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Biomedical subjects

D Beeson

Publications and source records attributed to D Beeson.

At least 55 records · Page 3Linked to original sources
PubMed1997

Autoantibodies detected to expressed K+ channels are implicated in neuromyotonia.

Antibody-mediated autoimmunity underlies a diverse range of disorders, particularly in the nervous system where the extracellular domains of ion channels and receptors are especially vulnerable targets. We present here a novel means of detecting autoantibodies where the genes of the suspected target proteins are known, and use it to detect specific autoantibodies in acquired neuromyotonia (Isaacs' syndrome), a disorder characterized by hyperexcitable motor nerves and sometimes by central abnormalities. We expressed different human brain voltage-gated potassium channels in Xenopus oocytes by injecting the relevant alpha-subunit complementary RNA, and detected antibody binding by immunohistochemistry on frozen sections. Antibodies were detected to one or more human brain voltage-gated potassium channel in 12 of 12 neuromyotonia patients and none of 18 control subjects. The results establish neuromyotonia as a new antibody-mediated channelopathy and indicate the investigative potential of this molecular immunohistochemical assay.

Autoantibodies↗
PubMed1997

Acetylcholine receptor expression in human extraocular muscles and their susceptibility to myasthenia gravis.

In myasthenia gravis (MG), extraocular muscle (EOM) weakness is often an initial and persisting symptom. It has been proposed that acetylcholine receptor (AChR) from EOM is antigenically different from AChR of other innervated muscles and that the presence of antibodies to fetal AChR expressed in EOM causes their weakness. We have (1) studied mRNA expression for each of the AChR subunits (alpha, beta, gamma, delta, and epsilon) in human muscle, including EOM, and (2) compared the binding of sera from ocular myasthenia gravis (OMG) patients with fetal (alpha2 beta gamma delta) and adult (alpha2 beta epsilon delta) human AChRs. RNase protection assays showed that expression of the AChR gamma-subunit (fetal-type) mRNA in EOM was comparable with that in other innervated muscle types. By contrast, epsilon-subunit (adult-type) mRNA was expressed at much higher levels in EOM than in other muscles studied. Moreover, some OMG sera bound specifically to adult AChR. These results do not support the contention that susceptibility of EOM in MG results from expression of fetal AChR and indicate that the inclusion of antigen from a source rich in adult AChR in the MG diagnostic assay will increase the yield of positive results in OMG patients.

Adolescent↗
PubMed1997

Genes at the junction--candidates for congenital myasthenic syndromes.

The neuromuscular junction is the site of several myasthenic (mys, muscle; aesthenia, weakness) disorders of autoimmune and genetic origin. The acquired autoimmune conditions are mainly adult-onset and caused by antibodies to specific neuronal and muscle ion channels, but can occur neonatally due to placental transfer of maternal antibodies. This review focuses on the rarer genetic conditions, called congenital myasthenic syndromes (CMS), that often present at birth. Mutations have yet to be characterized for familial infantile myasthenia, acetylcholinesterase deficiency and ACh-receptor deficiency; but genes encoding both structural and functional NMJ protiens should be considered. Other syndromes have recently been shown to involve defects in the functioning of the ACh receptor itself. In particular, eight different mutations have been reported in cases of the slow channel syndrome, a dominant condition associated with point mutations that generate single amino acid changes within the ACh receptor and result in prolonged channel activations. These investigations are providing new insights into the structure and function of the ACh receptor. Further studies of CMS should pave the way for analysis and treatment of disorders involving other synapses in the peripheral and central nervous system.

Acetylcholinesterase↗
PubMed1997

Mutations in different functional domains of the human muscle acetylcholine receptor alpha subunit in patients with the slow-channel congenital myasthenic syndrome.

Congenital myasthenic syndromes are a group of rare genetic disorders that compromise neuromuscular transmission. A subset of these disorders, the slow-channel congenital myasthenic syndrome (SCCMS), is dominantly inherited and has been shown to involve mutations within the muscle acetylcholine receptor (AChR). We have identified three new SCCMS mutations and a further familial case of the alpha G153S mutation. Single channel recordings from wild-type and mutant human AChR expressed in Xenopus oocytes demonstrate that each mutation prolongs channel activation episodes. The novel mutations alpha V156M, alpha T254I and alpha S269I are in different functional domains of the AChR alpha subunit. Whereas alpha T254I is in the pore-lining region, like five of six previously reported SCCMS mutations, alpha S269I and alpha V156M are in extracellular domains. alpha S269I lies within the short extracellular sequence between M2 and M3, and identifies a new region of muscle AChR involved in ACh binding/channel gating. alpha V156M, although located close to alpha G153S which has been shown to increase ACh binding affinity, appears to alter channel function through a different molecular mechanism. Our results demonstrate heterogeneity in the SCCMS, indicate new regions of the AChR involved in ACh binding/channel gating and highlight the potential role of mutations outside the pore-lining regions in altering channel function in other ion channel disorders.

Adolescent↗
PubMed1997

Congenital myasthenic syndromes.

Congenital myasthenic syndromes are a rare group of heterogeneous disorders affecting neuromuscular transmission. Recent identification and in-vitro functional analysis of some of the genetic mutations that cause these disorders correlates with previous electrophysiological, biochemical, pathological and therapeutic studies, and has advanced our understanding of neuromuscular transmission.

DNA Mutational Analysis↗
PubMed1996

Association of arthrogryposis multiplex congenita with maternal antibodies inhibiting fetal acetylcholine receptor function.

Arthrogryposis multiplex congenita (AMC), characterized by multiple joint contractures developing in utero, results from lack of fetal movement. Some cases are genetically determined, but AMC occasionally complicates pregnancy in patients with myasthenia gravis (MG) suggesting involvement of circulating maternal antibodies. We previously demonstrated antibodies that inhibited the function of fetal acetylcholine receptor (AChR) in one healthy woman with an obstetric history of recurrent AMC. Here we study sera from this woman, from one other with a similar history, and from three (one asymptomatic) whose babies had neonatal MG and AMC. All five maternal sera had high titers of antibodies that inhibited alpha-Bungarotoxin (alpha-BuTx) binding to fetal AChR, and their sera markedly inhibited fetal AChR function with little effect on adult AChR function. Moreover, in a further survey, 3 of 20 sera from anti-AChR negative AMC mothers inhibited fetal AChR function significantly at 1:100 dilution. These results demonstrate the role of antibodies to fetal AChR and perhaps other muscle antigens in some cases of AMC. More generally, they suggest that placental transfer of antibodies directed at fetal antigens should be considered as a cause of other recurrent fetal or perinatal disorders.

Adult↗
PubMed1996

Cloning of cDNA encoding human rapsyn and mapping of the RAPSN gene locus to chromosome 11p11.2-p11.1.

We have isolated and sequenced cDNA clones for the human 43-kDa acetylcholine receptor-associated protein rapsyn. The cDNA encodes a 412-amino-acid protein that has a predicted molecular mass of 46,330 Da and shows 96% sequence identity with mouse rapsyn. Analysis of PCR amplifications, first from somatic cell hybrids and subsequently from radiation hybrids, localizes the human RAPSN gene locus to chromosome 11p11.2-p11.1 in close proximity to ACP2.

Amino Acid Sequence↗
PubMed1996

Stable functional expression of the adult subtype of human muscle acetylcholine receptor following transfection of the human rhabdomyosarcoma cell line TE671 with cDNA encoding the epsilon subunit.

The human rhabdomyosarcoma cell line TE671 expresses the foetal subtype of muscle acetylcholine receptor (AChR). By transfecting TE671 cells with cDNA encoding the human muscle AChR epsilon subunit under the control of the cytomegalovirus promoter we have established a stable cell clone that, in addition, constitutively expresses the adult AChR subtype. Both subtypes are inserted into the plasma membrane and demonstrate their respective characteristic single channel properties. The level of expression of the adult AChR subtype is two- to three-fold higher than that of the foetal subtype. The new cell clone provides a relatively abundant source of human adult AChR for immunological and pharmacological investigations.

DNA, Complementary↗
PubMed1996

A transfected human muscle cell line expressing the adult subtype of the human muscle acetylcholine receptor for diagnostic assays in myasthenia gravis.

Immunoprecipitation of human acetylcholine receptor (AChR) is used in the diagnostic assay for myasthenia gravis (MG). We compared human AChR derived from TE671 cells, which express fetal-type AChR, with AChR from TE671-epsilon cells, which we have engineered to express adult-type AChR. Some low-titer MG sera distinguished strikingly between the two subtypes. Four out of seven MG sera that had equivocal titers in standard assays gave positive titers with TE671-epsilon AChR, whereas only one out of seven gave a positive titer with TE671 cells. The new cell line provides a greater concentration of adult AChR than can be obtained from normal human muscle and increases the sensitivity of the diagnostic assay.

Binding Sites↗
PubMed1995

Functional and non-functional isoforms of the human muscle acetylcholine receptor.

1. The properties of a recently identified isoform of the human muscle nicotinic acetylcholine receptor (AChR) alpha subunit (alpha +), which in muscle is expressed at similar levels to the alpha subunit, were investigated by both electrophysiological and biochemical approaches following expression in Xenopus laevis oocytes. The single-channel properties of adult (alpha 2 beta delta epsilon) and fetal (alpha 2 beta delta gamma) forms of the human AChR were also investigated. 2. The mean burst duration of adult channels (4.1 +/- 0.3 ms, mean +/- S.E.M., n = 5) is half that of fetal channels (7.9 +/- 0.6 ms, n = 4), while the single-channel conductance is larger (62.2 +/- 0.8 and 37.9 +/- 1.6 pS for adult and fetal channels, respectively), comparable to the developmental changes in single-channel properties observed for other mammalian species. 3. In contrast to the alpha isoform, the alpha + subunit does not bind 125I-labelled alpha-bungarotoxin or monoclonal antibodies directed against the AChR 'main immunogenic region' (MIR), illustrating why the alpha + subunit was first detected through screening of cDNA libraries. 4. By using site-directed mutagenesis to produce subunits that conferred different single-channel conductances on the AChR, we demonstrate that the alpha + isoform is not integrated into functional AChRs. 5. The mutagenesis experiments also revealed that the two alpha subunits within an AChR pentamer are not equivalent within the pore lining region.

Animals↗
PubMed1995

Arthrogryposis multiplex congenita with maternal autoantibodies specific for a fetal antigen.

Fetal arthrogryposis multiplex congenita (AMC) is characterised by non-progressive multiple joint contractures, which may result in fetal death, and is heterogeneous in origin. It can associate with maternal myasthenia gravis and autoantibodies to muscle acetylcholine receptor (AChR). We found maternal antibodies that selectively inhibit the fetal form of the AChR in a mother who herself had no features of myasthenia gravis. Maternal autoantibodies specific for fetal antigens could be an unrecognised cause of other congenital disorders.

Adult↗
PubMed1995

Antigen presentation by thymoma epithelial cells from myasthenia gravis patients to potentially pathogenic T cells.

Thymomas associate strongly with myasthenia gravis (MG). We now show that cultured thymoma epithelial cells can present synthetic acetylcholine receptor (AChR) peptides to HLA-sharing responder T cell lines/clones nearly as efficiently as blood mononuclear cells. Responses depended strictly on the specific antigen added. Processing of longer recombinant AChR polypeptides was clearly less efficient than by blood mononuclear cells, and was selectively abolished by preculture with chloroquine. The T cell responses depended on the presence of LFA-3 on the thymoma cells. This study demonstrates that thymoma epithelial cells have the capacity to stimulate T cells and perhaps, therefore, to autosensitize against AChR in vivo.

Antigen-Presenting Cells↗
PubMed1994

Immunogenicity of human recombinant acetylcholine receptor alpha subunit: cytoplasmic epitopes dominate the antibody response in four mouse strains.

In mysathenia gravis (MG) autoantibodies directed against acetylcholine receptors (AChR), at the neuromuscular junction lead to muscle weakness. These antibodies are directed against extracellular determinants, predominantly on the AChR alpha subunits. Similar antibodies can be induced in animals by immunisation with purified AChR, but immunisation of mice with recombinant human alpha subunit or its extracellular domain has produced conflicting results. To study further the immunogenicity of the human alpha subunit we immunised four inbred stains (C57B1/6, SJL, BALB/c, SWR) with almost full-length recombinant alpha subunit, r37-429, and looked at B cell epitopes by mapping with smaller recombinant fragments and synthetic peptides. The majority of anti-r37-429 antibodies bound to sequences within a region thought to be cytoplasmic, alpha 325-368, and reacted with human AChR. In two C57B1/6 sera, only, most antibodies were directed against an extracellular region, alpha 138-167, but the r37-429 used for immunisation of these two mice appeared to have lost the integrity of its cytoplasmic domain during preparation. Our results suggest that the antigenicity of the cytoplasmic region of the recombinant alpha subunit dominates the immune response in each of the four strains, and may even suppress the formation of antibodies to the extracellular domain. Moreover, although C57B1/6 and SJL mice were able to produce antibodies to alpha 138-167, these antibodies did not react with intact AChR, and none of the mice became weak.

Amino Acid Sequence↗