Thomas G. Burrage

Rabies virus binding at neuromuscular junctions

Morphological, immunocytochemical, biochemical, and immunological techniques have been used to describe rabies virus binding to a sub-cellular unit and molecular complex at the neuromuscular junction (NMJ). Early after infection in vivo, virus antigen and virus particles were found by immunofluorescence, electron microscopy and immunoelectron microscopy in regions of high density acetylcholine receptors (AChR) at NMJs. One monoclonal antibody (alpha-Mab) to the alpha subunit of the AChR blocked attachment of radio-labeled rabies virus to cultured muscle cells bearing high density patches of AChR. A sub-cellular structure, resembling an array of AChR monomers, bound both rabies virus antigens and alpha-Mab. By immunoblotting with electrophoretically transferred motor endplate proteins, rabies virus proteins and alpha-Mab bound to two proteins of 43 000 and 110 000 daltons. A rabies virus glycoprotein antibody detected virus antigen bound to the 110 000 dalton protein. An auto-immune (anti-idiotypic) response followed immunization of mice with rabies virus glycoprotein antigen; the antibody was directed to the 110 000 dalton protein. This auto-antibody altered the kinetics of neutralization by rabies virus antibody and induced the formation of rabies virus antibody after inoculation of mice. These results define, at the neuromuscular junction, a rabies virus receptor which may be part of the acetylcholine receptor complex.

Ultrastructural characterization of surface specializations containing high-density acetylcholine receptors on embryonic chick myotubes in vivo and in vitro

Abstract Surface specializations (patches) on myotubes from latissimus dorsi muscles of embryonic chick between 10 and 15 days of incubation and on 5-day-cultured chick thigh myotubes were characterized by heavy metal stains and the binding of horseradish peroxidase-labeled α-bungarotoxin (HRP-α-BTX) for the localization of acetylcholine receptors (AChR). The specializations consisted of slightly raised surface ridges (0.1 to 0.5 μm in width) and wider regions (2–5 μm) sometimes composed of a series of the smaller ridges. The specialized zones were characterized by an external coating of amorphous, fine-textured material, considerably more extensive than the sparse, developing basement lamina occurring elsewhere on the surface. On the internal surface and coextensive with the external layer, dense material was applied immediately adjacent to the plasma membrane. Cytoplasmic microfilaments and microtubules came into close proximity to the submembranous density. HRP-α-BTX binding occurred at specialized regions, indicating the presence of a high density of nicotinic AChR at these sites. Not all of the specialized regions nor the entire extent of the broader patches bound the conjugate. All regions binding the conjugate, however, had associated specializations. Reactive coated vesicles were associated with patch regions, indicating they may be involved in the transport of receptor-bearing membrane and coat material to the surface. The specialized regions binding HRP-α-BTX were usually widely separated on uninnervated surfaces. Reactive patches were invariably present at sites of nerve-muscle contact in vivo . In cultured myotubes, large reactive patches were more abundant and more extensive than in vivo . These regions correspond to the “hot spots” observed at the light-microscopic level. Reactive patches were most abundant on the lateral and basal surfaces of cells, on cell processes, and where cells contacted the substrate or other cells. These studies show that external and internal membrane specializations are associated with a high density of AChR in the absence of innervation. Constituents of these specializations may be involved in the localization or positioning of a high density of AChR at these sites or contain components which establish these regions as preferential sites of recognition or contact by the nerve.

Immunoelectron microscopic localization of rabies virus antigen in central nervous system and peripheral tissue using low-temperature embedding and protein A-gold

A protein A-gold technique was used in conjunction with low temperature embedding to visualize ultrastructurally mature virions and sites of viral replication in the brains of rabies virus-infected mice after peripheral inoculation of virus. The association of viral profiles and gold particles with synaptic membranes, microtubules and rough endoplasmic reticulum suggested a mechanism of rabies virus transport within the central nervous system. Early interactions of inflammatory cells with the virus inoculum were characterized by phagocytosis by non-degranulating neutrophils and mononuclear cells.

African swine fever virus infection in Ornithodoros ticks

African swine fever virus (ASFV) is an arbovirus which is vectored by soft ticks of the Ornithodoros spp. and in the sylvatic cycle infects wart hogs and bush pigs. ASFV infection of domestic swine causes a high mortality disease. On the other hand, ASFV infection of the tick can result in a high-titered and persistent infection depending upon the ASFV isolate and the tick combination. Recently, morphological, classical virology (titration) and recombinant ASFV have been used to study the cellular, molecular and genetic interactions that occur between ASFV and its host tick.

Distribution of Cell Surface Saccharides and Fibronectin on Cultured Chick Myotubes: Relationship to Acetylcholine Receptor Clusters

This study examines the distribution of glycoconjugates on the surface of cultured chick myotubes with a battery of lectins labeled with ferritin or horseradish peroxidase. In addition, the distribution of a specific glycoprotein, fibronectin, is investigated by immunocytochemistry. Particular attention is paid to the localization of these substances in specialized patches on the cell surface previously shown to contain a high density of acetylcholine receptors as demonstrated with horseradish peroxidase labeled alpha-bungarotoxin. The specialized patches are found to bind a greater amount of concanavalin A, ricin agglutinin I, and soy bean agglutinin and a lesser amount of wheat germ agglutinin than the general myotube surface. Limulus lectin is distributed over the entire cell surface while other lectins do not bind to any sites. The surface patches contain a high density of acetylcholine receptors as shown by double labeling with ferritin-labeled lectins and peroxidase labeled alpha-bungarotoxin. Fibronectin occurs in high concentration at the surface patches and is present over other regions of the cell surface as well. These results reveal differential patterns of distribution of glycoconjugates and fibronectin over the myotube surface. These regional differences may be related to the distribution of acetylcholine receptors or to recognition and attachment by the innervating nerve.

Hormonal Induction of a Heterogeneous Population of Storage Granules in GH4C1 Pituitary Tumor Cells

The mechanism by which endocrine cells regulate the amount of newly synthesized hormone that is sorted into storage granules is unknown.’ Model systems suitable for study have been lacking. We have developed a model system where the cellular content of a hormone and the number of storage granules can be regulated independently of hormone synthesis or degradation and have presented some of the details of this model here. GH,C, cells are a rat pituitary tumor cell strain that secretes prolactin and growth hormone, but stores very little of these hormones and contains almost no secretory granules. We plated these cells at low density (10 cells/mm*) and maintained them in DMEM:Ham’s F10 medium with 15% gelded horse serum. After three days, we treated the cells with 17P-estradiol (1 nM), insulin (300 nM), and epidermal growth factor (10 nM) for five days in the above medium and found that the cellular content of prolactin was increased by over 3@fold, but the accumulation of prolactin in the medium was increased only sixfold. Growth hormone storage and secretion were both decreased by the hormone treatment. To determine if the increase in cellular prolactin was accompanied by an increase in secretory granules, we compared the numbers of granules in electron micrographs of ultrathin sections from untreated GH,C, cells and from cells treated with the combined hormone regimen. Control cells contained 1.0 2 0.2 granules per cell section (n = 130), while hormone-treated cells contained 45.6 5 4.5 granules per cell section (n = 130), a nearly 50-fold increase in granule number (FIGS. 1A and 1B). To determine if all of the granules contained prolactin, ultrathin sections of Lowicryl-embedded GKC, cells were stained for prolactin by the Protein-A-gold technique. The density of immunoreactive sites for prolactin was determined in 208 granules from five separate cells. Only 75% of the granules stained for prolactin; most of the granules that did not stain were the less electron-opaque granules (FIGS. 2A and 2B).