Biomedical subjects

G P Richardson

Publications and source records attributed to G P Richardson.

At least 19 recordsLinked to original sources

PubMed2003

A receptor-like inositol lipid phosphatase is required for the maturation of developing cochlear hair bundles.

A screen for protein tyrosine phosphatases (PTPs) expressed in the chick inner ear yielded a high proportion of clones encoding an avian ortholog of protein tyrosine phosphatase receptor Q (Ptprq), a receptor-like PTP. Ptprq was first identified as a transcript upregulated in rat kidney in response to glomerular nephritis and has recently been shown to be active against inositol phospholipids. An antibody to the intracellular domain of Ptprq, anti-Ptprq, stains hair bundles in mice and chicks. In the chick ear, the distribution of Ptprq is almost identical to that of the 275 kDa hair-cell antigen (HCA), a component of hair-bundle shaft connectors recognized by a monoclonal antibody (mAb) that stains inner-ear hair bundles and kidney glomeruli. Furthermore, anti-Ptprq immunoblots a 275 kDa polypeptide immunoprecipitated by the anti-HCA mAb from the avian inner ear, indicating that the HCA and Ptprq are likely to be the same molecule. In two transgenic mouse strains with different mutations in Ptprq, anti-Ptprq immunoreactivity cannot be detected in the ear. Shaft connectors are absent from mutant vestibular hair bundles, but the stereocilia forming the hair bundle are not splayed, indicating that shaft connectors are not necessary to hold the stereocilia together; however, the mice show rapid postnatal deterioration in cochlear hair-bundle structure, associated with smaller than normal transducer currents with otherwise normal adaptation properties, a progressive loss of basal-coil cochlear hair cells, and deafness. These results reveal that Ptprq is required for formation of the shaft connectors of the hair bundle, the normal maturation of cochlear hair bundles, and the long-term survival of high-frequency auditory hair cells.

Animals↗

PubMed2002

Reduced climbing and increased slipping adaptation in cochlear hair cells of mice with Myo7a mutations.

Mutations in Myo7a cause hereditary deafness in mice and humans. We describe the effects of two mutations, Myo7a(6J) and Myo7a(4626SB), on mechano-electrical transduction in cochlear hair cells. Both mutations result in two major functional abnormalities that would interfere with sound transduction. The hair bundles need to be displaced beyond their physiological operating range for mechanotransducer channels to open. Transducer currents also adapt more strongly than normal to excitatory stimuli. We conclude that myosin VIIA participates in anchoring and holding membrane-bound elements to the actin core of the stereocilium. Myosin VIIA is therefore required for the normal gating of transducer channels.

Actins↗

PubMed2001

FM1-43 dye behaves as a permeant blocker of the hair-cell mechanotransducer channel.

Hair cells in mouse cochlear cultures are selectively labeled by brief exposure to FM1-43, a styryl dye used to study endocytosis and exocytosis. Real-time confocal microscopy indicates that dye entry is rapid and via the apical surface. Cooling to 4 degrees C and high extracellular calcium both reduce dye loading. Pretreatment with EGTA, a condition that breaks tip links and prevents mechanotransducer channel gating, abolishes subsequent dye loading in the presence of calcium. Dye loading recovers after calcium chelation with a time course similar to that described for tip-link regeneration. Myo7a mutant hair cells, which can transduce but have all mechanotransducer channels normally closed at rest, do not label with FM1-43 unless the bundles are stimulated by large excitatory stimuli. Extracellular perfusion of FM1-43 reversibly blocks mechanotransduction with half-blocking concentrations in the low micromolar range. The block is reduced by high extracellular calcium and is voltage dependent, decreasing at extreme positive and negative potentials, indicating that FM1-43 behaves as a permeant blocker of the mechanotransducer channel. The time course for the relief of block after voltage steps to extreme potentials further suggests that FM1-43 competes with other cations for binding sites within the pore of the channel. FM1-43 does not block the transducer channel from the intracellular side at concentrations that would cause complete block when applied extracellularly. Calcium chelation and FM1-43 both reduce the ototoxic effects of the aminoglycoside antibiotic neomycin sulfate, suggesting that FM1-43 and aminoglycosides enter hair cells via the same pathway.

Aminoglycosides↗

PubMed2001

The cell adhesion molecule BEN defines a prosensory patch in the developing avian otocyst.

The distribution of the cell adhesion molecule BEN in the developing chick inner ear is described. BEN is first detected in the otic placode at stage 11. As the placode begins to invaginate, BEN becomes concentrated in a ventromedial region extending from the anterior to the posterior end of the otic pit. BEN expression levels increase in this region as the pit closes to form the otocyst, and distinct boundaries become defined along the dorsal and ventral edges of the ventromedial band of BEN expression. BEN expression also becomes concentrated dorsally within the otic epithelium as the pit closes and is observed in the condensing otic ganglion. By stage 22, the ventromedial band of BEN expression splits into two distinct regions, a small caudal patch within which the posterior crista will develop, and a larger anterior patch. By stage 26, this larger anterior patch of cells expressing BEN becomes subdivided into five separate areas corresponding to the regions within which the anterior crista, the lateral crista, the utricle, the saccule, and both the basilar papilla and lagenar macula form. Hair cells only develop within these regions defined by BEN distribution. The data suggest that the ventromedial patch of BEN expression observed from stage 11 onwards defines a single sensory competent zone from which all sensory organs of the inner ear develop. BEN immunoreactivity in the inner ear declines after stage 38. In response to noise exposure, upregulation of BEN expression is mainly detected in regions of the posthatch papilla where the damage is severe and regenerating hair cells are not observed. The regenerating hair and supporting cells do not express BEN, highlighting a molecular difference between the processes of development and regeneration.

Activated-Leukocyte Cell Adhesion Molecule↗

PubMed2001

8-Carboxamidocyclazocine analogues: redefining the structure-activity relationships of 2,6-methano-3-benzazocines.

Unexpectedly high affinity for opioid receptors has been observed for a novel series of cyclazocine analogues where the prototypic 8-OH was replaced by a carboxamido group. For mu and kappa opioid receptors, the primary carboxamido derivative of cyclazocine ((+/-)-15) displayed high affinity (Ki=0.41 and 0.53 nM, respectively) nearly comparable to cyclazocine. A high enantiopreference ((2R,6R,11R)-) for binding was also observed. Compound (+/-)-15 also displayed potent antinociception activity in mice when administered icv.

Amides↗

PubMed2000

A targeted deletion in alpha-tectorin reveals that the tectorial membrane is required for the gain and timing of cochlear feedback.

alpha-tectorin is an extracellular matrix molecule of the inner ear. Mice homozygous for a targeted deletion in a-tectorin have tectorial membranes that are detached from the cochlear epithelium and lack all noncollagenous matrix, but the architecture of the organ of Corti is otherwise normal. The basilar membranes of wild-type and alpha-tectorin mutant mice are tuned, but the alpha-tectorin mutants are 35 dB less sensitive. Basilar membrane responses of wild-type mice exhibit a second resonance, indicating that the tectorial membrane provides an inertial mass against which outer hair cells can exert forces. Cochlear microphonics recorded in alpha-tectorin mutants differ in both phase and symmetry relative to those of wild-type mice. Thus, the tectorial membrane ensures that outer hair cells can effectively respond to basilar membrane motion and that feedback is delivered with the appropriate gain and timing required for amplification.

Acoustic Stimulation↗

PubMed1999

A missense mutation in myosin VIIA prevents aminoglycoside accumulation in early postnatal cochlear hair cells.

Myosin VIIA is expressed by sensory hair cells in the inner ear and proximal tubule cells in the kidney, the two primary targets of aminoglycoside antibiotics. Using cochlear cultures prepared from early postnatal Myo7a6J mice with a missense mutation in the head region of the myosin VIIA molecule we show that this myosin is required for aminoglycoside accumulation in cochlear hair cells. Hair cells in homozygous mutant Myo7a6J cochlear cultures have disorganized hair bundles, but are otherwise morphologically normal and transduce. However, and in contrast to hair cells from heterozygous Myo7a6J cultures, the homozygous Myo7a6J hair cells do not accumulate [3H]gentamicin and do not exhibit an ototoxic response on exposure to aminoglycoside. Possible roles for myosin VIIA in the process of aminoglycoside accumulation are discussed.

Animals↗

PubMed1999

The supporting-cell antigen: a receptor-like protein tyrosine phosphatase expressed in the sensory epithelia of the avian inner ear.

After noise- or drug-induced hair-cell loss, the sensory epithelia of the avian inner ear can regenerate new hair cells. Few molecular markers are available for the supporting-cell precursors of the hair cells that regenerate, and little is known about the signaling mechanisms underlying this regenerative response. Hybridoma methodology was used to obtain a monoclonal antibody (mAb) that stains the apical surface of supporting cells in the sensory epithelia of the inner ear. The mAb recognizes the supporting-cell antigen (SCA), a protein that is also found on the apical surfaces of retinal Müller cells, renal tubule cells, and intestinal brush border cells. Expression screening and molecular cloning reveal that the SCA is a novel receptor-like protein tyrosine phosphatase (RPTP), sharing similarity with human density-enhanced phosphatase, an RPTP thought to have a role in the density-dependent arrest of cell growth. In response to hair-cell damage induced by noise in vivo or hair-cell loss caused by ototoxic drug treatment in vitro, some supporting cells show a dramatic decrease in SCA expression levels on their apical surface. This decrease occurs before supporting cells are known to first enter S-phase after trauma, indicating that it may be a primary rather than a secondary response to injury. These results indicate that the SCA is a signaling molecule that may influence the potential of nonsensory supporting cells to either proliferate or differentiate into hair cells.

Animals↗

PubMed1999

Tectorin mRNA expression is spatially and temporally restricted during mouse inner ear development.

The tectorial and otolithic membranes are extracellular matrices that cover the sensory epithelia of the inner ear. They are required for mechanotransduction and may influence hair-cell development. The mRNA expression patterns for two major glycoproteins of these matrices, alpha- and beta-tectorin, were examined during mouse inner ear development to determine when and where these proteins are produced relative to hair cells and whether tectorin production is continuous or transient. Using in situ hybridisation, alpha- and beta-tectorin mRNAs are first detected in the basal end of the cochlea at embryonic day (E) 12.5, and the distinct patterns observed for each tectorin mRNA in the neonate become visible by E14.5. The neonatal expression patterns indicate that some cell types in the cochlea express both alpha- and beta-tectorin mRNAs, while other cells only express one tectorin mRNA. Although expressed early in development, alpha- and beta-tectorin mRNAs cannot be detected in the cochlea by postnatal day (P) 22. In the saccule and utricle, alpha-tectorin mRNA is detected at E12.5, but beta-tectorin mRNA is not observed until E14.5. Expression of alpha-tectorin mRNA ceases after P15, whereas beta-tectorin mRNA expression continues within the striolar region of the utricle until at least P150. The results show alpha- and beta-tectorin mRNAs are expressed during the early stages of inner ear development, prior to or concomitant with hair-cell differentiation, and before the appearance of hair bundles. The expression patterns suggest different cell types contribute to the formation of the various regions of the tectorial membrane. Although tectorin mRNAs are only expressed transiently during cochlear development, beta-tectorin mRNA is continuously expressed within the striolar region of the utricle.

Animals↗

PubMed1999

Chick alpha-tectorin: molecular cloning and expression during embryogenesis.

The avian and mammalian tectorial membranes both contain two non-collagenous glycoproteins, alpha and beta-tectorin. To determine whether variations in the primary sequences of the chick and mouse alpha-tectorins account for differences in subunit composition and matrix structure of the tectorial membranes in these two species, cDNAs spanning the entire open reading frame of chick alpha-tectorin were cloned and the derived amino acid sequence was compared with that of mouse alpha-tectorin. Chick alpha-tectorin shares 73% amino acid sequence identity with mouse alpha-tectorin and, like mouse alpha-tectorin, is composed of three distinct modules: an N-terminal region similar to the G1 domain of entactin, a central region that shares identity with zonadhesin and contains three full and two partial von Willebrand factor type D repeats, and a C-terminal region containing a zona pellucida domain. The central region of chick alpha-tectorin contains fewer potential N-glycosylation sites than that of mouse alpha-tectorin and is cleaved at two additional sites. Differences in the glycosylation and proteolytic processing of chick and mouse alpha-tectorin may therefore account for the variation observed in the composition and structure of the collagenase-insensitive matrices of the avian and mammalian tectorial membranes. In situ hybridisation and Northern blot analysis of chick inner ear tissue indicate that the spatial and temporal patterns of alpha and beta-tectorin mRNA expression in the developing chick inner ear are different, suggesting the two tectorins may each form homomeric filaments.

Amino Acid Sequence↗

PubMed1998

Mapping of the alpha-tectorin gene (TECTA) to mouse chromosome 9 and human chromosome 11: a candidate for human autosomal dominant nonsyndromic deafness.

alpha-Tectorin is one of the major noncollagenous components of the mammalian tectorial membrane in the inner ear. We have mapped the gene encoding alpha-tectorin to mouse chromosome 9 and human chromosome 11 in a known region of conserved synteny. Human YAC clones containing alpha-tectorin have been identified, demonstrating physical linkage to the anonymous marker D11S925. This places alpha-tectorin within the genetic interval that contains both the human nonsyndromic autosomal dominant deafness DFNA12 and the proximal limit of a subset of deletions within Jacobsen syndrome. Thus both DFNA12 and the hearing loss in some cases of Jacobsen syndrome may be due to haploinsufficiency for TECTA.

Animals↗

PubMed1998

Nonsyndromic hearing impairment is associated with a mutation in DFNA5.

Nonsyndromic hearing impairment is one of the most heterogeneous hereditary conditions, with more than 40 loci mapped on the human genome, however, only a limited number of genes implicated in hearing loss have been identified. We previously reported linkage to chromosome 7p15 for autosomal dominant hearing impairment segregating in an extended Dutch family (DFNA5). Here, we report a further refinement of the DFNA5 candidate region and the isolation of a gene from this region that is expressed in the cochlea. In intron 7 of this gene, we identified an insertion/deletion mutation that does not affect intron-exon boundaries, but deletes five G-triplets at the 3' end of the intron. The mutation co-segregated with deafness in the family and causes skipping of exon 8, resulting in premature termination of the open reading frame. As no physiological function could be assigned, the gene was designated DFNA5.

Adolescent↗

PubMed1998

Mutations in the human alpha-tectorin gene cause autosomal dominant non-syndromic hearing impairment.

The tectorial membrane is an extracellular matrix of the inner ear that contacts the stereocilia bundles of specialized sensory hair cells. Sound induces movement of these hair cells relative to the tectorial membrane, deflects the stereocilia, and leads to fluctuations in hair-cell membrane potential, transducing sound into electrical signals. Alpha-tectorin is one of the major non-collagenous components of the tectorial membrane. Recently, the gene encoding mouse alpha-tectorin (Tecta) was mapped to a region of mouse chromosome 9, which shows evolutionary conservation with human chromosome 11q (ref. 3), where linkage was found in two families, one Belgian (DFNA12; ref. 4) and the other, Austrian (DFNA8; unpublished data), with autosomal dominant non-syndromic hearing impairment. We determined the complete sequence and the intron-exon structure of the human TECTA gene. In both families, mutation analysis revealed missense mutations which replace conserved amino-acid residues within the zona pellucida domain of TECTA. These findings indicate that mutations in TECTA are responsible for hearing impairment in these families, and implicate a new type of protein in the pathogenesis of hearing impairment.

Alternative Splicing↗

PubMed1997

Myosin VIIA is required for aminoglycoside accumulation in cochlear hair cells.

Myosin VIIA is expressed by sensory hair cells and has a primary structure predicting a role in membrane trafficking and turnover, processes that may underlie the susceptibility of hair cells to aminoglycoside antibiotics. [3H]Gentamicin accumulation and the effects of aminoglycosides were therefore examined in cochlear cultures of mice with different missense mutations in the myosin VIIA gene, Myo7a, to see whether myosin VIIA plays a role in aminoglycoside ototoxicity. Hair cells from homozygous mutant Myo7ash1 mice, with a mutation in a nonconserved region of the myosin VIIA head, respond rapidly to aminoglycoside treatment and accumulate high levels of gentamicin. Hair cells from homozygous mutant Myo7a6J mice, with a mutation at a highly conserved residue close to the ATP binding site of the myosin VIIA head, do not accumulate [3H]gentamicin and are protected from aminoglycoside ototoxicity. Hair cells from heterozygotes of both alleles accumulate [3H]gentamicin and respond to aminoglycosides. Although aminoglycoside uptake is thought to be via apical surface-associated endocytosis, coated pit numbers on the apical membrane of heterozygous and homozygous Myo7a6J hair cells are similar. Pulse-chase experiments with cationic ferritin confirm that the apical endocytotic pathway is functional in homozygous Myo7a6J hair cells. Transduction currents can be recorded from both heterozygous and homozygous Myo7a6J hair cells, suggesting it is unlikely that the drug enters via diffusion through the mechanotransducer channel. The results show that myosin VIIA is required for aminoglycoside accumulation in hair cells. Myosin VIIA may transport a putative aminoglycoside receptor to the hair cell surface, indirectly translocate it to sites of membrane retrieval, or retain it in the endocytotic pathway.

Adenosine Triphosphate↗

PubMed1997

A quantitative comparison of mechanoelectrical transduction in vestibular and auditory hair cells of neonatal mice.

Vestibular hair cells (VHCs) and cochlear outer hair cells (OHCs) of neonatal mice were stimulated by a fluid jet directed at their stereociliary bundles. Relations between the force exerted by the jet, bundle displacement, and the resulting transducer current were studied. The mean maximum transducer conductance in VHCs (2.6 nS) was about half that of the OHCs (5.5 nS), with the largest recorded values being 4.1 nS and 9.2 nS, respectively. In some OHCs activity of a single, 112 pS transducer channel was observed, allowing an estimate of the maximum number of channels: up to 36 in VHCs and 82 in OHCs, corresponding to about one transducer channel per tip link. The VHC bundles required about 330 nm of tip displacement to activate 90% of the maximum transducer conductance, compared to 150 nm for the OHC bundles. This corresponded to 2 deg of rotation about their pivots for both, due to the greater length of the VHC bundles. The VHC bundles' translational stiffness was one-seventh of that of the OHCs. Conversion to rotational stiffness almost abolished this difference. Rotation of the hair bundle rather than translation determines the gating of the transducer channels, independent of bundle height or origin of the cells.

Animals↗

PubMed1997

The mouse tectorins. Modular matrix proteins of the inner ear homologous to components of the sperm-egg adhesion system.

The cDNA and derived amino acid sequences for the two major non-collagenous proteins of the mouse tectorial membrane, alpha- and beta-tectorin, are presented. The cDNA for alpha-tectorin predicts a protein of 239,034 Da with 33 potential N-glycosylation sites, and that of beta-tectorin a smaller protein of 36,074 Da with 4 consensus N-glycosylation sites. Southern and Northern blot analysis indicate alpha- and beta-tectorin are single copy genes only expressed in the inner ear, and in situ hybridization shows they are expressed by cells both in and surrounding the mechanosensory epithelia. Both sequences terminate with a hydrophobic COOH terminus preceded by a potential endoproteinase cleavage site suggesting the tectorins are synthesized as glycosylphosphatidylinositol-linked, membrane bound precursors, targeted to the apical surface of the inner ear epithelia by the lipid and proteolytically released into the extracellular compartment. The mouse beta-tectorin sequence contains a single zona pellucida domain, whereas alpha-tectorin is composed of three distinct modules: an NH2-terminal region similar to part of the entactin G1 domain, a large central segment with three full and two partial von Willebrand factor type D repeats, and a carboxyl-terminal region which, like beta-tectorin, contains a single zona pellucida domain. The central, high molecular mass region of alpha-tectorin containing the von Willebrand factor type D repeats has homology with zonadhesin, a sperm membrane protein that binds to the zona pellucida. These results indicate the two major non-collagenous proteins of the tectorial membrane are similar to components of the sperm-egg adhesion system, and, as such may interact in the same manner.

Alternative Splicing↗

PubMed1997

Antibodies to the sulphated, high molecular mass mouse tectorin stain hair bundles and the olfactory mucus layer.

Polyclonal antibodies were raised in chickens to the glycosylated forms of the high (H), medium (M) and low (L) molecular mass (MM) mouse tectorins. In the mouse cochlea, all three antibodies stained the tectorial membrane. Antibodies raised to HMM tectorin also stained the hair bundles of both inner and outer hair cells. A number of other mouse tissues were screened with the anti-tectorin antibodies to look for similar or antigenically related molecules. Staining was not observed in any other tissue type with the antibodies directed against the MMM and LMM tectorins. In the nose, the anti-HMM tectorin antibodies stained Bowman's glands and the mucus layer overlying the olfactory epithelium. The surface of the adjacent respiratory epithelium was not stained by these antibodies. HMM tectorin can be specifically radiolabelled by injecting neonatal mice with 35SO4 and undergoes a shift in electrophoretic mobility following treatment with keratanase, an endo-beta-galactosidase from Pseudomonas. However, when centrifuged on shallow CsCl gradients HMM tectorin has a buoyant density similar to that of glycoproteins and does not behave as a typical cartilage type proteoglycan. HMM tectorin does not react with mab 5D4, a monoclonal antibody that recognises keratan sulphate glycosaminoglycan from corneal and skeletal muscle proteoglycan. Unlike antibodies to HMM tectorin, mab 5D4 selectively stains the upper surface of the tectorial membrane, Hensen's stripe and the mucus layer overlying the respiratory epithelium. These studies indicate that the MMM and LMM tectorins may be unique to the cochlea, and that HMM may be a "light' keratan sulphate proteoglycan that is antigenically related to either the mucins or a more specific component of the olfactory mucus layer.

Animals↗

PubMed1996

Distribution of beta-tectorin mRNA in the early posthatch and developing avian inner ear.

Expression of beta-tectorin mRNA in the inner ear of the embryonic and early posthatch (PH) chick was studied by in situ hybridisation. In the PH chick, beta-tectorin mRNA is expressed in the basilar papilla, in the clear and the cuboidal cells that lie either side of the papilla, in the striolar regions of the maculae, and in two small groups of cells lying adjacent to the midline in the cristae of the anterior and posterior ampullae. Expression of beta-tectorin is not observed in the lateral ampulla. In the sensory epithelia of the PH chick in which beta-tectorin mRNA is detected, expression is restricted to the supporting cell population. During development of the cochlear duct, beta-tectorin expression begins between embryonic (E) days 5 and 6. At E6, expression is observed throughout the length of the duct but is highest at the distal end. By E7, the pattern of expression is reversed and is highest at the proximal end of the cochlea, suggesting that a wave of high beta-tectorin expression passes disto-proximally along the papilla during E6 and E7. Expression of beta-tectorin mRNA is not detected in the homogene cells at any stage during the development of the cochlear duct, indicating that these cells do not synthesise one of the two major proteins of the avian tectorial membrane. The distribution of supporting cells expressing beta-tectorin mRNA in the different epithelia was compared with the distribution of sensory cells that have type B hair bundles, those with shaft links restricted to basal regions of their stereocilia, and sensory cells that have type A bundles, those with shaft links all over the entire surface of their stereocilia. Hair cells with type A hair bundles are never found in association with supporting cells expressing beta-tectorin. Although there is a correspondence in the basilar papilla and the maculae of the utriculus and lagena between the distribution of supporting cells expressing beta-tectorin mRNA and hair cells with type B bundles, this correlation does not generalise to the other sensory epithelia.

Animals↗