Samuel C. Silverstein

The mechanisms of reovirus uncoating and gene activation in vivo.

Abstract Reovirus is concentrated in lysosomes soon after penetrating L cells. Hydrolases contained within the lysosomal compartment digest the outer layer of viral capsomeres but do not degrade the viral core proteins which form the subviral particle (SVP). Parental SVPs have been recovered from reovirus-infected cells. These SVPs are similar, but not identical, in morphology and protein composition to SVPs produced in vitro by chymotrypsin digestion of intact virus. Moreover, they catalyze the synthesis of ssRNA molecules which hybridize specifically with all ten segments of the double-stranded viral genome.

A fluorescence quenching technique using trypan blue to differentiate between attached and ingested glutaraldehyde-fixed red blood cells in phagocytosing murine macrophages

A fluorescence quenching method, using trypan blue, is described for quantifying the ingestion of either glutaraldehyde-fixed sheep red blood cells or antibody coated glutaraldehyde-fixed sheep red blood cells by murine peritoneal macrophages. This method is based on the observations that glutaraldehyde-fixed red blood cells fluoresce at about 585 nm when excited at 490 nm and that when trypan blue is in intimate contact with fixed red blood cells, the fluorescence is converted from a chartreuse to a red color. Thus, the ingestion of fixed erythrocytes by murine macrophages can be monitored by fluorescence microscopy after the addition of 1 mg/ml of trypan blue at the end of the assay. Extracellular glutaraldehyde-fixed red blood cells fluoresce a red color whereas the intracellular particles continue to fluoresce a chartreuse color. This method offers a simple and convenient technique for rapidly distinguishing between intracellular and extracellular glutaraldehyde-fixed red blood cells in macrophages.

Regulation of the reovirus RNA transcriptase by a viral capsomere protein

Abstract Reovirus subviral particles combine spontaneously in vitro with soluble viral proteins to form a particle which is similar to the intact virion in structure and buoyant density. The RNA transcriptase activity which is manifest in the subviral particle becomes masked in the reassembled virions. The viral protein responsible for these structural and functional alterations has been identified as the major outer capsomere protein, σ3.

Messenger activity in mammalian cell-free extracts of reovirus single-stranded RNA prepared in vitro

Abstract Reovirus ssRNA synthesized in vitro by a viral-specific RNA transcriptase displays messenger activity when incubated with supplemented S150 extracts and purified ribosomes prepared from L-cells. Pre-treatment of the ssRNA with HCHO enhances amino acid incorporation several-fold. 80S ribosomes and 60S and 40S ribosomal subunits are equally effective in supporting polypeptide synthesis.

Role of 2-deoxy-D-glucose in the inhibition of phagocytosis by mouse peritoneal macrophage

2-Deoxy-D-glucose inhibits Fc and complement receptor-mediated phagocytosis of mouse peritoneal macrophages. To understand the mechanism of this inhibition, we analyzed the 2-deoxy-D-glucose metabolites in macrophages under phagocytosis inhibition conditions and conditions of phagocytosis reversal caused by glucose, mannose and 5-thio-D-glucose, and compared their accumulations under these conditions. Macrophages metabolized 2-deoxy-D-glucose to form 2-deoxy-D-glucose 6-phosphate, 2-deoxy-D-glucose 1-phosphate, UDP-2-deoxy-D-glucose, 2-deoxy-D-glucose 1, 6-diphosphate, 2-deoxy-D-gluconic acid and 2-deoxy-6-phospho-D-gluconic acid. The level of bulk accumulation as well as the accumulation of any of these 2-deoxy-D-glucose metabolites did not correlate with changes in macrophage phagocytosis capacities caused by the reversing sugars. 2-Deoxy-D-glucose inhibited glycosylation of thioglycolate-elicited macrophage by 70-80%. This inhibition did not cause phagocytosis inhibition, since (1) the reversal of phagocytosis by 5-thio-D-glucose was not followed by increases in the incorporation of radiolabelled galactose, glucosamine, N-acetylgalactosamine or fucose; (2) cycloheximide at a concentration that inhibited glycosylation by 70-80% did not affect macrophage phagocytosis. The inhibition of protein synthesis by 2-deoxy-D-glucose similarly could not account for phagocytosis inhibition, since cycloheximide, when used at a concentration that inhibited protein synthesis by 95%, did not affect phagocytosis. 2-Deoxy-D-glucose lowered cellular nucleoside triphosphates by 70-99%, but their intracellular levels in the presence of different reversing sugars did not correlate with the magnitude of phagocytosis reversal caused by these sugars. The results show that 2-deoxy-D-glucose inhibits phagocytosis by a mechanism distinct from its usual action of inhibiting glycosylation, protein synthesis and depleting energy supplies, mechanisms by which 2-deoxy-D-glucose inhibits other cellular processes.

Membrane Receptors and the Regulation of Mononuclear Phagocyte Effector Functions

Mononuclear phagocytes form a body-wide system of cells (1) that originate from precursors in the bone marrow, circulate in the blood, and emigrate from the vascular compartment into the tissues where they spend the major portion of their life span. At the time of their emigration from the blood into the tissues mononuclear phagocytes are extremely immature cells. In the tissues they differentiate into their adult form, the macrophage. Macrophages in different tissues develop characteristic metabolic and structural properties. Thus alveolar macrophages develop a high capacity for oxidative metabolites (2), while splenic and peritoneal macrophages derive the major proportion of their metabolic energy from anerobic glycolysis (3). Inflammatory macrophages may develop into sheets of interlocking epithelioid cells, or fuse to form multinucleate giant cells (4). In the bones they fuse to produce osteoclasts (5).

THE INHIBITORY EFFECT OF 2-DEOXY-D-GLUCOSE ON Fc AND C3b RECEPTOR-MEDIATED PHAGOCYTOSIS IN PHAGOCYTIC CELLS

Publisher Summary This chapter discusses the inhibitory effect of 2-deoxy- D -glucose on Fc and third component of complement (C3b) receptor-mediated phagocytosis in phagocytic cells. The ability to phagocytize particulate materials is a function of many mammalian cells. Macrophages contain on their plasma membranes receptors for these ligands, the cleaved C3b and the Fc portion of immunoglobulin G (IgG). The ingestion of immunoglobulin G- or complement-coated particles proceeds by the sequential and circumferential interaction of these ligands with the Fc or complement receptors on the plasma membrane of the phagocyte. The ultrastructural examination of these preparations verified that the ingested lymphocytes were diffusely coated with anti-immunoglobulin that was labeled with the enzyme peroxidase to form an electron-dense reaction product. 2-Deoxy- D -glucose inhibits the phagocytosis of immunoglobulin G, or complement-coated sheep erythrocytes by mouse peritoneal macrophages, but does not inhibit the phagocytosis of latex or zymosan particles by the same cells. The capacity of 2-deoxy-O-glucose-treated macrophages to ingest latex and zymosan particles indicates that 2-deoxy-O-glucose does not have a toxic effect on the ingestion process.