[Show abstract][Hide abstract] ABSTRACT: Diagnosing middle ear disorders in neonates is a challenging task for both audiologists and otolaryngologists. Although high-frequency (1000 Hz) tympanometry and acoustic stapedial reflex tests are useful in diagnosing middle ear problems in this age group, they do not provide information about the dynamics of the middle ear in terms of its resonance frequency (RF) and mobility. The sweep frequency impedance (SFI) test can provide this information, which may assist in the diagnosis of middle ear dysfunction in neonates.
Journal of the American Academy of Audiology 04/2014; 25(4):343-54. · 1.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: OBJECTIVE: Early diagnosis and treatment of hearing disorders in neonates is highly effective for realization of linguistic competence and intellectual development. To objectively and quickly evaluate the dynamic characteristics of the middle ear, a sweep frequency impedance (SFI) meter was developed, which allowed the diagnosis of middle-ear dysfunctions in adults and children. However, this SFI meter was not applicable to neonates since the size of the measurement probe was too large. In the present study, therefore, the SFI meter was improved, i.e., the diameter of the probe was reduced to that of the neonatal external ear canal. By using this newly designed SFI meter, SFI tests were performed in healthy neonates. METHODS: A sound of the sweeping sinusoidal frequency between 0.1kHz and 2.0kHz in 0.02-kHz step intervals is presented to the ear canal by an SFI probe while the static pressure of the ear canal is kept constant. During this procedure, the sound pressure level (SPL) is measured. The measurements are performed at 50-daPa intervals of static pressure from 200daPa to -200daPa. RESULTS: Measurements were conducted in 10 ears of 9 neonates. The SPL showed two variations at 0.26±0.03kHz and 1.13±0.12kHz. Since the SPL is known to show a variation at frequencies from 1.0kHz to 1.6kHz due to the resonance of the middle ear in adults and children with normal hearing, the second variation is probably related to such resonance in neonates. The measurement of gel models, which mimics the neonatal external ear canal, showed a variation in SPL at around 0.5kHz. This implies that the source of the first variation may possibly be related to the resonance of the external ear canal wall. CONCLUSIONS: SFI tests revealed that there were two variations in the SPL curve in neonates, one at 0.26±0.03kHz and the other at 1.13±0.12kHz, the former and the latter being possibly related to the resonance of the external ear canal wall and that of the middle ear, respectively. This result suggests that the dynamic characteristics of the middle ear in neonates are different from those in adults.
International journal of pediatric otorhinolaryngology 01/2013; · 0.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Targeted delivery of treatment agents to the inner ear using nanoparticles is an advanced therapeutic approach to cure or alleviate hearing loss. Designed to target the outer hair cells of the cochlea, two 12-mer peptides (A(665) and A(666)) with affinity to prestin were identified following 3 rounds of sequential phage display. Two-round display with immobilized prestin protein was used to enrich the library for full-length prestin. The last round was performed using Cos-7 cells transiently transfected with a cCFP-prestin plasmid to display phages expressing peptides restrictive to the extracellular loops of prestin. The binding properties of A(665) and A(666) shown by flow cytometry demonstrated selectivity to prestin-expressing Chinese hamster ovary cells. PEG6K-b-PCL19K polymersomes covalently labelled with these peptides demonstrated effective targeting to outer hair cells in a rat cochlear explant study.
International Journal of Pharmaceutics 03/2012; 424(1-2):121-7. · 3.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cochlear hair cells convert sound vibration into electrical potential, and loss of these cells diminishes auditory function. In response to mechanical stimuli, piezoelectric materials generate electricity, suggesting that they could be used in place of hair cells to create an artificial cochlear epithelium. Here, we report that a piezoelectric membrane generated electrical potentials in response to sound stimuli that were able to induce auditory brainstem responses in deafened guinea pigs, indicating its capacity to mimic basilar membrane function. In addition, sound stimuli were transmitted through the external auditory canal to a piezoelectric membrane implanted in the cochlea, inducing it to vibrate. The application of sound to the middle ear ossicle induced voltage output from the implanted piezoelectric membrane. These findings establish the fundamental principles for the development of hearing devices using piezoelectric materials, although there are many problems to be overcome before practical application.
Proceedings of the National Academy of Sciences 11/2011; 108(45):18390-5. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the present study, purified prestin was observed by atomic force microscopy (AFM). First, the lipid bilayer was formed on a freshly cleaved mica disk by the deposition of small unilamellar lipid vesicles. Such bilayer was destabilized by incubation with a detergent. Afterward, purified prestin was added to the destabilized lipid bilayer. Finally, a high resolution image of the prestin-reconstituted lipid bilayer was acquired by AFM. As a result, densely embedded prestin with a diameter of 11.0±1.3 nm was visualized. From the obtained image, cytoplasmic side of prestin was found to form a ring-like structure with four peaks and one valley at its center.
[Show abstract][Hide abstract] ABSTRACT: Outer hair cell (OHC) motility is thought to be based on the voltage-dependent conformational changes of the motor protein prestin. However, little is known about its structure and function. In this study, the membrane topology of prestin was investigated by single molecule force spectroscopy using an atomic force microscope (AFM). The C-terminus of prestin was tagged with an Avi-tag and biotinylated. Prestin was then connected with a streptavidin-coated AFM cantilever via biotin-streptavidin binding. The prestin was pulled out from the plasma membrane by retracting the cantilever and force curves were obtained. Obtained force curves suggested the existence of 12 transmembrane domains of prestin.
[Show abstract][Hide abstract] ABSTRACT: The SLC26A4 gene encodes the transmembrane protein pendrin, which is involved in the homeostasis of the ion concentration of the endolymph of the inner ear, most likely by acting as a chloride/bicarbonate transporter. Mutations in the SLC26A4 gene cause sensorineuronal hearing loss. However, the mechanisms responsible for such loss have remained unknown. Therefore, in this study, we focused on the function of ten missense pendrin mutations (p.P123S (Pendred syndrome), p.M147V (NSEVA), p.K369E (NSEVA), p.A372V (Pendred syndrome/NSEVA), p.N392Y (Pendred syndrome), p.C565Y (NSEVA), p.S657N (NSEVA), p.S666F (NSEVA), p.T721M (NSEVA) and p.H723R (Pendred syndrome/NSEVA)) reported in Japanese patients, and analyzed their cellular localization and anion exchanger activity using HEK293 cells transfected with each mutant gene. Immunofluorescent staining of the cellular localization of the pendrin mutants revealed that p.K369E and p.C565Y, as well as wild-type pendrin, were transported to the plasma membrane, while 8 other mutants were retained in the cytoplasm. Furthermore, we analyzed whether salicylate, as a pharmacological chaperone, restores normal plasma membrane localization of 8 pendrin mutants retained in the cytoplasm to the plasma membrane. Incubation with 10 mM of salicylate of the cells transfected with the mutants induced the transport of 4 pendrin mutants (p.P123S, p.M147V, p.S657Y and p.H723R) from the cytoplasm to the plasma membrane and restored the anion exchanger activity. These findings suggest that salicylate might contribute to development of a new method of medical treatment for sensorineuronal hearing loss caused by the mutation of the deafness-related proteins, including pendrin.
Hearing research 12/2010; 270(1-2):110-8. · 2.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Prestin is the motor protein of cochlear outer hair cells and is essential for mammalian hearing. The present study aimed to clarify the structure of prestin by atomic force microscopy (AFM). Prestin was purified from Chinese hamster ovary cells which had been modified to stably express prestin, and then reconstituted into an artificial lipid bilayer. Immunofluorescence staining with anti-prestin antibody showed that the cytoplasmic side of prestin was possibly face up in the reconstituted lipid bilayer. AFM observation indicated that the cytoplasmic surface of prestin was ring-like with a diameter of about 11 nm.
[Show abstract][Hide abstract] ABSTRACT: Prestin is a key molecule for mammalian hearing. The present study investigated changes in characteristics of prestin by culturing prestin-transfected cells with salicylate, an antagonist of prestin. As a result, the plasma membrane localization of prestin bearing a mutation in the GTSRH sequence, which normally accumulates in the cytoplasm, was recovered. Moreover, the nonlinear capacitance of the majority of the mutants, which is a signature of prestin activity, was also recovered. Thus, the present study discovered a new effect of salicylate on prestin, namely, the promotion of the plasma membrane expression of prestin mutants in an active state.
[Show abstract][Hide abstract] ABSTRACT: The motor protein prestin in cochlear outer hair cells is a member of the solute carrier 26 family, but among the proteins of that family, only prestin can confer the cells with nonlinear capacitance (NLC) and motility. In the present study, to clarify contributions of unique amino acids of prestin, namely, Met-122, Met-225 and Thr-428, to the characteristics of prestin, mutations were introduced into those amino acids. As a result, NLC remained unchanged by both replacement of Met-122 by isoleucine and that of Thr-428 by leucine, suggesting that those amino acids were not important for the generation of NLC. Surprisingly, the replacement of Met-225 by glutamine statistically increased NLC as well as the motility of prestin-expressing cells without an increase in the amount of prestin expression in the plasma membrane. This indicates that Met-225 in prestin somehow adjusts NLC and the motility of prestin-expressing cells.
Biochemical and Biophysical Research Communications 10/2009; 389(4):569-74. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim of this study is to investigate the middle ear dynamic characteristics and their influence on TEOAEs in patients with middle ear disorders. The middle ear dynamic characteristics and TEOAE findings were investigated in 89 patients with middle ear disorders using the sweep frequency impedance (SFI) meter and the 1LO88 system, respectively. These patients were divided into six subcategories: tympanic membrane aberrations, otitis media with effusion, chronic otitis media, tympanic membrane perforation, otosclerosis, and ossicular chain dislocation. Details of the TEOAE frequency characteristics were compared with the individual's middle ear dynamic characteristics. TEOAE status as a function of hearing threshold and middle ear dynamic characteristics was also examined. The results showed that the middle ear dynamic characteristics in patients with middle ear disorders correlated with the TEOAE frequency characteristics and amplitudes. The hearing level and middle ear mobility were the controlling factors affecting TEOAE status. Using both non-linear and linear stimulus modes, larger TEOAE responses were obtained when the hearing level was better than 20 dB HL, and there was moderately good middle ear mobility. Moreover, TEOAEs were absent using the non-linear mode when the hearing level was worse than 30 dB HL, whereas with the linear mode, TEOAEs were recordable even with hearing losses of up to 40 dB HL in patients with middle ear disorders. A higher incidence of recordable TEOAEs was found in the subgroups with tympanic membrane abnormalities and secretory otitis media when compared with the other subgroups. No TEOAEs were recordable in patients with chronic otitis media.
El objetivo de este estudio fue investigar las características dinámicas del oído medio y su influencia en las TEOAEs en pacientes con trastornos del oído medio. Se investigaron las caracteristicas dinámicas del oído medio y los hallazgos en las TEOAEs en 89 pacientes con trastornos del oído medio, usando un impedanciómetro de barrido de frecuencias (SFI) y el sistema ILO99, respectivamente. Estos pacientes fueron divididos en seis subcategorías: aberraciones de la membrana timpánica, otitis media secretrora, otitis media crónica, perforación de la membrana timpánica, otoesclerosis y dislocación de la cadena osicular. Se compararon los detalles de las características frecuenciales de las TEOAEs con las características dinámicas del oído medio de cada individuo. También se examinó el status de las TEOAEs como función del umbral auditivo y las características dinámicas del oído medio. Los resultados muestran que las características dinámicas del oído medio en pacientes con trastornos del oído medio se correlacionan con la amplitud y con las caracteristicas frecuenciales de las TEOAE. El nivel auditivo y la movilidad del oído medio fueron los factores de control que afectaron el status de las TEOAE. Se obtuvieron mejores respuestas de TEOAEs usando tanto los modos de estimulación lineares como no lineares, cuando el nivel de audición era mejor que 20 dB HL y cuando había una moderadamente buena movilidad del oído medio. Mas aún, las TEOAEs estuvieron ausentes al usar el modo no linear, cuando el nivel auditivo era inferior a 30 dB HL mientras que con el modo linear, las TEOAEs eran registrables incluso con pérdidas auditivas superiores a 40 dB HL en pacientes con trastornos del oído medio. Se encontró una incidencia mayor de TEOAEs registrables en los subgrupos con anormalidades de la membrana timpánica o con otitis media secretoria que en otros subgrupos. No se pudieron registrar TEOAEs en pacientes con otitis media crónica.
International Journal of Audiology. 07/2009; 42(3).
[Show abstract][Hide abstract] ABSTRACT: Prestin is the motor protein of cochlear outer hair cells, which exhibit elongation and contraction in response to acoustic stimuli. In this study, the effects of mutations in unique amino acids, which among members of the SLC26 family were only present in prestin, on the characteristics of prestin were investigated. As a result, a mutation in Met-225 of prestin was found to increase nonlinear capacitance, which is a signature of prestin activity.
[Show abstract][Hide abstract] ABSTRACT: The high sensitivity of mammalian hearing is achieved by amplification of the motion of the cochlear partition. The origin of this cochlear amplification is the elongation and contraction of outer hair cells (OHCs) in response to acoustical stimulation. This motility is made possible by a membrane protein embedded in the lateral membrane of OHCs. The gene of this protein has been identified and termed prestin. We, herein, present a method for observation by atomic force microscopy (AFM) of prestin expressed in the Chinese hamster ovary (CHO) cell plasma membrane. To obtain a stable sample for AFM imaging in liquid, we used as an example in the protocol provide here, CHO cells transfected with prestin or FLAG-tagged prestin, and untransfected CHO cells. The cells attached to a substrate were subjected to ultrasonic waves generated from a sonicator probe so that the inside-out plasma membranes remained on the substrate. Prestin was immunostained with mouse anti-FLAG primary antibody and FITC-conjugated goat anti-mouse IgG secondary antibody. The lipid of the plasma membrane was labeled with fluorescence probes. The cytoplasmic faces of the cells were then observed in liquid by the tapping mode of AFM at low and high magnifications. More particle-like structures 8-12 nm in diameter were observed in the plasma membranes of the prestin-transfected CHO cells than in those of the untransfected CHO cells. Since the difference between these two types of cells is due to the existence of prestin, such particle-like structures in the prestin-transfected CHO cells are possibly constituted by prestin.
[Show abstract][Hide abstract] ABSTRACT: Accessories, watches, coins and other items containing metal sometimes cause contact dermatitis and metal allergy. Among metals, nickel in alloys is ionized by sweat on the surface of the skin and exhibits particularly marked irritancy and allergenicity. Although eosinophils play important roles in allergy, the effects of nickel on eosinophils have not been elucidated.
Eosinophils were prepared from the peritoneal cavity in rats immunized with Ascaris suum extract. Purified rat eosinophils were incubated in the presence of various kinds of metals including nickel. The viability of eosinophils was analyzed using a flow cytometer.
When rat eosinophils were incubated for 3 days in the presence of nickel chloride at 30-1,000 microM, the viability of eosinophils was decreased in a concentration-dependent manner. Nickel chloride at 300 muM significantly increased the percentage of annexin V+ PI- eosinophils. The population of annexin V+ PI- eosinophils was also increased by nickel sulfate, cobalt chloride and zinc sulfate. The binding of nickel ions to eosinophils was detected by flow cytometer.
Nickel ions bind to eosinophils and decrease the viability of eosinophils through the induction of apoptosis. Nickel ions may exhibit activity which modifies the function of eosinophils in allergy.
International Archives of Allergy and Immunology 02/2009; 149 Suppl 1:57-60. · 2.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Prestin, a membrane protein of the outer hair cells (OHCs), is known to be the motor which drives OHC somatic electromotility. Electron microscopic studies showed the lateral membrane of the OHCs to be densely covered with 10-nm particles, they being believed to be a motor protein. Imaging by atomic force microscopy (AFM) of prestin-transfected Chinese hamster ovary (CHO) cells revealed 8- to 12-nm particle-like structures to possibly be prestin. However, since there are many kinds of intrinsic membrane proteins other than prestin in the plasma membranes of OHCs and CHO cells, it was impossible to clarify which structures observed in such membranes were prestin. In the present study, an experimental approach combining AFM with quantum dots (Qdots), used as topographic surface markers, was carried out to detect individual prestin molecules. The inside-out plasma membranes were isolated from the prestin-transfected and untransfected CHO cells. Such membranes were then incubated with antiprestin primary antibodies and Qdot-conjugated secondary antibodies. Fluorescence labeling of the prestin-transfected CHO cells but not of the untransfected CHO cells was confirmed. The membranes were subsequently scanned by AFM, and Qdots were clearly seen in the prestin-transfected CHO cells. Ring-like structures, each with four peaks and one valley at its center, were observed in the vicinity of the Qdots, suggesting that these structures are prestin expressed in the plasma membranes of the prestin-transfected CHO cells.
Pflügers Archiv - European Journal of Physiology 09/2008; 457(4):885-98. · 4.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Acetylation and deacetylation of proteins occur in cells in response to various stimuli, and are reversibly catalyzed by histone acetyltransferase and histone deacetylase (HDAC), respectively. EoL-1 cells have an FIP1L1-PDGFRA fusion gene that causes transformation of eosinophilic precursor cells into leukemia cells. The HDAC inhibitors apicidin and n-butyrate suppress the proliferation of EoL-1 cells and induce differentiation into eosinophils by a decrease in the protein level of FIP1L1-PDGFRalpha without affecting the mRNA level for FIP1L1-PDGFRA. In this study, we analyzed the mechanism by which the protein level of FIP1L1-PDGFRalpha is decreased by apicidin and n-butyrate.
EoL-1 cells were incubated in the presence of the HDAC inhibitors apicidin, trichostatin A or n-butyrate. The protein levels of FIP1L1-PDGFRalpha and phosphorylated eIF-2alpha were determined by Western blotting. Actinomycin D and cycloheximide were used to block RNA synthesis and protein synthesis, respectively, in the chasing experiment of the amount of FIP1L1-PDGFRalpha protein.
When apicidin- and n-butyrate-treated EoL-1 cells were incubated in the presence of actinomycin D, the decrease in the protein level of FIP1L1-PDGFRalpha was significantly enhanced when compared with controls. In contrast, the protein levels were not changed by cycloheximide among these groups. Apicidin and n-butyrate induced the continuous phosphorylation of eIF-2alpha for up to 8 days.
The decrease in the level of FIP1L1-PDGFRalpha protein by continuous inhibition of HDAC may be due to the decrease in the translation rate of FIP1L1-PDGFRA.
International Archives of Allergy and Immunology 02/2008; 146 Suppl 1:7-10. · 2.25 Impact Factor