Disruption of the Golgi by brefeldin A (BFA) has been reported to block fast axonal transport and axonal growth. We used compartmented cultures of rat sympathetic neurons to investigate its effects on slow axonal transport. BFA (1 micro g/ml) applied to cell bodies/proximal axons for 6-20 h disrupted the Golgi, reversibly blocked axonal growth, and reversibly blocked anterograde transport of all proteins, including tubulin. The retrograde transport of nerve growth factor (NGF) was also blocked. The phosphorylation of Erk1 and Erk2 in response to NGF was unaffected after 6 h of treatment with BFA, suggesting that the block of axonal transport was specific and direct. Consistent with its principal site of action at the Golgi, no effects were observed when BFA was applied only to the distal axons. Block of fast anterograde and retrograde axonal transport is consistent with the role of the Golgi in supplying transport vesicles. Block of slow axonal transport was surprising, and further results indicated that transport of tubulin en route along the axon was arrested by application of BFA to the cell bodies, suggesting that a continuous supply of anterograde transport vesicles from the Golgi is required to maintain slow axonal transport of cytoskeletal proteins.

The proteins needed for growth and maintenance of the axon are generally believed to be synthesized in the cell bodies and delivered to the axons by anterograde transport. However, recent reports suggest that some proteins can also be synthesized within axons. We used [35S]methionine metabolic labeling to investigate axonal protein synthesis in compartmented cultures of sympathetic neurons from newborn rats. Incubation of distal axons for 4 hr with [35S]methionine resulted in a highly specific pattern of labeled axonal proteins on SDS-PAGE, with 4 prominent bands in the 43–55 kDa range. The labeled proteins in axons were not synthesized in the cell bodies, because they were also produced by axons after the cell bodies had been removed. Two of the proteins were identified by immunoprecipitation as actin and β-tubulin. Axons synthesized <1% of the actin and tubulin synthesized in the cell bodies and transported into the axons, and 75–85% inhibition of axonal protein synthesis by cycloheximide and puromycin failed to inhibit axonal elongation. Nonetheless, the specific production by axons of the major proteins of the axonal cytoskeleton suggests that axonal protein synthesis arises from specific mechanisms and likely has biological significance. One hypothetical scenario involves neurons with long axons in vivo in which losses from turnover during axonal transport may limit the availability of cell body synthesized proteins to the distal axons. In this case, a significant fraction of axonal proteins might be supplied by axonal synthesis, which could, therefore, play important roles in axonal maintenance, regeneration, and sprouting.

In the process of myogenesis, cadherins are thought to be involved in the initial cell-cell recognition and possible initiation of myoblast fusion to form multinucleated myotubes. Of the cadherins, M-cadherin, but not N-cadherin, is down-regulated upon inhibition of myogenesis, suggesting that M-cadherin may be a key receptor involved in myogenesis. M-cadherin binds in a calcium-dependent manner, and depletion of divalent cations inhibits myoblast fusion. We analyzed the regulation of M-cadherin protein and mRNA levels in primary rat myogenic cultures in the presence and absence of divalent cations. In untreated cultures M-cadherin was localized to various myogenic cell-cell contacts. M-cadherin protein and mRNA levels showed a peak at day 2 after the initiation of growth. When divalent cations were removed from the cell culture medium, myoblast fusion was inhibited and immunocytochemical analysis revealed a failure of M-cadherin to localize to cell-cell contacts. Analysis of M-cadherin protein and mRNA in fusion-inhibited cultures still revealed a peak at day 2. However, by day 3, M-cadherin protein levels in the fusion-inhibited cultures were reduced in both the detergent-soluble and -insoluble fractions in comparison with the untreated cultures. Interestingly, beta-catenin, a protein associated with cadherins, was frequently observed at intercellular contacts in the fusion-inhibited cultures. We could also show that the intracellular levels of beta-catenin protein remained constant regardless of the presence or absence of divalent cations. In summary, the dynamic regulation of M-cadherin in muscle-fusion-related events is an indication of the importance of M-cadherin for myoblast fusion and myogenic differentiation.

Beta2-adrenergic receptors (beta2AR) are present on both lymphocytes and skeletal muscle cells. Antibodies and T cells that react with these receptors are present in patients with myasthenia gravis (MG). Immune reactivity against the beta2AR may thus modify both the immune and the muscle functions in MG. In this study, we analysed the density and affinity of beta2AR on peripheral blood mononuclear cells using a radioligand binding assay. The density (Bmax) of the receptor on cells from patients with MG was significantly lower than that on cells from patients with other neurological disorders and healthy individuals. The affinity (Kd) of the receptor and the concentration of the second messenger, cAMP, in the cells did not differ between the groups. Serum antibodies against beta2AR were demonstrated in 22% of 27 MG patients vs. 0% of 26 healthy controls. Incubation of cells with serum or purified IgG containing antibodies against the beta2AR resulted in a decline in ligand binding of the receptor in samples from three out of five patients. Thus, this study suggests that a downregulation of the beta2AR may occur in MG. This downregulation might be of importance in the patho-genesis of the disease and its symptoms.

Eighteen percent of patients with myasthenia gravis (MG) have serum antibodies against a synthetic peptide corresponding to the second extracellular loop of the human beta(2)AR (residues 172-197). In this study we examined T and B cell responses to the peptide, using assays to detect individual cells secreting interferon-gamma (IFN-gamma) and IL-4 or antibodies against the peptide, and by measuring thymidine incorporation in response to the peptide. The peptide from the beta(2)AR induced cytokine secretion from blood mononuclear cells in 67% of MG patients, compared with 14-28% of the control groups. Cells secreting antibodies binding to the peptide were present in 54% of MG patients and in 19-28% of controls. The numbers of beta(2)AR-reactive cells were higher in MG patients than in controls. Peptide-induced increase in thymidine incorporation in cells was also more frequently demonstrated in patients (26%) compared with controls (about 10%). Activation of cells was dependent on monocytes and on MHC class II DR antigen. Based on the pattern of the cytokine secretion induced, beta(2)AR-reactive T cells comprise both T helper type-1 and type-2 subsets. In addition, control peptide-reactive T and B cells were much less frequently demonstrated in the patients, and the number of such cells did not differ between the groups. Our results show that beta(2)AR-reactive cells are present in most patients with MG. Such autoreactive antibodies and cells might play a role in the pathogenesis of the disease by influencing the function of skeletal muscle and immune systems.

Although autoantibodies against the nicotinic acetylcholine receptor are the characteristic feature of the autoimmune disease myasthenia gravis (MG), no strong correlation is found between the autoantibody titer and the degree of clinical severity. Numerous studies have attempted to detect the presence of other autoantibody populations that might have a role in the pathology of the disease. We report, for the first time, that 18% of the MG patients we screened have antibodies in their serum to a peptide corresponding to the second extracellular loop of the human β2-adrenergic receptor (residues 172–197). Affinity purified antibodies to the β2-adrenergic receptor peptide 172–197 reacted with the human β2-adrenergic receptor protein obtained from transfected E. coli cell membrane extracts, but did not cross-react with the human AChR. Sufficient material was obtained from nine MG patients and it was found that the gamma globulin fraction from these patients immunoprecipitated the receptor, and that affinity purified IgG to peptide 172–197 competed for receptor binding with the β-antagonist iodo-cyanopindolol. Using truncated peptides or amino acid modification procedures, no immunodominant B-cell epitope could be detected within region 172–197. Thus, a subpopulation of MG patients possesses anti-β2-adrenergic receptor antibodies which are a distinct set of autoantibodies with possible pharmacological activity.

In myasthenia gravis the production of anti-acetylcholine receptor antibodies is modulated by acetylcholine receptor-specific T cells. Most B- and T-cell epitopes are located on the alpha-subunit of the receptor. In order to map the fine specificity of the antigen-specific T cells in myasthenia gravis, T-cell stimulation in response to 70 hexapeptides was studied in 24 patients and 24 healthy individuals. The hexapeptides overlapped with one amino acid and represented residues 10-84 of the NH2-terminal part of the alpha-subunit of the receptor. The IFN-gamma secretion from single T cells was used to detect T-cell stimulation.A significant difference in the T-cell response to several of the peptides was found between patients and healthy controls. The majority of the hexapeptides induced T-cell stimulation in at least one of the patients. Peptide-induced T-cell stimulation was evident in all but one of the patients. The results indicate that different epitopes and multiple T-cell clones are involved in the T-cell recognition of the acetylcholine receptor.

A pair of identical twins, 47 years of age, who have been discordant for myasthenia gravis for 15 years were studied with regard to clinical status, neuromuscular function, and presence and properties of myasthenia specific autoantibodies. The autoantibody repertoire was tested in serum, as produced by peripheral lymphocytes in culture and as revealed by B cell lines. The healthy twin had no clinical signs of myasthenia and no signs of impaired neuromuscular function on electrophysiological tests. The autoantibody repertoire and the avidity of the anti-receptor antibodies were similar in both individuals. Epstein-Barr virus transformation of peripheral lymphocytes revealed a higher incidence of B cells committed to make autoantibodies in the healthy twin than in her myasthenic sister.

Fluctuations in idiotypic and anti-idiotypic Ab levels over time in two myasthenia gravis patients were found to vary either inversely with one another or in relation to one another. A pair of 46-year-old female twins in which one of the twins developed myasthenia gravis while the other remained healthy were also studied. Ab specificities and immunoglobulin specificities available in the B cell repertoire were found to be similar in both twins.

The network theory predicts that a subpopulation within the antiidiotypic (anti-Id) antibody response will be the internal image of the priming stimulus. In myasthenia gravis, a portion of the anti-acetylcholine-receptor (anti-AChR) antibody repertoire is directed against the ligand-binding site. These antibodies would be expected to elicit an "anti-idiotype" which is the internal image of the receptor-binding site and hence may also bind cholinergic ligands. We have utilized this theoretical specificity to isolate anti-Id antibodies with AChR-like ligand-binding properties from the serum of 4 myasthenia gravis patients using a choline affinity column. Affinity-purified antibodies from one patient were characterized and found to exhibit binding properties similar to the AChR for various cholinergic ligands.

In myasthenia gravis, a portion of the anti-acetylcholine receptor antibody repertoire is directed against the ligand binding site. The network theory predicts the presence of an anti-Id which resembles the binding site of the receptor and which may also bind cholinergic ligands. We have utilized this theoretical specificity to isolate these anti-Id from the serum of one myasthenia gravis patient using a choline affinity column. These antibodies exhibited binding properties similar to the receptor for various cholinergic ligands and were found to be predominantly of IgG subclass 3.

Myeloma immunoglobulins, once thought to be without any immunological function, are now known to be reactive with many antigens, including self components. We have screened 149 monoclonal immunoglobulin samples and found 14 (9%) to react with the human acetylcholine receptor (AChR). Such anti‐AChR antibodies are often associated with the autoimmune disease myasthenia gravis (MG). The anti‐AChR binding of the myeloma components was restricted to the F(ab′) 2 fragment and the affinities were similar to anti‐AChR antibodies isolated from MG patients. Despite the presence of anti‐AChR antibodies none of the patients exhibited any symptoms of MG.

Annals of the New York Academy of SciencesVolume 540, Issue 1 p. 520-522 Probing for the Main Immunogenic Region of the Human Acetylcholine Receptor H. ENG, H. ENG Department of Medicine Karolinska Hospital Stockholm, SwedenSearch for more papers by this authorH. JÖRNVALL, H. JÖRNVALL Departments of Medical Chemistry, Karolinska Institute Stockholm, SwedenSearch for more papers by this authorM. CARLQUIST, M. CARLQUIST Department of Medicine Karolinska Hospital Stockholm, SwedenSearch for more papers by this authorA. K. LEFVERT, A. K. LEFVERT Department of Medicine Karolinska Hospital Stockholm, Sweden Departments of Biochemistry, Karolinska Institute Stockholm, SwedenSearch for more papers by this author H. ENG, H. ENG Department of Medicine Karolinska Hospital Stockholm, SwedenSearch for more papers by this authorH. JÖRNVALL, H. JÖRNVALL Departments of Medical Chemistry, Karolinska Institute Stockholm, SwedenSearch for more papers by this authorM. CARLQUIST, M. CARLQUIST Department of Medicine Karolinska Hospital Stockholm, SwedenSearch for more papers by this authorA. K. LEFVERT, A. K. LEFVERT Department of Medicine Karolinska Hospital Stockholm, Sweden Departments of Biochemistry, Karolinska Institute Stockholm, SwedenSearch for more papers by this author First published: November 1988 https://doi.org/10.1111/j.1749-6632.1988.tb27157.xAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume540, Issue1Advances in NeuroimmunologyNovember 1988Pages 520-522 RelatedInformation