Biomedical subjects

A F Ryan

Publications and source records attributed to A F Ryan.

At least 19 recordsLinked to original sources

PubMed1996

Role of transcription factors Brn-3.1 and Brn-3.2 in auditory and visual system development.

The neurally expressed genes Brn-3.1 and Brn-3.2 (refs 1-6) are mammalian orthologues of the Caenorhabditis elegans unc-86 gene that constitute, with Brn-3.0 (refs 1-3,8,9), the class IV POU-domain transcription factors. Brn-3.1 and Brn-3.2 provide a means of exploring the potentially distinct biological functions of expanded gene families in neural development. The highly related members of the Brn-3 family have similar DNA-binding preferences and overlapping expression patterns in the sensory nervous system, midbrain and hindbrain, suggesting functional redundancy. Here we report that Brn-3.1 and Brn-3.2 critically modulate the terminal differentiation of distinct sensorineural cells in which they exhibit selective spatial and temporal expression patterns. Deletion of the Brn-3.2 gene causes the loss of most retinal ganglion cells, defining distinct ganglion cell populations. Mutation of Brn-3.1 results in complete deafness, owing to a failure of hair cells to appear in the inner ear, with subsequent loss of cochlear and vestibular ganglia.

Animals↗

PubMed1996

Identification of a novel myosin heavy chain gene expressed in the rat larynx.

Based on reactivity to antibodies against known myosin heavy chains, expression of a novel fast myosin heavy chain (MHC) gene was suspected in the thyroarytenoid (TA) muscle of the rat larynx. The 3' ends of MHC transcripts in the TA were amplified by RT-PCR using a primer to a highly conserved MHC sequence and to the poly(A) tail. The resultant products were cloned and fourteen PCR products were screened by dot-blotting with oligonucleotides specific for known skeletal muscle MHC genes. A clone that reacted weakly to the 2B oligo was sequenced and found to encode a novel fast MHC transcript, termed 2L, that appears to represent an eighth vertebrate skeletal muscle MHC gene. By homology analysis, the 2L sequence is most similar to the extraocular MHC, suggesting a possible evolutionary relationship between MHCs associated with the branchial arches.

Amino Acid Sequence↗

PubMed1996

Basic fibroblast growth factor (FGF-2) protects rat cochlear hair cells in organotypical culture from aminoglycoside injury.

Given the evidence that basic fibroblast growth factor (FGF-2) can protect neural and retinal cells from degeneration, we evaluated the potential of this growth factor to protect sensory cells in the inner ear. When sensory cells of the organ of Corti are exposed to aminoglycoside antibiotics such as neomycin either in vivo or in vitro, significant ototoxicity is observed. The in vitro cytotoxic effects of neomycin are dose and time dependent. In neonatal rat organ of Corti cultures, complete inner and outer hair cell destruction is observed at high (mM) concentrations of neomycin while inner hair cell survival and severely damaged outer hair cells are noted at moderate (microM) concentrations, with a maximal effect observed after 2 days of culture. Approximately 50% of cochlear outer hair cells are lost at a dose of 35 microM neomycin, and most surviving cells show disorganized stereocilia. Inner hair cells show primarily disorganization of their stereocilia. A significant protective effect is observed when the organ of Corti is pre-treated with FGF-2 (500 ng/ml) for 48 hours, and then FGF-2 is included with neomycin in the culture medium. A greater extent of outer hair cell survival and a significant decrease in stereociliary damage are noted with FGF-2. However, disorganization of inner hair cell stereocilia is unaffected by FGF-2. The protective effect of FGF-2 is specific, since interleukin-1B, nerve growth factor, tumor necrosis factor, and epidermal growth factor are ineffective, while retinoic acid and transforming growth factor alpha show only a moderate protective effect. These results confirm the potential of molecules like FGF-2 for preventing cell death due to a variety of causes.

Animals↗

PubMed1995

Distribution of non-NMDA glutamate receptor mRNAs in the developing rat cochlea.

In situ hybridization was used to document the distribution of mRNA encoding six subunit isoforms of non-N-methyl D-aspartic acid (NMDA) glutamate receptors (GluR1, GluR2, GluR3, GluR4, GluR5 and GluR6) in the inner ears of embryonic, postnatal and adult rats. GluR2 and GluR3 expression in the spiral ganglion appeared well before birth, and reached adult levels several days before the onset of function in the cochlea. In the spiral limbus, expression of GluR2 and GluR3 mRNA reached very high levels at around the time of birth, then declined after a few days. Low levels of GluR1, GluR4 and GluR6 expression were detected in various tissues of the cochlea during development. In the adult cochlea, GluR expression was limited to GluR2 and GluR3 mRNAs in the spiral ganglion neurons and GluR2 mRNA in fibrocytes of the spiral limbus, a non-neural tissue. The ontogenetic expression of additional GluR subunit genes and their appearance in different cochlear tissues could reflect different roles for these genes during development, or less precise regulation of gene expression within the GluR family. In particular, the very high levels of GluR gene expression in the spiral limbus during the perinatal period support a non-neural function, perhaps as cell surface receptors during tissue differentiation.

Animals↗

PubMed1995

Identification of a short form of the P2xR1-purinoceptor subunit produced by alternative splicing in the pituitary and cochlea.

A truncated form of the P2xR1 purinoceptor subunit (which we designate P2xR1-2) was detected in rat pituitary gland and the secretory epithelial tissue (stria vascularis) of the cochlea using RT-PCR of solid-phase cDNA libraries. PCR products corresponding to the P2xR1 purinoceptor subunit (1) were obtained from vas deferens, brain and microdissected cochlear sensory epithelial tissues including organ of Corti, sacculus and crista ampullaris. Cloning and sequencing revealed that the P2xR1-2 product included an 85-bp insertion in a region corresponding to a novel C-terminal end of the second membrane spanning domain and continuing as the cytoplasmic domain. A stop codon sequence after the first 51 bp of the insert effectively truncates this subunit, reducing the final cytoplasmic domain by 90% compared with the previously published P2xR1(-1) sequence, thereby reducing the overall peptide by approximately 25%. The region of the receptor lost in the truncated version coded for a number of serine/proline rich regions which may act as potential intracellular regulatory sites.

Alternative Splicing↗

PubMed1995

Expression of acidic FGF mRNA in rat auditory brainstem during postnatal maturation.

In situ hybridization was used to investigate the mRNA distribution of acidic and basic fibroblast growth factor (aFGF and bFGF) in the auditory brainstem of neonatal and adult rats. bFGF mRNA was not detected at any age. In adult rats, aFGF mRNA was strongly expressed in the principal neurons of the anteroventral and posteroventral cochlear nuclei, but not in the octopus cells. In the dorsal cochlear nucleus, aFGF mRNA was seen only in scattered smaller vertical cells. aFGF was strongly expressed in the nucleus of the trapezoid body and in all periolivary cell groups, but not in the medial and lateral olivary nuclei. No expression was observed in the lemniscal nuclei or in the central nucleus of the inferior colliculus, but large neurons in the external zone of the colliculus were labeled. Developmentally, low levels of aFGF expression appeared in the cochlear nuclei and olivary nuclei between P0 and P6. This expression increased rapidly during the onset of hearing, between P10 and P14, and reached adult level by P14-P17. Labeling in collicular neurons appeared slightly later. The results suggest that the appearance of strong aFGF mRNA expression is related to the onset of function.

Aging↗

PubMed1995

Detection of mRNA encoding guanylate cyclase A/atrial natriuretic peptide receptor in the rat cochlea by competitive polymerase chain reaction and in situ hybridization.

Expression of mRNA encoding guanylate cyclase A (GC-A)/atrial natriuretic peptide (ANP) receptor in the rat cochlea was examined by polymerase chain reaction (PCR) and in situ hybridization (ISH). After reverse-transcription, PCR amplification, subcloning, and sequencing, we found that GC-A mRNA with sequence identical to that previously cloned from the rat brain (Chinkers et al., 1989) was expressed in the rat spiral ligament as well as in the spiral ganglion. However, GC-A mRNA was not detected in the stria vascularis. Competitive PCR using internal standard DNAs indicated that the expression of GC-A in the cochlea occurred at a level approximately 16 times less than that measured in kidney cortex. ISH histochemistry using a 35S-labeled antisense riboprobe showed the highest level of expression of GC-A mRNA to be in oligodendrocytes of the cochlear nerve root. The results suggest that ANP may play a role in the cochlear nerve function.

Amino Acid Sequence↗

PubMed1995

Fundamental immune mechanisms of the brain and inner ear.

Because of the blood-brain and blood-labyrinthine barriers, the brain and inner ear were once thought to be immunoprivileged sites. Although these barriers provide protection from inflammatory damage to the delicate structures of the organs, both sites have since been shown to be capable of active immune responses when appropriately stimulated. In the inner ear, perisacular tissue around the endolymphatic sac hosts resident lymphocytes and serves as a site of immunosurveillance. Lymphocytes also enter the inner ear from the circulation, and in the cochlea this occurs via the spiral modiolar vein. Immune responses can protect the labyrinth from infection, but they can also cause bystander injury. Moreover, the cochlea can itself become the target of immune responses that damage hearing. Such autoimmune sensorineural hearing loss can be site specific, with the primary manifestation of the disorder being hearing loss and dysequilibrium. Some of these cases can be diagnosed by antibody or lymphocyte responses to inner ear antigens. Alternately, systemic autoimmune disorders can result in inner ear dysfunction as part of a broader spectrum of disease. Both forms of immune-mediated inner ear dysfunction may respond to immunosuppressive therapies, including steroids, cytotoxic agents, and plasmapheresis.

Animals↗

PubMed1995

Expression of immunoregulatory cytokines during acute and chronic middle ear immune response.

In both patients and experimental animals, immunoglobulin G (IgG) has been found to dominate acute otitis media with effusion (OME), whereas IgA tends to be present in chronic but not in acute OME. To determine whether local immunoregulation could account for this difference, the expression of cytokines associated with the production of different antibody isotypes was investigated in experimental acute and chronic OME. Mice were systemically immunized and then challenged transtympanically, once to produce an acute OME or once per week for 6 weeks to produce chronic OME. Hybridization with molecular probes for cytokine genes showed that cells producing interleukin-2 (IL-2) and IL-4, but not IL-5, were present during acute OME. In chronic OME, IL-2-positive (IL-2+) and IL-4+ cells were less prevalent, but IL-5+ cells were numerous. These finding support a model by which locally produced IL-2 and IL-4 augment IgG production in acute OME, whereas, IL-5 contributes to increased IgA production in chronic OME.

Acute Disease↗

PubMed1994

2-Deoxyglucose uptake patterns in response to pure tone stimuli in the aged rat inferior colliculus.

The tonotopic map of the inferior colliculus (IC) of aged rats (25 months old) was examined to determine whether age-related changes known to occur in the cochlea are reflected in the 2-deoxyglucose (2-DG) uptake pattern of the IC. Because aged animals have hearing losses, auditory brainstem response thresholds were measured. Animals with threshold shifts of no greater than 30 dB relative to young animals were used. Animals were injected with radiolabeled 2-DG and stimulated with continuous pure tones presented at 70 dB above the behavioral thresholds for young animals at either 1, 4 or 32 kHz for one hour in a sound attenuated booth. The stimulus sound pressure levels were chosen to achieve comparable sensation levels between the young and aged animals. The tonotopic map of the IC in aged rats was different from that reported previously for young animals (Ryan et al., 1988), in that, the regions stimulated by 1 and 4 kHz were shifted towards the higher frequencies and the uptake areas were twice as broad for the aged animals as for the young animals. The observed 2-DG uptake patterns are consistent with an activation pattern of a high intensity stimulus and a loss of responsive elements in the cochlear apex. Similar broad and shifted bands of activated tissue may contribute to difficulties in auditory perception in aged humans with increased thresholds and sound amplification.

Acoustic Stimulation↗

PubMed1994

Nicotinic acetylcholine receptor subunits expressed in rat cochlea detected by the polymerase chain reaction.

Poly(A)+ RNA was extracted from rat cochleae using guanidinium thiocyanate and oligo(dT)-cellulose, and converted into cDNA by reverse transcriptase using an oligo(dT) primer. Oligonucleotides complementary to conserved 5' and 3' regions of alpha and beta subunits of the neuronal nicotinic acetylcholine receptor subunit (nAChR) family were then used as primers to screen the cochlear cDNA via the polymerase chain reaction (PCR) procedure. PCR products of approximately 900 bp length, purified by agarose gel electrophoresis, were nick translated to produce [32P]-dCTP labelled probes for Southern Blot screening of nAChR cDNAs. Of the four alpha and three beta subunits screened, only alpha 5 and beta 4 nAChR cDNAs hybridized. The alpha 5 PCR product was cloned and sequenced and proved to be identical to published sequence for alpha 5. The detection of alpha 5 and beta 4 nAChR subunit expression in cochlear tissue supports previous electrophysiological and immunocytochemical evidence for nAChR-mediated centrifugal control of hearing function.

Animals↗

PubMed1994

Protection from noise-induced hearing loss by prior exposure to a nontraumatic stimulus: role of the middle ear muscles.

Recent evidence suggests that prior exposure to a moderate-level acoustic stimulus can reduce damage due to later exposure to the same stimulus at high intensity [Canlon et al., Hear. Res. 34, 197-200 (1988)]. To test the role of the middle ear muscles (MEMs) in this phenomenon, Mongolian gerbils were conditioned by exposure to a two-octave band of noise (1414-5656 Hz) at 81 dB SPL for 3 weeks. Either immediately afterward, or following a one week rest period, they were exposed to the same stimulus at 110 dB SPL for one hour. The ABR thresholds of these animals were compared to those seen in animals exposed at 110 dB SPL without conditioning. The MEMs of one ear in each subject were cut, to determine their role in any noise trauma protection effects. In the unoperated ears, conditioning without a recovery period did not alter the effects of the 110 dB stimulus. Conditioning followed by a one week recovery period reduced both temporary (TTS) and permanent (PTS) threshold shift. MEM section had no effect on either TTS or PTS in unconditioned subjects, and did not alter the reduction in TTS or PTS seen with conditioning. It is concluded that the noise trauma resistance provided by acoustic conditioning is not mediated by the MEMs.

Acoustic Stimulation↗

PubMed1994

Acidic and basic FGF mRNA expression in the middle ear mucosa during experimental acute and chronic otitis media.

Fibroblast growth factors (FGFs) induce the proliferation and differentiation of cells of mesodermal and neuroectodermal origin. Using in situ hybridization, messenger ribonucleic acid encoding acidic FGF, basic FGF and FGF receptor 1 (FGFR1) were localized in the middle ear mucosa of experimental animals with acute and chronic immune-mediated otitis media with effusion (OME). Basic FGF-labeled cells were seen in the subepithelial connective tissue layer (SE) preferentially near the epithelial basement membrane. Acidic FGF-labeled cells were seen in the SE, preferentially near blood vessels and occasionally in the cellular middle ear effusion (CE). FGFR1-labeled cells were seen in the SE and in the CE. The distribution of labeled cells in the middle ear suggests that basic FGF is produced by fibroblasts, acidic FGF is produced by leukocytes, and FGFR1 is produced by both fibroblasts and leukocytes. A role is proposed for these peptides in the proliferation and maintenance of the middle ear submucosa during otitis media.

Acute Disease↗

PubMed1993

Cloning and in situ hybridization of type 2A and 2B rat skeletal muscle myosin tail region: implications for filament assembly.

Changes in fast myosin expression play a critical role in skeletal muscle adaptation. Two fast myosin isoforms, type 2A and type 2B, are commonly expressed by fast muscle fibers but their sequences have not been determined to allow mRNA expression studies. A complete set of rat skeletal muscle myosins was amplified by PCR of cDNAs derived from skeletal muscle mRNA, cloned in a TA cloning vector, and sequenced. Specificity was demonstrated by in situ hybridization against skeletal muscle and myosin protein identification using monoclonal antibodies. Two novel sequences were cloned: A type 2A myosin which consisted of a 642 bp segment from the 3' end and a type 2B myosin which consisted of a 624 bp segment also from the 3' end. This region encodes that portion of the myosin molecule implicated in the control of filament assembly. The two fast myosins showed 88% homology in the open reading frame and 95% homology at the amino acid level. Based on this homology, it is unlikely that selective myosin filament assembly occurs during muscle fiber type transformation between type 2A and 2B.

Amino Acid Sequence↗

PubMed1993

Fibronectin-like immunoreactivity of the basilar membrane of young and aged rats.

Dysfunction of cochlear mechanics has been hypothesized to be a source of age-related hearing loss and the basilar membrane mass and stiffness contribute to normal cochlear mechanics. Fibronectin, a large, extracellular matrix protein and a major component of the basilar membrane, may contribute to both the mass and stiffness of the membrane. Mesothelial cells underlying the basilar membrane may produce the fibronectin and also contribute to the mass of the membrane. Changes in either the fibronectin or the mesothelial cells might, therefore, have an effect on cochlear mechanics. In order to assess basilar membrane changes in aged animals, young adult (2-4 months) and aged (24-26 months) Sprague-Dawley rats were evaluated for the presence of fibronectin-like protein and mesothelial cells. The basilar membrane in the young animals had strong fibronectin-like immunoreactivity throughout its length. The old animals, on the other hand, showed normal fibronectin immunoreactivity in the basilar membrane of the basal turn, but little or no reactivity in the apical cochlear turn. The number of mesothelial cells was reduced throughout the length of the membrane in aged animals, with the greatest loss in the basal turn (60% fewer cells). These two degenerative changes, which appear to be independent of each other, may contribute to the observed threshold shifts in aged cochleas.

Aging↗

PubMed1993

Cloning genes from an inner ear cDNA library.

A rat inner ear complementary DNA (cDNA) library containing 1.9 x 10(6) recombinants was constructed and evaluated. Inserts averaged 2.0 (+/- 2.1) kilobases in length. A subset of inserts was screened for site of expression. Two cDNA transcripts were isolated based on cochlear expression restricted to the spiral ganglion. One transcript showed a high degree of homology to several long interspersed DNA elements, neuron-specific nuclear transcripts thought to be involved in gene regulation. The second transcript showed no homology to known sequences and appears to encode a neuron-specific protein of about 248 amino acids. The library can be used to identify proteins important for inner ear function and disease.

Animals↗

PubMed1993

Acidic and basic FGF mRNA expression in the adult and developing rat cochlea.

In situ hybridization was used to document the distribution of mRNA encoding acidic and basic fibroblast growth factor (aFGF and bFGF) in the rat cochlea from embryonic day (E) 16 to postnatal day (P) > 60. bFGF mRNA was not detected in the cochlea at any age. In the adult, aFGF mRNA was strongly expressed in spiral ganglion (SG) neurons, and this expression increased from base to apex. The stria vascularis (SV) and spiral prominence (SP) showed lesser expression which was equal in all turns. Developmentally, low level expression of aFGF mRNA was first seen in the SG at E-20, and remained low until P-4. Expression increased from P-6 to P-14, when adult levels were reached. aFGF mRNA was also observed in the developing hair cells of all turns at E-20. This expression increased after birth but disappeared after P-6. Expression in the SV and SP was first noted at E-20 and reached adult levels by P-16 and P-10, respectively. High levels of aFGF mRNA in the adult SG suggest that aFGF is important for the maintenance of SG neuron function and structure. aFGF in hair cells during the first postnatal week may be involved in the establishment of cochlear innervation.

Animals↗

PubMed1993

Growth factors during proliferation of the middle ear mucosa.

The presence of growth factors during otitis media with effusion (OME), induced by antigenic stimulation of the middle ear, was explored in guinea pigs. There was a significant increase in heparin-binding proliferative activity detected in mucosal tissue. This heparin-binding activity was identified as basic fibroblast growth factor (FGF) using several criteria. First, the majority of this activity was eluted from heparin-Sepharose gels with 1.3-1.8 M NaCl, consistent with basic FGF. Second, these fractions are potent stimulators of endothelial cell proliferation, characteristic of basic FGF. Third, the fractions contain basic FGF-like immunoreactivity based on radioimmunoassay. Finally, Western blotting of tissue extracts reveals the presence of an 18 kDa protein that is indistinguishable from basic FGF. We conclude that basic FGF plays a major role in the proliferation of the middle ear mucosa during OME.

Animals↗