO or by the deletion of any of the other reagents. A 6 million-fold excess of chloride ions inhibits iodination by no more than 50%. Incorporation of up to 1 x 10⁶ iodide atoms into a single erythrocyte membrane results in no significant cell lysis. The incorporated label is exclusively in tyrosine residues as monoiodotyrosine. 10–15% of the trichloroacetic acid-precipitable radioactivity can be extracted with lipid solvents but is present as either labeled protein or ¹²⁵I. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized membrane proteins reveals only two labeled protein bands out of the 15 present, and the presence of 50-1 x 10⁶ iodide atoms per ghost does not alter this pattern. Component a has a molecular weight of 110,000, is carbohydrate poor, and represents 40% of the total label. Component b has an apparent molecular weight of 74,000, contains all of the demonstrable sialic acid, and accounts for 60% of the total label. Trypsinization of iodinated, intact red cells results in the disappearance of only component b, the appearance of labeled glycopeptides in the medium, and the absence of smaller, labeled peptides remaining in the membrane. Pronase treatment hydrolyzes component b in a similar fashion, but also cleaves component a to a 72,000 mol wt peptide which is retained in the membrane. A combination of protease treatment and double labeling with ¹²⁵I and ¹³¹I does not reveal the appearance of previously unexposed proteins.     

Fluorine-19 nuclear magnetic resonance studies of the structure of 5-fluorouracil-substituted Escherichia coli transfer RNA

19F nuclear magnetic resonance has been used to study fully active Escherichia coli tRNA1Val in which 5-fluorouracil has replaced more than 90% of all uracil and uracil-derived modified bases. The 19F spectrum of the native tRNA contains resolved resonances for all 14 incorporated 5-fluorouracils. These are spread over a 6 ppm range, from 1.8 to 7.7 ppm downfield of the standard free 5-fluorouracil. The 19F resonances serve as sensitive monitors of tRNA conformation. Removal of magnesium or addition of NaCl produces major, reversible changes in the 19F spectrum. Most affected is the lowest field resonance (peak A) in the spectrum of the native tRNA. This shifts 2-3 ppm upfield as the Mg2+ concentration is lowered or the NaCl concentration is raised. Thermal denaturation of the tRNA results in a collapse of the spectrum to a single broad peak centered at 4.7 ppm. Study of the pH dependence of the 19F spectrum shows that five incorporated fluorouracils with 19F signals in the central, 4-5.5 ppm, region of the spectrum, peaks C, D, E, F, and H, are accessible to titration in the pH 4.5-9 range. All have pKa's close to that of free 5-fluorouridine (ca. 7.5). Evidence for a conformation change in the tRNA at mildly acidic pHs, ca. 5.5, is also presented. Four of the titratable 5-fluorouracil residues, those corresponding to peaks D, E/F, and H in the 19F spectrum of fluorine-labeled tRNAVal1, are essentially completely exposed to solvent as determined by the solvent isotope shift (SIS) on transfer of the tRNA from H2O to 2H2O. These are also the 5-fluorouracils that readily form adducts with bisulfite, a reagent that reacts preferentially with pyrimidines in single-stranded regions. On the basis of these results, resonances D, E, F, and H in the middle of the 19F spectrum are attributed to 5-fluorouracils in non-base-paired (loop) regions of the tRNA. Evidence from the ionic strength dependence of the 19F spectrum and arguments based on other recent studies with fluorinated tRNAs support earlier suggestions [Horowitz, J., Ofengand, J., Daniel, W. E., & Cohn, M. (1977) J. Biol. Chem. 252, 4418-4420] that the resonances at lowest field correspond to tertiary hydrogen-bonded 5-fluorouracils. Consideration of ring-current effects and the preferential perturbation of upfield 19F resonances by the cyclophotoaddition of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen, which is known to react most readily with pyrimidines in double-stranded regions, permits initial assignment of upfield resonances to 5-fluorouracils in helical stems.(ABSTRACT TRUNCATED AT 400 WORDS)     

Seed Dormancy in Red Rice : III. Response to Nitrite, Nitrate, and Ammonium Ions

Sodium nitrite at 10 millimolar breaks dormancy of dehulled red rice (Oryza sativa). While germination is light independent, low pH conditions (pH 3) are required for maximum response. Water and buffer controls at pH 3 remain dormant. The response to nitrite occurs at 25 and 30°C but is reduced at 20°C, although nondormant seeds germinate readily at this temperature. The contact time for response to nitrite is less than 2 h at the start of imbibition. Seeds imbibed first in water show reduced germination when subsequently transferred to nitrite. Dehulled seeds show little or no response to nitrate and ammonium ions.

Intact seeds remain dormant in the presence of nitrite or nitrate unless partially dry-afterripened. The pH dependence of nitrite sensitivity is reduced in intact, afterripening seeds. In highly dormant seeds, vacuum infiltration experiments suggest that the hull restricts uptake of nitrite.

     

Regulatory mutations affecting ornithine decarboxylase activity in Saccharomyces cerevisiae.

We isolated several strains of Saccharomyces cerevisiae containing mutations mapping at a single chromosomal gene (spe10); these strains are defective in the decarboxylation of L-ornithine to form putrescine and consequently do not synthesize spermidine and spermine. The growth of one of these mutants was completely eliminated in a polyamine-deficient medium; the growth rate was restored to normal if putrescine, spermidine, or spermine was added. spe10 is not linked to spe2 (adenosylmethionine decarboxylase) or spe3 (putrescine aminopropyltransferase [spermidine synthease]). spe 10 is probably a regulatory gene rather than the structural gene for ornithine decarboxylase, since we isolated two different mutations which bypassed spe10 mutants; these were spe4, an unliked recessive mutation, and spe40, a dominant mutation linked to spe10. Both spe4 and spe40 mutants exhibited a deficiency of spermidine aminopropyltransferase (spermine synthase), but not of putrescine aminopropyltransferase. This suggests that ornithine decarboxylase activity is negatively controlled by the presence of spermidine aminopropyltransferase.     

Mechanism of the melibiose porter in membrane vesicles of Escherichia coli

The melibiose transport system of Escherichia coli catalyzes sodium--methyl 1-thio-beta-D-galactopyranoside (TMG) symport, and the cation is required not only for respiration-driven active transport but also for binding of substrate to the carrier in the absence of energy and for carrier-mediated TMG efflux. As opposed to the proton--beta-galactoside symport system [Kaczorowski, G. J., & Kaback, H. R. (1979) Biochemistry 18, 3691], efflux and exchange of TMG occur at the same rate, implying that the rates of the two processes are limited by a common step, most likely the translocation of substrate across the membrane. Furthermore, the rate of exchange, as well as efflux, is influenced by imposition of a membrane potential (delta psi; interior negative), suggesting that the ternary complex between sodium, TMG, and the porter may bear a net positive charge. Consistently, energization of the vesicles leads to a large increase in the Vmax for TMG influx, with little or no change in the apparent Km of the process. It is proposed that the sodium gradient (Na+out < Na+in) and the delta psi (interior negative) may affect different steps in the overall mechanism of active TMG accumulation in the following manner: the sodium gradient causes an increased affinity for TMG on the outer surface of the membrane relative to the inside and the delta psi facilitates a reaction involved with the translocation of the positively charged ternary complex to the inner surface of the membrane.     

Surgical Treatment of Atherosclerotic Lesions of the Subclavian Artery

Of eight patients with atherosclerotic lesions (seven occlusive, one aneurysmal) of the subclavian artery, five were operated upon because of the subclavian steal and three for severe ischemia of the hand and fingers.Removal or bypass of these lesions was uniformly successful in relieving symptoms. In most cases transcervical carotid-subclavian saphenous vein bypass graft is the treatment of choice, provided no carotid obstruction exists or, if there is obstruction, it can be dealt with at operation.