Biomedical subjects
Publications and source records attributed to C M Becker.
STATEMENT OF PROBLEM: Many clinical situations are suitably treated with cantilevered implant-supported cemented fixed restorations. PURPOSE: This article details the indications for the restorative dentist to use a cantilever fixed prosthesis after insertion of ITI dental implants. CONCLUSION: The use of implants to support cantilevered fixed partial dentures has been successful in selected clinical situations.
Missense mutations as well as a null allele of the human glycine receptor alpha1 subunit gene GLRA1 result in the neurological disorder hyperekplexia [startle disease, stiff baby syndrome, Mendelian Inheritance in Man (MIM) #149400]. In a pedigree showing dominant transmission of hyperekplexia, we identified a novel point mutation C1128A of GLRA1. This mutation encodes an amino acid substitution (P250T) in the cytoplasmic loop linking transmembrane regions M1 and M2 of the mature alpha1 polypeptide. After recombinant expression, homomeric alpha1(P250T) subunit channels showed a strong reduction of maximum whole-cell chloride currents and an altered desensitization, consistent with a prolonged recovery from desensitization. Apparent glycine binding was less affected, yielding an approximately fivefold increase in Ki values. Topological analysis predicts that the substitution of proline 250 leads to the loss of an angular polypeptide structure, thereby destabilizing open channel conformations. Thus, the novel GLRA1 mutant allele P250T defines an intracellular determinant of glycine receptor channel gating.
NMDA receptors (NR) are encoded by a family of genes including those of the NR1 and NR2A-D subunits. In situ hybridization has revealed that NR1, comprising eight splice variants, is ubiquitously expressed in the central nervous system (CNS) while the expression of NR2 isoforms is restricted to particular CNS regions. We report on the cellular and ultrastructural distribution of the NR2B polypeptide in rat telencephalon. In the telencephalon, the hippocampus represented the most intensively immunolabeled region. Here, predominantly the CA pyramidal neurons were heavily stained. Intense immunoreactivity (IR) was also detected in cortical neurons, in particular in pyramidal-like ones of layers II/III and V. On the ultrastructural level, the NR2B subunit was present not only in synaptic complexes where it usually was present in postsynaptic sites but in addition could be located at extrasynaptic sites. Furthermore, preliminary evidence indicates a presynaptic location of NR2B in some rare cases. NR2B antigen distribution is consistent with that of corresponding transcripts. Indeed, NR2B immunoreactivity coincides largely with that for NR1, indicating that both subunits are coexpressed in numerous cortical and hippocampal neurons.
The neuronal glycine receptor is a ligand-gated chloride channel composed of ligand binding alpha and structural beta polypeptides. Homology screening of a human fetal brain cDNA library resulted in the identification of two alternative splice variants of the glycine receptor alpha3 subunit. The amino acid sequence predicted for the alpha3L variant was largely identical to the corresponding rat subunit. In contrast, the novel splice variant alpha3K lacked the coding sequence for 15 amino acids located within the cytoplasmic loop connecting transmembrane spanning region 3 (TM3) and TM4. Using P1 artificial chromosome (PAC) clones, the structure of the GLRA3 gene was elucidated and its locus assigned to human chromosomal bands 4q33-q34 by fluorescence in situ hybridization. Two transcripts of 2.4 and 9 kilobases, corresponding to alpha3L and alpha3K, respectively, were identified and found to be widely distributed throughout the human central nervous system. Structural analysis of the GLRA3 gene revealed that the alpha3K transcript resulted from a complex splice event where excision of the novel exon 8A comprising the alternative sequence of 45 base pairs coincides with the persistence of a large intronic sequence in the 3'-untranslated region. Functional expression in HEK 293 cells of alpha3L and alpha3K subunits resulted in the formation of glycine-gated chloride channels that differed significantly in desensitization behavior, thus defining the cytoplasmic loop as an important determinant of channel inactivation kinetics.
The glycine receptor of the human CNS comprises ligand-binding alpha 1 and structural beta subunits encoded by the GLRA1 and GLRB genes, respectively. Screening of a human hippocampal cDNA library resulted in the identification of the novel subunit transcript beta B, differing in the 5'-UTR. Analysis of the genomic organization of GLRB showed that the coding region is distributed over nine exons, highly homologous to the GLRA1 gene. By in situ hybridization, the chromosomal localization of GLRB was refined to band 4q31.3. Based on the identical phenotypes of mouse lines carrying mutant alleles of the alpha 1 and beta subunit genes, GLRB was assumed to be a candidate gene for those cases of hyperekplexia that cannot be associated with mutations of GLRA1. Therefore, flanking intronic sequences were determined, and DNA samples from more than 30 index patients were subjected to SSCP screening of the entire GLRB coding region. A polymorphism in exon 8 was found both in the normal population and in families affected by hyperekplexia, although no coding mutation was detectable.
Molecular cloning has introduced an unexpected diversity of neurotransmitter receptors. In this study we review the types, the localization and possible synaptic function of the inhibitory neurotransmitter receptors in the mammalian retina. Glycine receptors (GlyRs) and their localization in the mammalian retina were analyzed immunocytochemically. Specific antibodies against the alpha 1 subunit of the GlyR (mAb2b) and against all subunits of the GlyR (mAb4a) were used. Both antibodies produced a punctate immunofluorescence, which was shown by electron microscopy to represent clustering of GlyRs at synaptic sites. Synapses expressing the alpha 1 subunit of the GlyR were found on ganglion cell dendrites and on bipolar cell axons. GlyRs were also investigated in the oscillator mutant mouse. The complete loss of the alpha 1 subunit was compensated for by an apparent upregulation of the other subunits of the GlyR. GABAA receptors (GABAARs) and their retinal distribution were studied with specific antibodies that recognize the alpha 1, alpha 2, alpha 3, beta 1, beta 2, beta 3, gamma 2 and delta subunits. Most antibodies produced a punctate immunofluorescence in the inner plexiform layer (IPL) which was shown by electron microscopy to represent synaptic clustering of GABAARs. The density of puncta varied across the IPL and different subunits were found in characteristic strata. This stratification pattern was analyzed with respect to the ramification of cholinergic amacrine cells. Using intracellular injection with Lucifer yellow followed by immunofluorescence, we found that GABAARs composed of different subunits were expressed by the same ganglion cell, however, they were clustered at different synaptic sites. The distribution of GABAC receptors was studied in the mouse and in the rabbit retina using an antiserum that recognizes the rho 1, rho 2 and rho 3 subunits. GABAC receptors were found to be clustered at postsynaptic sites. Most, if not all of the synapses were found on rod and cone bipolar axon terminals. In conclusion we find a great diversity of glycine and GABA receptors in the mammalian retina, which might match the plethora of morphological types of amacrine cells. This may also point to subtle differences in synaptic function still to be elucidated.
The N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptors comprises a family of highly homologous subunits which assemble into oligomeric protein complexes. Alterations in subunit composition are developmentally regulated, leading to functionally distinct receptor populations. Here, the contribution of the subunit NR2B to NMDA receptor complex formation was analysed in neonatal rat brain, employing polyclonal antibodies raised against NR2B-specific synthetic peptides. By hydrodynamic size fractionation of the solubilized receptor protein and chemical cross-linking, NR2B antigen was found to be associated with several protein species of up to 690 kDa molecular weight. These observations show NR2B to be part of a multimeric receptor complex. Fractionation of cortex homogenates from E18 rat embryos on sucrose density gradients revealed NR2B polypeptide to be highly enriched in axonal growth cones. A similar distribution was found by fluorescence microscopy of immature hippocampal neurons, showing a preferential accumulation of NR2B antigen in axonal growth cones and varicosities. In mature cells, NR2B antigen displayed a punctated distribution pattern with redistribution to somato-dendritic spheres. The association of NR2B with axonal growth cones and processes of immature neurons suggests a role of NMDA receptors in the regulation of neurite outgrowth and migration.
The inhibitory glycine receptor is a member of the ligand-gated ion channel superfamily. It mediates inhibitory synaptic transmission in mammalian spinal cord and brainstem. Structure and function of the receptor, as well as its chromosomal localization and genetic structure, have been extensively studied. While hereditary and acquired receptor dysfunctions can be identified, selective and specific modulation of receptor function is still lacking. The preponderance of current literature regarding the inhibitory glycine receptor raises the prospect that adequate methods for the treatment of glycine receptor-mediated disorders might be developed.
This article reviewed clinical conditions that assist selection of specific design decisions for the tooth preparation for cast metal restorations. This decision should result in a conservative restoration for a given clinical situation. A paradigm is included to assist the dentist in the decision-making process.
This article presents a review of the evaluation of the temporary splint leading up to the present-day acid-etched composite, wire-reinforced, internal splint called the "A-splint." Nine general uses for the A-splint in the modern dental practice and some common problems are presented.
Mice homozygous for the recessive mutation oscillator (Glra1(spd-ot)) suffer from a complex motor disorder leading to death within three weeks after birth. Symptoms of this disorder mimic poisoning by strychnine, the antagonist of the inhibitory glycine receptor. The syndrome has previously been correlated to a 7 base pair microdeletion within the Glra1 gene (chromosome 11) encoding the alpha1-subunit of the adult glycine receptor isoform. As shown by [3H]strychnine binding and western blot analysis employing subunit-specific antibodies, spinal cord of homozygous oscillator mice was totally devoid of alpha1-polypeptide, characterizing the Glra1(spd-ot) gene as a functional null allele of Glra1. Moreover, tissue levels of the postsynaptic anchoring protein gephyrin were drastically reduced in the Glra1(spd-ot)/Glra1(spd-ot) genotype. In contrast, immunoanalysis revealed a persisting low-level expression of non-alpha1 glycine receptor polypeptides. Spinal glycine receptor content was also significantly reduced in the +/Glra1(spd-ot) genotype. This reduction coincided with increased acoustic startle responses in heterozygous animals consistent with haplotype insufficiency of glycine receptor function. Lethality of the murine null allele Glra1(spd-ot) contrasts with the situation in the human, where homozygosity for a GLRA1 null allele produces the phenotype of the non-lethal disorder hyperekplexia (startle disease; stiff baby syndrome). This suggests a disparate regulation of glycine receptor subunit genes and/or diverse compensatory pathways in mice and humans.
Mutations in inhibitory glycine receptor (GlyR) subunit genes are associated with neuromotor diseases in man and mouse. To use the potential of the mouse mutants as animal models of human disease, we altered GlyR levels in mutant mice and studied their phenotype. A transgene coding for the beta subunit of the rat GlyR was introduced into the genetic background of the spa mutation, which is characterized by low endogenous expression levels of the beta subunit and a dramatic neuromotor phenotype. The resulting transgenic mice expressed the beta subunit mRNA at intermediate levels, and their phenotype was rescued. This provides formal proof for the casual relationship between GlyR beta gene mutation and motor disease, and indicates that a low level of beta gene expression (25% of normal) is sufficient for proper functioning of glycinergic synapses.
An exaggerated startle response caused by mutations of the alpha 1 subunit gene of the inhibitory glycine receptor (GLRA1) is the key symptom of human hyperekplexia or startle disease. The recessive mouse mutant spasmodic (spd) carries a missense mutation in the corresponding murine Glra1 gene which reduces the affinity of agonists for the mutant receptor. This mutant has been regarded as an animal model with which to investigate the molecular basis of hyperekplexia and related motor disorders. The recessive mouse mutant spastic (spa) carries an insertional mutation in the glycine receptor beta subunit gene (Glrb) that results in aberrant splicing and, consequently, in a reduced number of functionally intact receptors. The resulting phenotype is similar to that of spasmodic. This study measured the acoustic startle response of spasmodic and spastic mice under different stimulus conditions, in order to test for sensorimotor processing deficits in these animals. Both mutants show increased startle responses to acoustic stimuli of different intensities compared with wild-type animals.
We previously demonstrated that the inhibitory neurotransmitter glycine induced membrane currents in glial cells from rat spinal cord. In this present study, the patch-clamp technique was combined with the reverse transcription-mediated PCR to analyze the glycine receptor-subunit expression in individual glial cells of rats age 3-18 days. Using the patch-clamp technique in the whole-cell configuration, glial cells were identified by their membrane current pattern and tested for responsiveness to glycine. Subsequently, the cytoplasm was harvested followed by reverse transcription of total cytoplasmic RNA. Subunit-specific cDNA fragments were amplified and analyzed by agarose gel electrophoresis, Southern blotting, and sequencing. In all cell types investigated, transcripts of the alpha1 subunit, but not of alpha 2 or alpha 3 subunits, were detected. In addition, about one-half the glial cells analyzed contained beta-subunit mRNA. These results illustrate that glial cells of rat spinal cord express functional glycine receptors in contrast to cultured glial cells. Glial cells are in intimate contact with synaptic regions making it likely that these nonneuronal receptors may be activated during glycinergic transmission and may trigger yet unknown responses in the glial cells.
"Rural-urban migration has been modeled by both demographers and economists since the 1960s. Little regard has been given by either discipline for the other's models.... The purpose of this paper is to address this void in the African context. We examine three hypotheses: (1) that variables explaining the net urban in-migration rates vary with the age of the migrants; (2) that changes in the availability of services in urban areas [are] a factor in migration; and (3) that cohort structures (age pyramids) are also part of the explanation."
Dominant missense mutations in the human glycine receptor (GlyR) alpha 1 subunit gene (GLRA1) give rise to hereditary hyperekplexia. These mutations impair agonist affinities and change conductance states of expressed mutant channels, resulting in a partial loss of function. In a recessive case of hyperekplexia, we found a deletion of exons 1-6 of the GLRA1 gene. Born to consanguineous parents, the affected child is homozygous for this GLRA1(null) allele consistent with a complete loss of gene function. The child displayed exaggerated startle responses and pronounced head-retraction jerks reflecting a disinhibition of vestigial brain-stem reflexes. In contrast, proprio- and exteroceptive inhibition of muscle activity previously correlated to glycinergic mechanisms were not affected. This case demonstrates that, in contrast to the lethal effect of a null allele in the recessive mouse mutant oscillator (Glra1 spd-ot), the loss of the GlyR alpha 1 subunit is effectively compensated in man.
Subacute necrotizing encephalomyelopathy (Leigh's syndrome) is a rare neurodegenerative disease in the adult. The precise metabolic defect is unknown, but abnormalities of a mitochondrial enzyme system related to cytochrome-c oxidase or pyruvate dehydrogenase are described. The clinical picture usually consists of an altered breathing pattern, oculomotor paralysis, other signs of cranial nerve dysfunction, ataxia, myoclonic jerks, nystagmus, generalized seizures, optic atrophy and demyelinating peripheral neuropathy. Hypopnea leads to CO2-retention with consecutive loss of consciousness demanding mechanical ventilation. Respiratory failure is the most frequent cause of death. Here we describe two patients with adult onset Leigh's syndrome and we discuss the longterm treatment strategies including vitamin B1 and CPAP mask.
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