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C M Becker

Publications and source records attributed to C M Becker.

At least 19 recordsLinked to original sources
PubMed1992

Development of glycine receptor alpha subunit in cultivated rat spinal neurons: an immunocytochemical study.

Ontogenesis of the inhibitory glycine receptor was studied up to 12 days in vitro in spinal neurons placed previously in culture at embryonic day 14. The alpha subunit of the receptor was detected using standard and confocal immunofluorescence and a specific monoclonal antibody. The immunostaining was compared to that of synaptophysin, a synaptic vesicle antigen, which was taken as an index of synaptic maturity. Glycine receptors could be detected intracellularly, and not at the cellular surface in some cells as early as 2-3 days in vitro (DIV) prior to any synaptic contact. At 4-5 DIV, the number of cells which expressed the immunoreactivity and the fluorescence intensity increased. At this stage, spherical fluorescent blobs started to migrate in the neurites. From 6 DIV, the glycine receptor alpha subunit was detected at the neuronal surface and was organized in clusters whose number increased progressively with time. From 7 DIV, the intrasomatic immunoreactivity decreased, and at day 12, the pattern of labelling was similar to that observed in the adult spinal cord. A diffuse presence of the receptor at the surface of neurons could never be visualised, and when detected, the glycine receptors were always clustered. Thus, the increasing expression of clusters of glycine receptors at the neuronal surface was paralleled by that of synaptophysin in neuritic varicosities. These data suggest that transport of glycine receptors to the plasmamembrane and the formation of aggregates occurs simultaneously to synaptogenesis.

Animals↗
PubMed1992

Isoform-selective deficit of glycine receptors in the mouse mutant spastic.

The mutant mouse spastic (spa) develops a characteristic motor disorder about 2 weeks after birth, with symptoms resembling sublethal poisoning by the glycinergic antagonist strychnine. Correspondingly, adult homozygotic mutants (spa/spa) exhibit a severe reduction of inhibitory glycine receptors in spinal cord and brain. Here we show that the spastic mutation selectively interferes with the postnatal accumulation of the adult isoform of the glycine receptor protein, whereas perinatal expression of the neonatal receptor isoform is not detectably affected. Heterologous expression in X. laevis oocytes of poly(A)+ RNA and Northern blot analysis indicate normal levels of glycine receptor alpha 1 subunit transcripts in spinal cord of adult spastic mutants. Thus, the age-dependent manifestation of spastic symptoms after birth reflects a selective effect of the mutation on the developmental expression of the adult glycine receptor isoform.

Age Factors↗
PubMed1991

Glycine receptor immunoreactivity in rat and human cerebral cortex.

The distribution of the inhibitory glycine receptor was studied in rat and human cerebral cortex using a monoclonal antibody (MAb 4a) directed against the ligand-binding subunit. Significant amounts of glycine receptor antigen were found in forebrain structures such as caudatum and neocortex, although cortical levels were significantly below those seen in spinal cord. Immunohistochemically, glycine receptors were preferentially localized to the apical dendrites of pyramidal neurons in layers III and V. Ultrastructurally, these sites corresponded to synaptic neuronal contacts. Immunoreactivity was found in neuronal perikarya, dendrites and postsynaptic membranes which may correspond to sites of intracellular synthesis, transport and membrane incorporation of the glycine receptor. These immunological data corroborate previous pharmacological studies suggesting the existence of glycinergic transmission in mammalian cerebral cortex.

Aged↗
PubMed1991

Mapping of antigenic epitopes on the alpha 1 subunit of the inhibitory glycine receptor.

The inhibitory glycine receptor (GlyR) is a ligand-gated chloride channel protein that occurs in developmentally regulated isoforms in the vertebrate central nervous system. Monoclonal antibodies (mAbs) against the GlyR distinguish neonatal and adult GlyR proteins by identifying distinct alpha subunit variants within these receptor isoforms. Here, bacterially expressed fusion proteins of the rat GlyR alpha 1 subunit were used to localize the major antigenic epitopes of this protein within its N-terminal 105 amino acids. Synthetic peptides allowed further fine mapping of two mAb binding domains. MAb 2b, specific for the adult alpha 1 subunit, bound to a peptide corresponding to amino acids 1-10, whereas mAb 4a, which recognizes both neonatal and adult GlyR isoforms, reacted with a peptide representing residues 96-105 of the alpha 1 polypeptide. These data define unique and common antigenic epitopes on GlyR alpha subunit variants.

Animals↗
PubMed1991

Conservation of antigenic epitopes of the inhibitory glycine receptor in rodent and goldfish CNS.

Monoclonal antibodies against the inhibitory glycine receptor of rat spinal cord were used to identify corresponding receptor polypeptides in goldfish CNS. Both Western blot analysis and quantitative receptor immunoassays revealed crossreacting antigens in goldfish brain membranes. A polypeptide of 46 kDa molecular weight is immunologically related to the 48 kDa alpha subunit of the mammalian receptor. Similarly, a large receptor-associated protein of 93 kDa is present both in goldfish and mammals. Throughout the goldfish CNS, glycine-displaceable [3H]strychnine binding codistributes with the alpha subunit protein as determined immunologically. Glycine receptor contents were highest in goldfish medulla oblongata, medium in optic tectum and mesencephalon, whereas little or no receptor was detected in cerebellum, olfactory bulb, and spinal cord. Immunohistochemistry confirmed that the alpha subunit antigen and the 93 kDa protein were located in the plasma membrane of neurons and concentrated in small clusters found on the soma and dendrites. These data indicate that immunological properties and cellular distribution of glycine receptors are conserved from fish to mammals.

Animals↗
PubMed1990

The inhibitory glycine receptor: a ligand-gated chloride channel of the central nervous system.

The postsynaptic glycine receptor (GlyR) is a major inhibitory chloride channel protein in the central nervous system. The affinity-purified receptor contains polypeptides of 48 kDa, 58 kDa, and 93 kDa. The 48-kDa (alpha) and 58 kDa (beta) subunits span the postsynaptic membrane in a pentameric arrangement to form the anion channel of the receptor. The 93-kDa polypeptide is cytoplasmically localized and may have an anchoring function. Molecular cloning revealed that different structural characteristics are shared by the membrane-spanning subunits of the GlyR and those of other ligand-gated ion channel proteins. Developmental regulation of the GlyR is characterized by alterations in antagonist binding, heterogeneity of alpha subunits, and increased levels of the 93-kDa polypeptide. Glycine receptor function can be reconstituted by expression of cloned alpha subunits in heterologous cell systems. Positive charges found at the presumed mouths of the GlyR channel appear to be important determinants of ion selectivity. These data establish the anion-conducting GlyR as a homolog of other ligand-gated ion channel proteins and suggest that the diversity of these channels originates from divergent evolution of a primordial channel protein early in phylogeny.

Amino Acid Sequence↗
PubMed1990

Alpha subunit variants of the human glycine receptor: primary structures, functional expression and chromosomal localization of the corresponding genes.

Two cDNAs encoding variants (alpha 1 and alpha 2) of the strychnine binding subunit of the inhibitory glycine receptor (GlyR) were isolated from a human fetal brain cDNA library. The predicted amino acid sequences exhibit approximately 99% and approximately 76% identity to the previously characterized rat 48 kd polypeptide. Heterologous expression of the human alpha 1 and alpha 2 subunits in Xenopus oocytes resulted in the formation of glycine-gated strychnine-sensitive chloride channels, indicating that both polypeptides can form functional GlyRs. Using a panel of rodent-human hybrid cell lines, the gene encoding alpha 2 was mapped to the short arm (Xp21.2-p22.1) of the human X chromosome. In contrast, the alpha 1 subunit gene is autosomally located. These data indicate molecular heterogeneity of the human GlyR at the level of alpha subunit genes.

Amino Acid Sequence↗
PubMed1990

Disorders of the inhibitory glycine receptor: the spastic mouse.

The mutant mouse spastic suffers from a motor disorder of autosomal recessive inheritance which is characterized by tremor, myoclonic episodes, and a disturbed righting response. The most prominent alteration in the mutant is a substantial deficit of postsynaptic glycine receptor channels resulting in a dramatic reduction of glycinergic synaptic inhibition. Function and structure of the glycine receptor protein appear unaffected, which argues for a regulatory rather than a structural effect of the spastic mutation. It appears that other alterations in the spastic mouse are secondary to this fundamental disturbance in the balance of excitatory and inhibitory impulses. In particular, a significant increase in GABAA receptors of the lower parts of the CNS may serve a compensatory function, counteracting in part losses of glycinergic inhibition. Pharmacological experiments indeed show that facilitation of GABAA receptor-mediated inhibition alleviates symptoms of the spastic motor disorder. The recent cDNA cloning of glycine receptor subunits should help define the molecular mechanism by which the spastic gene causes the glycine receptor deficit.

Animals↗
PubMed1989

A simplified solid-phase assay for the quantitation of native membrane proteins. Application to the measurement of EGF receptor induction by dexamethasone.

Direct application of membrane fractions to a nitrocellulose support without previous solubilization, denaturation or fixation of the antigen permits the quantitation of antibodies binding to denaturation sensitive epitopes. In experiments reported here, this method was used to determine the rate of EGF receptor expression on squamous carcinoma cell lines. The assay revealed that dexamethasone treatment leads to an eight-fold increase of EGF receptor protein expression on C4-I cervical carcinoma cells. The data, when compared to the results obtained in immunoprecipitation experiments, suggest that this simple protocol yields reliable and precise quantitative data. The simplicity of the method permits simultaneous testing of large sample numbers with various antibodies.

Carcinoma, Squamous Cell↗
PubMed1989

Functional chloride channels by mammalian cell expression of rat glycine receptor subunit.

Cultured human cells were transfected with cloned rat glycine receptor (GlyR) 48 kd subunit cDNA. In these cells glycine elicited large chloride currents (up to 1.5 nA), which were blocked by nanomolar concentrations of strychnine. However, no corresponding high-affinity binding of [3H]strychnine was detected in membrane preparations of the transfected cells. Analysis by monoclonal antibodies specific for the 48 kd subunit revealed high expression levels of this membrane protein. After solubilization, the 48 kd subunit behaved as a macromolecular complex when analyzed by sucrose density centrifugation. Approximately 50% of the solubilized complex bound specifically to a 2-aminostrychnine affinity column, indicating the existence of low-affinity antagonist binding sites on most of the expressed GlyR protein. Thus, the 48 kd strychnine binding subunit efficiently assembles into high molecular weight complexes, resembling the native spinal cord GlyR. However, formation of functional receptor channels of high affinity for strychnine occurs with low efficiency.

Amino Acid Sequence↗
PubMed1989

Primary cultures of mouse spinal cord express the neonatal isoform of the inhibitory glycine receptor.

Expression of the inhibitory glycine receptor complex was investigated in primary cultures of fetal mouse spinal cord using sensitive immunomethods. In these cells, glycine receptor is predominantly of the neonatal isoform characterized by a low affinity for the antagonist strychnine. It contains a ligand binding subunit that differs from that of the adult receptor in antigenic epitopes and apparent molecular weight. Whereas in vivo the neonatal receptor isoform is completely replaced by the adult isoform within 3 weeks after birth, this exchange of subtypes is not seen in culture. The increased expression of the cytoplasmic glycine receptor-associated polypeptide of 93 kd occurring after birth is also seen under culture conditions. Purification of glycine receptor from cultures yielded polypeptides of 49 kd and 93 kd, suggesting that the membrane-spanning core of the neonatal receptor may be a homooligomer composed of 49 kd subunits. About half of the 49 kd subunit is cleaved by trypsinization of the cultures, indicating a predominant cell surface localization of the receptor. Pulse-labeling experiments revealed the 49 kd subunit to be a metabolically stable glycoprotein (half-life approximately 2 days). After its synthesis, a transition time of 30-45 min is required for acquisition of a strychnine binding conformation.

Animals↗
PubMed1989

Sensitive immunoassay shows selective association of peripheral and integral membrane proteins of the inhibitory glycine receptor complex.

The inhibitory glycine receptor of mammalian spinal cord is a ligand-gated chloride channel that, on affinity purification, contains two subunits of 48-kilodalton (kD) and 58-kD molecular mass in addition to an associated 93-kD protein. Ligand-binding 48-kD subunit and 93-kD protein were quantified in the CNS of the adult rat using a newly developed dot receptor assay (detection limit less than or equal to 1 fmol/assay) which employs monoclonal antibodies specific for glycine receptor polypeptides. The 93-kD protein was found to codistribute at a fixed stoichiometry with the 48-kD subunit throughout the CNS of the rat. Moreover, the 93-kD protein cofractionated with the ligand-binding subunit on solubilization and affinity chromatography or immunoprecipitation. However, both proteins were separated on sucrose gradient centrifugation of detergent extracts of spinal cord membranes in accord with earlier observations on purified receptor. These data prove that the 93-kD polypeptide is selectively associated with the membrane core of the strychnine-sensitive glycine receptor. The regional distribution of glycine receptor polypeptides was also determined in the CNS of the spastic rat mutant. In contrast to hereditary spasticity in mouse and cattle, no reduction of glycine receptors was found in the spastic rat.

Animals↗
PubMed1988

Glycine receptor heterogeneity in rat spinal cord during postnatal development.

Two different isoforms of the inhibitory glycine receptor were identified during postnatal development of rat spinal cord. A neonatal form characterized by low strychnine binding affinity, altered antigenicity, and a ligand binding subunit differing in mol. wt (49 kd) from that of the adult receptor (48 kd) predominates at birth (70% of the total receptor protein). Separation from the adult form could be achieved by either use of a selective antibody or glycine gradient elution of 2-aminostrychnine affinity columns. Both isoforms co-purify with the mol. wt 93 kd peripheral membrane protein of the postsynaptic glycine receptor complex.

Animals↗
PubMed1988

Fractionation of synaptophysin-containing vesicles from rat brain and cultured PC12 pheochromocytoma cells.

Synaptophysin is a transmembrane glycoprotein of neuroendocrine vesicles. Its content and distribution in subcellular fractions from cultured PC12 cells, rat brain and bovine adrenal medulla were determined by a sensitive dot immunoassay. Synaptophysin-containing fractions appeared as monodispersed populations similar to synaptic vesicles in density and size distribution. Membranes from synaptic vesicles contained approximately 100-times more synaptophysin than chromaffin granules. In conclusion, synaptophysin is located almost exclusively in vesicles of brain and PC12 cells which are distinct from dense core granules.

Adrenal Medulla↗
PubMed1987

Bovine fibrinogen aggregates: electron microscopic observations of quasi-globular structures.

Electron microscopic observations of bovine fibrinogen preparations were made following negative staining with uranyl acetate and phosphotungstic acid. Preparations were first incubated at pH 6.6, gamma/2 0.3 and 0 degrees C to develop fibrinogen aggregates at concentrations approaching equilibrium levels. The fraction of fibrinogen present as aggregates, designated the product mass fraction (Pmf), was determined by gel filtration on Bio-Gel A-1.5m. Electron micrographs of preparations with significant Pmf revealed partially closed quasi-globular structures of variable size. Fibrinogen aggregates could be dissociated by incubating preparations at 2 mg/ml, 37 degrees C for 18h, with a resulting Pmf less than 0.01. In these dissociated systems, monomers were observed with no apparent aggregate forms. At intervals during the approach to equilibrium, the size distributions of aggregates present were estimated by Bio-Gel A-15m filtration. A thermodynamic analysis incorporating these and previous data led to the following model specifications. Fibrinogen aggregates develop by monomer addition, followed by periodic reactions among the constituent members of an aggregate. These intra-aggregate reactions produce a non-linear structure, most likely begin after the association of four monomers, and on the average then follow the addition of every other monomer. In this manner, quasiglobular aggregates containing up to about 28 constituent monomers may develop as equilibrium is approached.

Animals↗