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Biomedical subjects

G G Lunt

Publications and source records attributed to G G Lunt.

At least 55 records · Page 3Linked to original sources
PubMed1987

GABA binding to receptor sites in locust supraoesophageal ganglia.

The binding of 4-amino-n-[2,3-3H]butyric acid (GABA) to receptor sites in the supraoesophageal ganglia of the locust Schistocerca gregaria is reported. Binding is saturable with a Kd of 30 nM and a Bmax of 150 fmol/mg protein. Binding is sodium-independent with a pH optimum of 6.8 and the pharmacological properties of the site suggest a receptor rather than an uptake or transport protein. The assay is being utilised in a comparative study of the binding sites of the GABA receptor and the enzyme 4-aminobutyrate: 2-oxoglutarate amino-transferase (EC 2.6.1.19, GABA-T). GABA binds to at least 4 proteins in the nervous system of vertebrates: the GABAA and GABAB receptors, GABA-T the enzyme involved in the GABA shunt, and the GABA transport system. In the invertebrates the status of these GABA-binding proteins is less well established. There are reports of a GABA receptor complex resembling the GABAA receptor; GABA-T activity has been reported and we have recently purified the enzyme from locust ganglia; it is assumed that GABA uptake systems are present in invertebrates. Proteins with different functions which specifically bind the same ligand are interesting from an evolutionary point of view. Are they distinct gene products or is the sub-unit of the receptor which binds GABA an enzyme which has lost the ability to bind pyroxidal phosphate? Do either receptor or enzyme differ significantly from their mammalian counterparts?

Animals↗
PubMed1987

The neurotoxin histrionicotoxin interacts with the putative ion channel of the nicotinic acetylcholine receptors in the central nervous system.

Perhydrohistrionicotoxin at micromolar concentrations blocked the nicotine-evoked transmitter release from perfused striatal (dopaminergic) and hippocampal (cholinergic) nerve terminals. Perhydrohistrionicotoxin failed to compete with [3H]nicotine for its high-affinity binding site in rat brain, suggesting that the action of this toxin on central nicotinic receptors is noncompetitive. From the dose-response curve, 50% inhibition of nicotine-evoked striatal dopamine release occurred at 5 microM perhydrohistrionicotoxin, a value similar to that obtained in frog sartorius muscle and Electrophorus electroplax. This close agreement may suggest that the ionic channel of the presynaptic nicotinic acetylcholine receptor of brain neurons has similar properties to those of the peripheral receptor.

Acetylcholine↗
PubMed1987

Immunological factors in neuronal degeneration with particular reference to motor neurone disease.

Cellular and humoral immunoreactivity to neuronal antigens was investigated in patients with motor neurone disease (MND). Lymphocytes from patients with MND and normal healthy controls were cultured with a membrane fraction prepared from cultured spinal cord neurones. 4 out of 14 patients with MND and 0 out of 9 normal controls showed a significantly increased stimulation index. An enzyme-linked immunoabsorbent assay (ELISA) was established to detect antibodies to synaptic membrane fraction prepared from human motor cortex. Sera from MND patients showed a significantly increased immunoglobulin binding with respect to normal control sera. Antineuronal antibody production by MND lymphocytes was studied by using Epstein-Barr virus transformation followed by fusion with a mouse myeloma cell line. Antibody-producing clones were isolated. This procedure would allow a more detailed analysis of the antineuronal antibody production in MND.

Cell Transformation, Viral↗
PubMed1986

Subcellular fractionation and distribution of cholinergic binding sites in fetal human brain.

Conventional subcellular fractionation techniques have been applied to human fetal brain (13-15 weeks gestation) and the fractions have been characterized by assaying for marker enzymes, cholinergic binding sites and electron microscopy. Fractionation of the homogenate resulted in a nuclear pellet (P1), a crude mitochondrial pellet (P2) and a supernatant (S2). Further resolution of the P2 fraction by density gradient centrifugation resulted in two bands at the gradient interfaces and a pellet. The P2 and subsequently the P2B fraction contained intact plasma membrane profiles as judged by the predominance of adenylate cyclase activity and the presence of occluded lactate dehydrogenase which constituted over 70% of the total activity in these fractions. Morphological examination of the gradient fractions revealed that the P2B fraction contains membrane bound structures which resemble synaptosomes prepared from neonatal rat brain. These structures have a granular matrix in which mitochondria and frequently, neurofilaments were observed. Very few synaptic vesicles were present and there was no evidence for post synaptic attachments. The cholinergic markers choline acetyltransferase, acetylcholinesterase and receptor sites defined by quinuclidinyl benzilate and alpha-bungarotoxin binding were enriched in fractions P2 and P2B which contained the bulk of nerve ending particles. This enriched preparation of fetal synaptosomes may be valuable for functional studies on pre-synaptic terminals in developing brain.

Brain↗
PubMed1986

Decreased autologous mixed lymphocyte reaction in myasthenia gravis.

Myasthenia gravis is an autoimmune disease in which the autoantigen, the acetylcholine receptor at the neuromuscular junction, is well characterized. As with many other autoimmune diseases, however, the basic cause of immune malfunction is unknown. The autologous mixed lymphocyte reaction (AMLR) involves the proliferation of T lymphocytes when co-cultured with autologous non-T cells and may reflect in vivo mechanisms of immune control. We have measured the AMLR in 22 patients with myasthenia gravis and found the magnitudes of the peak responses to be significantly depressed compared to those of 41 normal healthy controls. Proliferative responses of T cells from myasthenic patients to the mitogens, Concanavalin A and Phytohaemagglutinin-P, were also found to be significantly depressed relative to controls. These abnormal immune cell responses can be, in part, interpreted in terms of defective suppressor cell functions in myasthenia gravis.

Acetylcholinesterase↗
PubMed1985

Cultured rat spinal cord neurons: interaction with motor neuron disease immunoglobulins.

Conditions have been developed for the culture of rat spinal cord neurons in serum-free media supplemented with hormones and growth factors. Neurons were identified by immunofluorescence-labeled anti-neurofilament antibody, and their growth was monitored by assay of choline acetyltransferase and cholinesterase activities. Activities of these enzymes were considerably higher than those of comparable cultures in serum supplemented media in which there were visibly many more nonneuronal cells. Serum immunoglobulins from patients with motor neuron disease showed enhanced binding to rat spinal cord cells maintained in both serum-supplemented and serum-free media, as compared with those from normal healthy individuals. Enhanced binding was more marked with the latter cells, presumably because of the higher proportion of neuronal cells in these cultures. Serum immunoglobulins from patients with other neurologic disorders showed a similar binding to that of the normal controls. The results demonstrate the presence of an immune response to spinal cord cell membrane components in patients with motor neuron disease, although whether the response is primary or secondary in the disease process remains unclear.

Animals↗
PubMed1985

Complement-dependent toxicity of serum from myasthenic patients to muscle cells in culture.

The toxicity of myasthenic sera to rat myotubes in monolayer culture was examined by measuring the release of [Me-3H]carnitine from pre-loaded cells. In the presence of guinea pig complement, heat-inactivated serum samples from 9 out of 13 myasthenic patients showed clear myotoxicity, in contrast to 0 out of 11 normal controls and 0 out of 6 polymyositis patients. Neither heat-inactivated sera alone nor guinea pig complement sera alone showed myotoxicity. Removal of anti-acetylcholine receptor (anti-AChR) antibodies from a myasthenic serum sample by affinity absorption led to loss of myotoxicity. Myotoxicity of myasthenic sera could, in most cases, be confirmed by light microscopy. These results support the idea that complement-mediated cell damage, initiated by anti-AChR antibodies, contributes to post-synaptic membrane degeneration in myasthenia gravis.

Animals↗
PubMed1983

Partial purification and characterisation of an acetylcholine receptor with nicotinic properties from the supraoesophageal ganglion of the locust (Schistocerca gregaria).

An alpha-bungarotoxin-binding component has been partially purified from the supraoesophageal ganglion of the locust, (Schistocerca gregaria). The component binds alpha-bungarotoxin with a Kd of about 1.7 nM and this value changes little throughout the purification procedure. The specific binding activity ranges from 1.18 pmol alpha-bungarotoxin bound/mg protein for the membrane-bound site up to a maximum of 230 pmol bound/mg protein for the partially purified component. The pharmacological properties of the membrane-bound site are predominantly nicotinic. Affinity labelling of the binding species with 4-(N-maleimido)-[3H]benzyltrimethylammonium suggests that the binding is associated with a peptide of Mr 58000. Polyacrylamide gel electrophoresis of the partially purified of binding component shows three major bands corresponding to Mr of 60000, 41000 and 25000. We suggest that the binding component can be tentatively identified as a nicotinic acetylcholine receptor.

Animals↗
PubMed1981

Glutamate receptor binding in insects and mammals.

High affinity stereospecific binding sites for L-glutamate have been reported in several regions of mammalian brain. The binding sites in the hippocampus and cerebellum have been studied more extensively than binding in other brain regions. The hippocampal and cerebellar binding sites show similar properties with respect to their pharmacology and their independence of Na+. There is evidence, particularly good in the case of hippocampus, of mechanisms that may regulate the availability of the binding sites in both brain areas. Some progress has been made with the isolation of the hippocampal binding site but the protein has not been extensively characterised. In the case of insect muscle, high-affinity stereospecific binding of L-glutamate to whole membrane preparations, to detergent-solubilised membranes and to isolated proteolipids has been reported. Much greater variability in the binding characteristics is seen than is the case with the mammalian brain preparations. Preliminary experiments suggest that at least four distinct binding sites may be present on insect muscle. The complete characterisation of glutamate binding sites is at present precluded by a lack of potent agonists and antagonists. However, recent advances in the pharmacological classification of receptor sites for the excitatory amino acids in mammalian brain could provide sufficient information to permit the identification of the binding sites as synaptic receptors. Invertebrate toxins whose site of action is the insect neuromuscular junction may well prove to be useful tools with which to isolate and characterise the synaptic receptor proteins.

Animals↗
PubMed1981

The isolation and characterisation of the nicotinic acetylcholine receptor from human skeletal muscle.

Nicotinic acetylcholine receptor protein has been purified from human skeletal muscle by a procedure involving extraction in non-ionic detergent followed by affinity purification on immobilised alpha-toxin. Purified receptor preparations had specific activities of 0.5-3.5 mumol alpha-bungarotoxin binding sites/g protein and sedimented as a single 125I-alpha-bungarotoxin-binding species in sucrose-density-gradient centrifugation with s20,w = 9.5 S. The purified protein focussed as a single sharp band at pH 5.1 when complexed to 125I-alpha-bungarotoxin. Polyacrylamide gel electrophoresis of the purified receptor under denaturing conditions showed two major protein bands with Mr 42 000 and 66 000 respectively with the occasional appearance of minor components of Mr 56 000 and 85 000. Only the 42 000-Mr band was labelled with the affinity reagent, 4-(N-maleimido)[3H]-benzyltrimethylammonium. The purified receptor bound 125I-alpha-bungarotoxin and d-tubocurarine with Kd values of 0.5 nM and 0.25 microM respectively. It behaved similarly to unpurified detergent-extracted human receptor in the radioimmunoassay for anti-(human acetylcholine receptor) antibodies and when injected into rabbits caused increased levels of the latter antibodies but did not cause experimental autoimmune myasthenia gravis.

Animals↗
PubMed1981

An assessment of radioimmunoassay procedures for determination of anti-acetylcholine receptor antibodies in the sera of patients with myasthenia gravis.

A reproducible radioimmunoassay procedure for the determination of anti-acetylcholine receptor antibodies in the sera of patients with myasthenia gravis is described and examined in detail. The assay combines features of a number of methods previously outlined and allows repeat determinations of antibody titre in a given myasthenic serum sample with coefficient of variation 6%. The mean +/- standard deviation for normal human serum anti-acetylcholine receptor antibodies was found by this procedure to be 0.024 +/- 0.033 nmol/l alpha-bungarotoxin binding sites whereas the range for myasthenic patients was 0-139.14 nmol/l with a mean value of 7.55 nmol/l alpha-bungarotoxin binding sites.

Acetylcholine↗
PubMed1980

Interrelationship of concanavalin-A-binding and antigenic sites on the acetylcholine receptor from Torpedo marmorata.

A preparation of purified 125I-labelled acetylcholine receptor was shown to bind to concanavalin A and to be totally bound by rabbit antiserum to Torpedo acetylcholine receptor. Pre-incubation of the receptor with F(ab')2 and Fab fragments from antibodies against Torpedo acetylcholine receptor, or with corresponding fragments from control immunoglobulin G showed that subsequent binding of the receptor to concanavalin A was specifically inhibited to a maximum of approximately 25% by the immune fragments. Treatment of acetylcholine receptor with periodate or with glycosidases apparently destroyed or removed carbohydrate residues without affecting the antigenicity of the receptor as assessed by radioimmunoassay. These results suggest that although there is a steric interrelatonship between the antigenic and concanavalin-A-binding sites of the receptor the latter sites do not contain its major antigenic determinants.

Acetylcholine↗