2-adrenergic receptors (2 AR) are present on both lymphocytes and skeletal muscle cells. Antibodi... more 2-adrenergic receptors (2 AR) 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 2 AR may thus modify both the immune and the muscle functions in MG. In this study, we analysed the density and affinity of 2 AR on peripheral blood mononuclear cells using a radioligand binding assay. The density (B max) 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 2 AR 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 2 AR 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 2 AR may occur in MG. This downregulation might be of importance in the pathogenesis of the disease and its symptoms.
SUMMARY Eighteen percent of patients with myasthenia gravis (MG) have serum antibodies against a ... more SUMMARY Eighteen percent of patients with myasthenia gravis (MG) have serum antibodies against a synthetic peptide corresponding to the second extracellular loop of the human β2AR (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-γ) and IL-4 or antibodies against the peptide, and by measuring thymidine incorporation in response to the peptide. The peptide from the β2AR 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 β2AR-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...
The proteins needed for growth and maintenance of the axon are generally believed to be synthesiz... more 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 [ 35 S]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 [ 35 S]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 -tubulin. Axons synthesized Ͻ1% 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 recog... more 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.
2-adrenergic receptors (2 AR) are present on both lymphocytes and skeletal muscle cells. Antibodi... more 2-adrenergic receptors (2 AR) 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 2 AR may thus modify both the immune and the muscle functions in MG. In this study, we analysed the density and affinity of 2 AR on peripheral blood mononuclear cells using a radioligand binding assay. The density (B max) 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 2 AR 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 2 AR 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 2 AR may occur in MG. This downregulation might be of importance in the pathogenesis of the disease and its symptoms.
SUMMARY Eighteen percent of patients with myasthenia gravis (MG) have serum antibodies against a ... more SUMMARY Eighteen percent of patients with myasthenia gravis (MG) have serum antibodies against a synthetic peptide corresponding to the second extracellular loop of the human β2AR (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-γ) and IL-4 or antibodies against the peptide, and by measuring thymidine incorporation in response to the peptide. The peptide from the β2AR 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 β2AR-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...
The proteins needed for growth and maintenance of the axon are generally believed to be synthesiz... more 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 [ 35 S]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 [ 35 S]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 -tubulin. Axons synthesized Ͻ1% 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 recog... more 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.
Uploads
Papers by Hubert Eng