INTRODUCTIONhemispheres from 16 to 18-day old rat embryo were treated with a solution of 0 .125% (wt/vol) trypsin for 5 min, and the resulting cell suspension was aspirated through a Pasteur pipette to complete the dissociation . The cells were centrifuged at 400 g for 5 min and the supernate was discarded . The pellet was then resuspended in the growth medium (13), and the suspension was centrifuged for I min at 200 g . The supernate containing the dissociated single cells was transferred to a sterile bottle, final cell concentration was adjusted to 0 .1 0 .3 x 106 cells per I ml medium, and 3 ml of the above suspension was placed into polylysine-coated Petri dishes . After 30 min incubation at 37üC in an atmosphere of 95% air, 5% CO 2 , the medium containing the nonadhered cells was removed and replaced by fresh medium which was then changed every 3-4 days, depending on its acidity . Phase-contrast microscopy was employed for observation of cultures during their growth period .

Expression of human Cu/Zn superoxide dismutase (SOD) with activity comparable to the human erythrocyte enzyme was achieved in Escherichwa coli by using a vector containing a thermoinducible X PL promoter and a ,8-lactamase-derived ribosomosal binding site. The recombinant human SOD was found in the cytosol of disrupted bacteria and represented >10% of the total bacterial protein. The enzyme was purified to homogeneity by salt precipitation, gel filtration chromatography, and ion exchange chromatography. The active enzyme was obtained in high yield only when 1 mol of copper and 1 mol of zinc were incorporated into each mol of subunit during bacterial growth or by reconstitution of the apoenzyme. Human Cu/Zn SOD produced in bacteria has an apparent subunit molecular mass of 19 kDa on NaDodSO4/polyacrylamide gels. The native enzyme behaves as a dimer of 32 kDa as determined by gel filtration. Sequence analysis of the NH2 terminus revealed that the first 14 amino acids corresponded to authentic human SOD except that the NH2-terminal alanine was not acetylated. Thus, the bacterial processing system readily removes the NH2-terminal methionine residue from recombinant human SOD. and thus provide a defense against oxygen toxicity. There are three known forms of SOD that contain different metalsnamely, iron, manganese, or both copper and zinc. All of these catalyze the same reaction with high efficiency, and all operate by a similar mechanism in which the metal is the catalytic factor in the active site. These enzymes fall into several evolutionary groups. The Fe-containing SODs are found primarily in prokaryotic cells, while Cu/Zn SODs have been demonstrated in all higher eukaryotes. Mn SODs exist throughout the phylogenetic range, from microorganisms to humans (reviewed in ref. 4).Since every biological macromolecule can serve as a target for the damaging action of the abundant oxygen radical, interest has evolved in the therapeutic potential of SOD. A wide range of clinical applications has been suggested. These include prevention of oncogenesis and tumor promotion, reduction of the cytotoxic and cardiotoxic effects of anticancer drugs (5), anti-inflammatory action (6), and protection against reperfusion damage of ischemic tissues (7). In addition, there is much interest in studying the effects of SOD on the aging process (8).The exploration of the therapeutic potential of human SOD has been hindered by its limited availability. The enzyme is a dimeric metalloprotein composed of identical noncovalently linked subunits, each of 16 kDa and containing one atom of copper and one atom of zinc (9). Each subunit is composed of 153 amino acids of known sequence (10, 11). Recently, a cDNA clone containing the entire coding region of human SOD was isolated and sequenced (12, 13). The gene coding for human SOD was introduced by us into an efficient bacterial expression vector. We report here the production of gram quantities of enzymatically active human Cu/Zn SOD in Escherichia coli. MATERIALS AND METHODSBacterial Gro...

Human monoclonal antibodies have been generated from heterohybridomas obtained by fusing mouse myeloma cells with peripheral lymphocytes from patients with active Graves disease. This report characterizes four antibodies as presumptive thyrotropin receptor antibodies because they specifically inhibit thyrotropin binding and competitively inhibit thyrotropin-induced cAMP levels in human thyroid cells. Two of these antibodies, 208F7 and 206H3, are representative of autoimmune stimulators in Graves disease sera because they stimulate thyroid function in all assays, including the mouse bioassay; their ability to inhibit thyrotropin-induced cAMP increases in thyroid cells competitively is complemented by more than additive agonism at low (10 pM) thyrotropin concentrations. These stimulating antibodies interact more potently with human thyroid ganglioside preparations than with bovine thyroid or brain gangliosides; in contrast, they are poor inhibitors of 125I-labeled thyrotropin binding to liposomes containing the glycoprotein component of the human thyrotropin receptor. Antibodies 129H8 and 122G3 appear to be representative of inhibiting or "blocking" antibodies in Graves disease sera. Thus they have no intrinsic stimulatory action in assays of thyroid function but rather inhibit thyrotropin activity in the assays tested. These two antibodies do not react with human thyroid gangliosides but are strong inhibitors of thyrotropin binding to liposomes containing the high-affinity glycoprotein component from human, bovine, and rat thyroid membranes. The data unequivocally establish the pluritopic nature of the immunoglobulins in Graves disease and relate individual components or determinants of the thyrotropin receptor structure with specific autoimmune immunoglobulins.

The appearance of myelinated axons and establishment of synaptic contracts in dissociated cells from rat embryo cerebral hemispheres grown on polylysine precoated surfaces were studied by light and electron microscopy. Typical myelin lamellae ensheathing axons were observed as early as the eighth day. Immature synaptic profiles exhibiting slight membrane thickening and few synaptic vesicles were observed after seven days. At 3 weeks, mature synapses containing many synaptic vesicles and the typical irregular membrane thickening could be seen. It is suggested that the presence of polylysine may provide a support which enhances the attachment and subsequent establishment of contacts between neuronal and glial cells, resulting in the early onset of myelin formation.

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Energy-dependent internalization of 125I-labeled tetanus toxin into cultured neural cells is shown to follow an energy-independent binding process. A three-step model, involving receptor-mediated binding followed by sequestration and internalization is proposed. In the first step, binding of toxin is enhanced in appearance under low ionic strength medium, at 0-4 degrees C; it is suppressed, however, with increasing incubation temperature under physiological salt concentrations. Cell-bound toxin is displaced by approximately 35.5% when high-salt medium (physiological concentrations) is added to cells at 0-4 degrees C; the effect is further amplified at 37 degrees C. Addition of disialoganglioside GD1b (1-5 micrograms/ml) also lowers the amount of cell-associated toxin. The fraction of 125I-labeled toxin retained by the cells after exposure to high-salt medium at 0-4 degrees C or after addition of GD1b is operationally defined as sequestered toxin. This second step, characterized by a stable association of the toxin with the neural cells, is affected by both physiological salt and by 37 degrees C conditions. Lastly, an energy-dependent phenomenon of firm association of tetanus toxin with neural cells, compatible with internalization, is described. The toxin residing in this fraction is bioactive and cannot be removed by salts, gangliosides, or by treatment with protease or neuraminidase. Binding, sequestration, and internalization are mutually dependent, as they are all blocked by pretreatment of cells with neuraminidase and by an enhanced energy-independent sequestration event, which results in enhanced tetanus toxin internalization by an energy-dependent process.

nef genes from human immunodeficiency virus type 1 isolates BH10 and LAV1 (lymphadenopathy-associated virus type 1) were expressed in Escherichia coli under the deo operon promoter. The two proteins found in the soluble compartment of the bacterial lysate were purified by ion-exchange column chromatography to apparent homogeneity. Determination of the amino-terminal sequence revealed glycine as the first amino acid in the Nef protein, indicating removal of the initiator methionine during expression in E. coli. Under native conditions, the recombinant Nef protein is a monomer of 23 kilodaltons. In denaturing polyacrylamide gels, however, BH10 and LAV1 Nef proteins migrate as 28 and 26 kilodaltons, respectively. GTP binding and GTPase activity were monitored during Nef protein purification. These activities did not copurify with the recombinant Nef protein from either the BH10 or the LAV1 isolate. Purified recombinant BH10 Nef protein was used as an immunogen to elicit mouse monoclonal antibodies. A series of monoclonal antibodies were obtained which reacted with sequences at either the amino or carboxy terminus of Nef. In addition, a conformational epitope reacting with native BH10, but not LAV1, Nef was isolated.

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