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J M Lindstrom

Publications and source records attributed to J M Lindstrom.

At least 73 records · Page 4Linked to original sources
PubMed1985

Characterization of a component in chick ciliary ganglia that cross-reacts with monoclonal antibodies to muscle and electric organ acetylcholine receptor.

Chick ciliary ganglion neurons have previously been shown to contain a component that shares an antigenic determinant with the "main immunogenic region" of the alpha-subunit in nicotinic acetylcholine receptor from skeletal muscle and electric organ. Ultrastructural studies of antibody binding in the ganglion have shown that the cross-reacting antigen exposed on the surface of the neurons is located predominantly in synaptic membrane. Here we show that the neuronal antigen can be identified in detergent extracts of ciliary and sympathetic ganglia, but not in extracts of heart, liver, spinal cord, retina, or dorsal root ganglia. In the ciliary ganglion the component is present as an integral membrane constituent, and, when detergent solubilized, it sediments as a 10 S species and binds to concanavalin A. The component is distinct from the alpha-bungarotoxin-binding site on the neurons since toxin-binding sites and antibody-binding sites can be precipitated separately in ganglion extracts. The component reaches peak levels per ganglionic protein between embryonic days 8 and 12. These are some of the properties expected for the nicotinic acetylcholine receptor on ciliary ganglion neurons.

Animals↗
PubMed1985

Cytotoxic granules from killer cells: specificity of granules and insertion of channels of defined size into target membranes.

The channel-forming polyperforins P1 and P2 are thought to be formed from the contents of dense core vesicles of cytolytic effector cells. To test this hypothesis, granules from various cytotoxic effector cells were assayed for cytolytic activity on nucleated or unnucleated targets. The results show that in general, granules from cytolytic effector cells are cytolytic, whereas granules from noncytotoxic cells are not. Cytotoxicity of granules is not specific, but there appears to be a preference in that nucleated targets are lysed better than are erythrocytes by granules from T killer or natural killer cells. Granules from CTLL-2, however, preferentially lyse erythrocyte targets. This cell line has been in culture for a long period of time and has lost its cytotoxicity. We tested whether granules from CTLL-2 caused formation of transmembrane pores in erythrocyte target membranes. We found that granule- and complement-induced lesions have similar pore sizes. They are big enough to allow the total release of alpha-bungarotoxin, an 8000 Mr polypeptide with dimensions of 4 X 2.5 nm. Larger molecules are released partially or not at all. Under acidic conditions (pH 5.4) granules do not permeabilize target membranes. This may suggest a pH-dependent control mechanism in the formation, insertion, or function of polyperforin channels, in addition to a previously recognized Ca2+-dependent mechanism. Permeabilization of lipid vesicles by granules was studied to explore what the molecular requirements for channel insertion into membranes may be. Release of alpha-bungarotoxin induced by granules was observed in liposomes made of soybean lipid with or without cholesterol, suggesting that no membrane component other than lipid is required for the insertion of polyperforins, and that the action of polyperforins does not require other mechanisms in the target cell. When pure lecithin from soybean and egg, or synthetic phosphatidylcholines were used, slower release or no release of macromolecules was observed. We suggest that some kind of lipid specificity is required for perforin action. This may be related to the hydrophobic region of the lipid bilayer rather than to the polar portion, because different lecithins with varying fatty acid composition gave similar results.

Animals↗
PubMed1985

The occurrence of long openings in the purified cholinergic receptor channel increases with acetylcholine concentration.

To establish the relationship between cholinergic ligand occupancy and channel open states, we recorded single-channel currents activated by different acetylcholine (ACh) concentrations from Torpedo californica ACh receptors reconstituted in lipid bilayers. Inspection of single-channel records shows that the frequency of occurrence of long openings increases with ACh concentration. Analysis of the probability distribution of open dwell times indicates that the ACh receptor channel has two distinct channel open states, short- and long-lived. The frequency of occurrence of the long openings over the short increased with ACh concentration, whereas the corresponding time constants were virtually unaltered. The extent of agonist occupancy at the ACh-binding sites in the purified cholinergic receptor appears, therefore, to correlate with an increased probability of the long-lived open state. These results are consistent with the notion that the two open-channel states arise from different extents of ligand occupancy at the receptor molecule.

Acetylcholine↗
PubMed1985

Myasthenia gravis--current concepts.

An edited summary of an Interdepartmental Conference arranged by the Department of Medicine of the UCLA School of Medicine, Los Angeles. The Director of Conferences is William M. Pardridge, MD, Associate Professor of Medicine. Current findings indicate that autoimmune myasthenia gravis is an acquired immune complex disorder of neuromuscular transmission in voluntary striated muscle. There is a break in immunologic tolerance leading to blocking and degradation of acetylcholine receptors, together with widening of the synaptic cleft associated with partial destruction, simplification and shortening of the postjunctional membrane. Thymic hyperplasia and thymoma may be present. A decremental response to nerve-muscle stimulation, blocking and jitter on single-fiber electromyography and circulating antibodies to acetylcholine receptor are detectable in most patients with generalized weakness. Although the cause of this abnormal immunologic mechanism remains to be discovered, anticholinesterases, corticosteroids, immunosuppressants, plasmapheresis or thymectomy (individually or in combination) provide control and better prognosis in most patients.

Acetylcholine↗
PubMed1984

Shared antigenic determinant between the Electrophorus acetylcholine receptor and a synaptic component on chicken ciliary ganglion neurons.

Monoclonal antibodies raised against purified acetylcholine receptor from muscle and electric organ were tested for cross-reaction with surface components on chicken ciliary ganglion neurons. Indirect immunofluorescence indicated that antibodies to a determinant in the "main immunogenic region" of the receptor bind to the neurons in culture. Ultrastructural studies on 16-day embryonic ganglia, using horseradish peroxidase-conjugated monoclonal antibody, revealed that most of the conjugate labeling was associated with synaptic membrane on the neurons. A lesser amount of labeling was associated with the short processes extending from the neuronal somata in the region of preganglionic innervation. The labeling was blocked by coincubation with unlabeled antibodies of appropriate specificity and not by nonimmune serum. The pattern of labeling was clearly different from that previously found for a horseradish peroxidase conjugate of alpha-bungarotoxin: the toxin conjugate bound extensively to the short processes but not to synaptic membrane on the neurons. The synaptic antigen identified here by the cross-reacting antibodies is a candidate for the synaptic acetylcholine receptor on chicken ciliary ganglion neurons.

Animals↗
PubMed1984

Structure and transmembrane nature of the acetylcholine receptor in amphibian skeletal muscle as revealed by cross-reacting monoclonal antibodies.

A collection of 126 monoclonal antibodies (mAbs) made against acetylcholine receptors (AChRs) from the electric organs of Torpedo californica or Electrophorus electricus was tested for cross-reactivity with AChRs in cryostat sections of skeletal muscle from Rana pipiens and Xenopus laevis by indirect immunofluorescence. 49 mAbs (39%) cross-reacted with AChRs from Rana, and 25 mAbs (20%) cross-reacted with AChRs from Xenopus. mAbs specific for each of the four subunits of electric organ AChR (alpha, beta, gamma, delta) cross-reacted with AChRs from each amphibian species. mAbs cross-reacting with Xenopus AChRs were, with one exception, a subset of the mAbs cross-reacting with Rana AChRs. The major difference detected between the two species was in binding by mAbs specific for the main immunogenic region (MIR) of the alpha-subunit. Whereas 22 of 33 anti-MIR mAbs tested cross-reacted with Rana AChRs, only one of these mAbs cross-reacted with Xenopus AChRs. Some (32) of the cross-reacting mAbs were tested for binding to AChRs in intact muscle. 21 of these mAbs bound to AChRs only when membranes were made permeable with saponin. Electron microscopy using immunoperoxidase or colloidal gold techniques revealed that these mAbs recognize cytoplasmic determinants and that mAbs that do not require saponin in order to bind AChRs in intact muscle recognize extracellular determinants. These results suggest that AChRs in skeletal muscle of Rana and Xenopus are composed of subunits corresponding to the alpha-, beta-, gamma-, and delta-subunits of AChRs from fish electric organs. The subunit specificity of mAbs whose binding was examined by electron microscopy suggests that parts of each subunit (alpha, beta, gamma, delta) are exposed on the cytoplasmic surface and that, as in AChRs from fish electric organs and mammalian muscle, the MIR on alpha-subunits of Rana AChRs is exposed on the extracellular surface.

Animals↗
PubMed1984

Multisubunit structure and amino-terminal sequences of piscine muscle acetylcholine receptor.

The nicotinic acetylcholine receptor (AcChR) has been purified from both the electric organ and the muscle of the fish Electrophorus electricus. Upon SDS gel electrophoresis muscle AcChRs appeared to contain four main polypeptides whose molecular weights were similar but not identical to the molecular weights of the four peptides present in the electric organ AcChR. Each of these peptides has been isolated and their amino-terminal sequences have been determined. The AcChRs from muscle were found to be composed of four homologous proteins of apparent molecular weight 40,500, 50,000, 56,000 and 63,000, respectively. The subunit of Mr 40,500 is present in two copies for each AcChR molecule, while the other three components are present in one copy. No difference was found between the sequenced segments of corresponding subunits from muscle and from electric organ AcChR, suggesting that AcChRs in different tissues of the same animal are products of identical genes. The Electrophorus AcChR subunits are highly homologous with the corresponding subunits of Torpedo californica AcChR.

Amino Acid Sequence↗
PubMed1984

Autoantibodies to platelet glycoprotein IIb/IIIa and to the acetylcholine receptor in a patient with chronic idiopathic thrombocytopenic purpura and myasthenia gravis.

The coexistence of chronic idiopathic thrombocytopenic purpura and myasthenia gravis has been infrequently reported. We report another case and show the coexistence of autoantibodies to the platelet glycoprotein IIb and IIIa complex and the acetylcholine receptor. Autoantibody levels were followed during 8 weeks of treatment with cyclophosphamide, vincristine, and prednisone; the concentration of both autoantibodies fell during treatment but without measureable clinical improvement.

Adult↗
PubMed1984

Specific killing of lymphocytes that cause experimental autoimmune myasthenia gravis by ricin toxin-acetylcholine receptor conjugates.

Experimental autoimmune myasthenia gravis (EAMG) is an autoimmune disease in which antibodies to acetylcholine receptor (AChR) cause loss of AChR from muscle, thereby impairing neuromuscular transmission. Here we report the use of a hybrid molecule that contains ricin toxin, irreversibly coupled to AChR to specifically suppress the immune response to AChR in vitro. Lymph node cell cultures from rats with EAMG pretreated with ricin toxin-AChR conjugates exhibited suppressed T helper cell proliferation and B cell antibody synthesis in response to the subsequent addition of AChR. Nonspecific toxicity of the conjugates was measured by suppression of the T cell proliferative response to the mitogen concanavalin A and the antigen keyhole limpet hemocyanin (KLH), and B cell antibody production to KLH. We have evaluated different pretreatment conditions and ricin toxin covalently coupled to AChR in different molar ratios to optimize specific immunosuppression. By varying the number of ricin molecules covalently bound to AChR in the immunotoxin, we were able to minimize the nonspecific toxicity while still maintaining specific killing of AChR-reactive lymphocytes. Furthermore, B cells were more susceptible to specific killing than were the T cells. The specific immunosuppression was potentiated by performing the pretreatment with immunotoxin in the presence of chloroquine. Chloroquine raises lysosomal pH and probably delays the degradation of immunotoxin in the cell. It should be noted that ricin toxin was covalently coupled to AChR by using a novel, non-reducible reaction. These in vitro results suggest that it may be feasible to use immunotoxin molecules to specifically suppress this autoimmune response in vivo.

Adjuvants, Immunologic↗
PubMed1984

Spectrotypic analysis of antibodies to acetylcholine receptors in experimental autoimmune myasthenia gravis.

We have studied the isoelectric focusing pattern of antibodies expressed in rats with experimental autoimmune myasthenia gravis (EAMG) induced by immunization with acetylcholine receptors (AChR) purified from Torpedo californica. Sera or tissue eluates were obtained at intervals in the course of disease and subjected to isoelectric focusing. Subsequently, the focused antibodies were detected by autoradiography of gels labelled with 125I-alpha-bungarotoxin conjugated AChR. Reverse electrofocusing was used to separate complexes of antibody and AChR formed in vivo, thereby allowing detection of the full spectrotype (banding pattern). As little as 1.1 X 10(-12) moles of monoclonal antibodies (MoAbs) to AChR yielded distinct bands of radiolabelled antigen binding by this technique. The anti-AChR MoAbs studied showed a multitude of bands localized in neutral to alkaline position. The clonotypes expressed in late post-immunization sera were compared to early sera. The spectrotypes of immunized Lewis and Brown Norway rats were not identical. In early sera most of the isoelectric focusing bands were specific for T. californica AChR, whereas in late sera further expansion of the repertoire produced bands that reacted with rat muscle AChR as well. The focused bands that bound rat AChR also bound T. californica AChR. The anti-AChR antibodies eluted from muscles of rats with EAMG showed similar binding patterns to anti-receptor antibodies in rats' sera. These results indicate that the antibody specificities detected in serum are the same specificities which are effective in binding to muscle AChR in vivo. Minor specificities of serum anti-receptor antibodies are not disproportionally represented in the antibodies actually bound at the neuromuscular junction in EAMG.

Animals↗
PubMed1983

Purification of acetylcholine receptors from the muscle of Electrophorus electricus.

Muscle from the electric eel Electrophorus electricus contains acetylcholine receptors at 50 times the concentration of normal mammalian muscle and fully one-tenth the concentration of receptors in its electric organ tissue. Receptor is organized much more diffusely over the surface of Electrophorus muscle cells than is the case in normally innervated mammalian skeletal muscle. Receptor was purified from Electrophorus muscle by affinity chromatography on cobra toxin-agarose and found to contain subunits which correspond immunochemically to the alpha, beta, gamma, and delta subunits of receptor from electric organ tissue of Torpedo californica. Receptor purified from Electrophorus muscle appears virtually identical with receptor purified from Electrophorus electric organ tissue.

Animals↗
PubMed1983

Comparison of the subunit structure of acetylcholine receptors from muscle and electric organ of Electrophorus electricus.

The acetylcholine receptors of the electric organ and muscle tissues of Electrophorus electricus are composed of alpha, beta, gamma, and delta subunits. Receptor subunits from the two tissues were compared by peptide mapping with monoclonal antibodies, an affinity-labeling reagent, and a lectin to characterize particular peptide fragments. These experiments indicate that the corresponding receptor subunits from the two tissues are extensively homologous or identical throughout their amino acid sequences. Small differences in the electrophoresis of peptide fragments of alpha subunits between the two tissues occurred on fragments which bound labeled lectin. These results suggest that the acetylcholine receptors in electric organ and muscle tissues of Electrophorus differ in structure only by minor posttranslational modifications perhaps involving carbohydrate.

Animals↗
PubMed1982

Structural similarities between acetylcholine receptors from fish electric organs and mammalian muscle.

Acetylcholine receptors from fish electric organ tissue and mammalian muscle were compared by peptide mapping. The alpha subunits from receptors of Torpedo californica and Electrophorus electricus electric organ tissue were digested with V8 protease and the resulting fragments separated on polyacrylamide gels and stained for protein or for carbohydrate. 125I-Labeled alpha subunits of acetylcholine receptors from Electrophorus electric organ tissue and bovine muscle were digested with V8 protease, and the resulting fragments were also separated on polyacrylamide gels. Intact receptors from both the fish electric organs and mammalian muscle were labeled with [4-(N-maleimido) benzyl]tri[3H]-methylammonium iodide which binds specifically to the acetylcholine binding site on alpha subunits, and the isolated alpha subunits were subjected to the same peptide mapping procedure. The fragment patterns produced were stained for protein and fluorographed to identify active site containing polypeptides. None of these peptide mapping approaches revealed extensive homologies between alpha subunits. Intact and V8 proteolyzed sodium dodecyl sulfate denatured receptors from Torpedo and Electrophorus electric organs and bovine muscle were electrophoretically transferred to diazophenyl thioether paper and probed with antisera to Torpedo receptor subunits and two monoclonal antibodies. Unique fragment patterns were obtained with each antisubunit serum. A fragment of the same size was derived from the beta subunit of each acetylcholine receptor and was shown to specifically bind the same monoclonal antibody in each case. These results indicate that only in the beta subunits from all of the species examined is a large sequence nearly identical. However, it is likely that corresponding receptor subunits from receptors of all of these species have extensive structural homologies.

Animals↗
PubMed1982

Structure of the acetylcholine receptor and specificities of antibodies to it in myasthenia gravis.

Acetylcholine receptors in skeletal muscle and fish electric organs are intrinsic membrane proteins whose function is to bind acetylcholine released from the nerve ending and trigger the opening of a cation-specific channel in the postsynaptic membrane, thereby facilitating transmission of the nerve signal to the muscle. Investigations from several laboratories indicate that acetylcholine receptors from fish electric organs are composed of four homologous glycoprotein subunits of apparent relative molecular masses (Mr) approximating 40, 50, 57 and 64 x 10(3) designated, respectively, alpha, beta, gamma and delta. These subunits are present in receptor monomers in the mole ratio alpha 2 beta gamma delta. Receptor purified from skeletal muscle appears to have a similar structure. The alpha subunits are unknown. It is known that the cation channel regulated by acetylcholine binding is located within the receptor monomer. Experimental autoimmune myasthenia gravis (EAMG) is induced by immunizing animals with purified receptor. The mechanisms by which neuromuscular transmission is impaired in this model are very similar to those in myasthenia gravis (MG). Although there are many immunogenic determinants on receptors, and EAMG can be induced in rats by any of the denatured subunits, there is a main immunogenic region at which most of the antibodies to native receptors are directed. The main immunogenic region is a conformationally dependent part of the external surface of alpha subunits other than the acetylcholine-binding site or the attached carbohydrate. Antisera from MG patients are also directed primarily at this region. No correlation was detected between the specificities of antibodies to receptor in patients' sera and the severity of their weakness.

Animals↗
PubMed1982

The antigenic structure of the acetylcholine receptor from Torpedo californica.

The immunological structure of the acetylcholine receptor (AChR) from the electric organ of Torpedo californica was studied using a large number of monoclonal antibodies which were initially selected for their abilities to bind to intact AChRs. The monoclonal antibodies were tested for their ability to bind to denatured AChR subunits labeled with 125I. Antibodies derived from rats immunized with individual denatured subunits or a mixture of subunits of Torpedo AChR reacted well in the assay. A much smaller proportion of antibodies derived from rats immunized with native Torpedo AChR or native AChR from Electrophorus electricus electric organ, bovine muscle, or human muscle reacted with denatured subunits of Torpedo AChR. Many monoclonal antibodies reacted with more than one subunit, but they always reacted best with the subunit used for immunization. Those monoclonal antibodies that bound to intact subunits were mapped more precisely by their ability to bind characteristic fragments of each subunit generated by proteolysis with Staphylococcal V8 protease. These fragments were analyzed by SDS polyacrylamide gel electrophoresis, and monoclonal antibodies that precipitated the same fragment pattern were placed in groups. By this method, we define a minimum of 28 determinants on Torpedo AChR.

Animals↗
PubMed1982

Immunopathology of acetylcholine receptors in myasthenia gravis.

It is now clear that the muscular weakness and fatigability seen in MG result from an antibody-mediated immune response to AChR. The mechanisms by which antibodies impair transmission are moderately well understood and detection of antibodies in patient's sera is a reliable diagnostic test for the disease. The spectrum of antibody specificities produced in MG is also beginning to be understood, largely through the use of antibodies produced in the experimental model EAMG. Treatment for MG continues to rely heavily on the symptomatic relief afforded by acetylcholinesterase inhibitors. However, the recent recognition of the autoimmune nature of MG has led to increased emphasis on immunosuppression and antibody removal with some beneficial effects. Despite all that has been learned, the level of ignorance has just been pushed back one step--from the neuromuscular junction to the immune system. What initiates the immune response to AChR in MG and how to specifically suppress this aberrant response remain completely unknown.

Acetylcholinesterase↗