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Introduction and objectives Acquired sensorineural hearing loss (SNHL) has become a critical societal issue in recent years. SNHL is considered a risk factor for type 2 diabetes mellitus (T2DM). Metformin is commonly used to treat T2DM. However, its effects on SNHL have not been reported yet. Hence, this study aimed to evaluate the association between the use of metformin and SNHL incidence. Patients and methods In this retrospective matched-cohort study, the medical records of 1219 patients with T2DM aged18 years from our hospital's inpatient database from January 1, 2012, to December 31, 2019, were examined, and matched cohorts were generated (76 patients receiving metformin and 76 not receiving metformin). A multivariable logistic regression analysis was performed to investigate the factors influencing the incidence of SNHL. Results After adjustment by propensity matching, multivariable logistic regression analysis revealed that the non-use of metformin increased the risk of developing SNHL (odds ratio, 0.26; 95% confidence interval, 0.07-0.93; p = 0.03). Conclusions This study demonstrated an association between the use of metformin and a reduced incidence of SNHL among patients with T2DM.

In contrast to mammals, the avian cochlea, specifically the basilar papilla, can regenerate sensory hair cells, which involves fate conversion of supporting cells to hair cells. To determine the mechanisms for converting supporting cells to hair cells, we used single-cell RNA sequencing during hair cell regeneration in explant cultures of chick basilar papillae. We identified dynamic changes in the gene expression of supporting cells, and the pseudotime trajectory analysis demonstrated the stepwise fate conversion from supporting cells to hair cells. Initially, supporting cell identity was erased and transition to the precursor state occurred. A subsequent gain in hair cell identity progressed together with downregulation of precursor-state genes. Transforming growth factor beta receptor 1-mediated signaling was involved in induction of the initial step, and its inhibition resulted in suppression of hair cell regeneration. Our data provide new insights for understanding fate conversion from supporting cells to hair cells in avian basilar papillae.

Post-radiation fibrosis of the vocal folds is thought to cause vocal impairment. However, the mechanism by which this occurs has been poorly documented, probably because of the lack of an appropriate experimental animal model. The purpose of this study was to establish a simple and reproducible mouse model of laryngeal radiation to investigate the development of vocal fold fibrosis over time. C57BL/6 mice individually placed in a lead shield were irradiated with a single dose of 20 Gy. At 1, 2, and 6 months after irradiation, larynges were harvested and subjected to histological examination and gene expression analysis. Irradiated vocal folds showed time-dependent tissue contraction and increased collagen deposition, with no significant difference in the changes in hyaluronic acid levels. Transcriptional analysis revealed upregulated expressions of TGF-β1 and iNOS at 6 months, but downregulated expressions of Acta2, Col1a1, Col3a1, and MMP8. Moreover, elevated TGF-β1 and reduced downstream gene expression levels indicated the existence of an inhibitory factor over the TGF-β/Smad pathway. Discrepancies in histological and transcriptional studies of collagen might suggest that radiation-induced vocal fold fibrosis could be caused by the elongated turnover of collagen. Overall, we established a mouse model of radiation-induced vocal fold fibrosis using a simple protocol. Further investigations are warranted to elucidate the pathogenesis of irradiation-induced fibrosis in vocal folds.

Macrophages aid in wound healing by changing their phenotype and can be a key driver of fibrosis. However, the contribution of macrophage phenotype to fibrosis following vocal fold injury remains unclear. Peroxisome proliferator-activated receptor-γ (PPARγ) is expressed mainly by macrophages during early wound healing and regulates the macrophage phenotype. This study aimed to evaluate the effects of pioglitazone, a PPARγ agonist, on the macrophage phenotype and fibrosis following vocal fold injury in rats. Pioglitazone was injected into the rats' vocal folds on days 1, 3, 5, and 7 after injury, and the vocal fold lamina propria was evaluated on days 4 and 56 after injury. Moreover, THP-1-derived macrophages were treated with pioglitazone, and the expression of pro-inflammatory cytokines under lipopolysaccharide/interferon-γ stimulation was analyzed. The results revealed that pioglitazone reduced the expression of Ccl2 both in vivo and in vitro. Furthermore, pioglitazone decreased the density of inducible nitric oxide synthase+ CD68+ macrophages and inhibited the expression of fibrosis-related factors on day 4 after injury. On day 56 after injury, pioglitazone inhibited fibrosis, tissue contracture, and hyaluronic acid loss in a PPARγ-dependent manner. These results indicate that PPARγ activation could inhibit accumulation of inflammatory macrophages and improve tissue repair. Considered together, these findings imply that inflammatory macrophages play a key role in vocal fold fibrosis.

In the context of acquired sensorineural hearing loss (SNHL), cochlear hair cells have long been thought to be among the most vulnerable elements in mammalian cochleae. However, recent studies have indicated that the synaptic connection between inner hair cells (IHC) and spiral ganglion neurons (SGN) can be an important target for the treatment of SNHL. Our previous studies in patients with sudden SNHL demonstrated delayed and gradual hearing recovery following topical application of insulin-like growth factor 1 (IGF-1), suggesting that not only protective but also regenerative mechanisms may account for hearing recovery after treatment with IGF-1. We then hypothesized that IGF-1 has the potential to drive the regeneration of IHC–SGN synapses. To test this hypothesis, we investigated the effects of IGF-1 on IHC–SGN synapses using cochlear explant cultures from postnatal day 2 mice that had been damaged by exposure to the excitatory amino acids N-methyl- d -aspartate and kainate. Cochlear explants that lost IHC–SGN synapses upon exposure to excitatory amino acids were cultured with exogenous IGF-1 for an additional 48 h. We observed increased numbers of IHC–SGN synapses after exogenous IGF-1 application. Pharmacological inhibition of the IGF-1 receptor attenuated the restoration of IHC–SGN synapses by exogenous IGF-1. These findings indicated that IGF-1 induces regeneration of IHC–SGN synapses in cochlear explant cultures from postnatal day 2 mice. Therefore, in a future study we will perform in vivo experiments using adult mice to ascertain the effects of IGF-1 on the regeneration of IHC–SGN synapses.

Hearing loss has become one of the most common disabilities worldwide. The synaptic connections between inner hair cells (IHCs) and spiral ganglion neurons have specialized synaptic constructions, termed ribbon synapses, which are important for auditory function. The ribbon synapses in the cochlea are quite vulnerable to various insults. As such, the maintenance of ribbon synapses is important for ensuring hearing function. Insulin-like growth factor 1 (IGF1) plays a critical role in the development and maintenance of the cochlea and has the potential to protect cochlear hair cells from various insults. In this study, we examined the role of IGF1 in the maintenance of ribbon synapses in cochlear explants of postnatal day four mice. We cultured cochlear explants with an IGF1 receptor antagonist, JB1, which is an IGF1 peptide analog. Results showed that exposure to JB1 for 24 h resulted in the loss of ribbon synapses. After an additional 24-h culture without JB1, the number of ribbon synapses spontaneously recovered. The application of exogenous IGF1 showed two different aspects of ribbon synapses. Low doses of exogenous IGF1 promoted the recovery of ribbon synapses, while it compromised the spontaneous recovery of ribbon synapses at high doses. Altogether, these results indicate that the paracrine or autocrine release of IGF1 in the cochlea plays a crucial role in the maintenance of cochlear ribbon synapses.

We report on validating a mitochondrial gene therapeutic strategy using fibroblasts derived from patients with an A1555G point mutation in mitochondrial DNA coding 12S ribosomal RNA (rRNA (12S)). Wild-type rRNA (12S) as a therapeutic RNA was encapsulated in a mitochondrial targeting liposome, a MITO-Porter (rRNA-MITO-Porter), and an attempt was made to deliver the MITO-Porter to mitochondria of the diseased cells. It was confirmed that the rRNA-MITO-Porter treatment significantly decreased the ratio of the mutant rRNA content. Moreover, it was shown that the mitochondrial respiratory activities of the diseased cells were improved as the result of the mitochondrial transfection of the rRNA-MITO-Porter.

The hybrid or electric-acoustic stimulation cochlear implant is indicated in patients with a residual hearing at low frequencies. It provides electric and acoustic stimulation for compensating for high- and low-frequency sounds, respectively. However, the implantation procedure damages the cochlea, resulting in loss of the residual-hearing and diminished effects of the acoustic-hearing in several patients. To prevent hearing loss after implantation, corticosteroids have been used clinically although their effects are limited. As an alternative to corticosteroids, insulin-like growth factor 1 (IGF1) has shown potent effects in various types of cochlear injury. In this study, the effects of IGF1 on hearing preservation were examined after cochlear implantation to a normal-hearing guinea pig model. The electrode was inserted in an atraumatic way through the round window membrane of guinea pigs with the application of a gelatin-sponge soaked with IGF1 or saline. The auditory brainstem response (ABR) was recorded pre-operatively, immediately after cochlear implantation, and 7, 14, 28, and 56 days after electrode insertion. In comparison to the control group, the IGF1-treated group showed better hearing preservation at low frequencies, 7 days after surgery. IGF1 application was effective at low frequencies (2 and 4 kHz) throughout the period of examination. Histological studies revealed that outer hair cell numbers, in the IGF1-treated group, were maintained in the cochlear region responsible for low-frequency hearing (upper midbasal turn) and that there was less fibrous tissue formation around the electrode. Both the outer hair cell counts and the extent of fibrosis significantly correlated with the ABR threshold shifts at low frequencies. These results indicate that IGF1 might attenuate loss of low-frequency hearing after cochlear implantation, suggesting its possible clinical use in soft surgeries involving cochlear implants with electric-acoustic stimulation for hearing preservation.

The mechanosensory hair cells of the inner ear are required for hearing and balance and have a distinctive apical structure, the hair bundle, that converts mechanical stimuli into electrical signals. This structure comprises a single cilium, the kinocilium, lying adjacent to an ensemble of actin-based projections known as stereocilia. Hair bundle polarity depends on kinociliary protocadherin-15 (Pcdh15) localization. Protocadherin-15 is found only in hair-cell kinocilia, and is not localized to the primary cilia of adjacent supporting cells. Thus, Pcdh15 must be specifically targeted and trafficked into the hair-cell kinocilium. Here we show that kinocilial Pcdh15 trafficking relies on cell type-specific coupling to the generic intraflagellar transport (IFT) transport mechanism. We uncover a role for fibroblast growth factor receptor 1 (FGFR1) in loading Pcdh15 onto kinociliary transport particles in hair cells. We find that on activation, FGFR1 binds and phosphorylates Pcdh15. Moreover, we find a previously uncharacterized role for clathrin in coupling this kinocilia-specific cargo with the anterograde IFT-B complex through the adaptor, DAB2. Our results identify a modified ciliary transport pathway used for Pcdh15 transport into the cilium of the inner ear hair cell and coordinated by FGFR1 activity.

Various tissues possess tissue-specific stem/progenitor cells, including the inner ears. Stem/progenitor cells of the inner ear can be isolated as so-called otospheres from differentiated cells using a sphere forming assay. Although recent studies have demonstrated the characteristics of otospheres to some extent, most of the features of these cells are unknown. In this report, we describe the findings of transcriptome analyses with a cDNA microarray of otospheres derived from the cochleae of the inner ears of neonatal mice in order to clarify the gene expression profile of otic stem/progenitor cells. There were common transcription factors between otospheres and embryonic stem cells, which were supposed to be due to the stemness of otospheres. In comparison with the cochlear sensory epithelium, the otospheres shared characteristics with the cochlea, although several transcription factors specific for otospheres were identified. These transcription factors are expected to be essential for maintaining the characteristics of otospheres, and appear to be candidate genes that promote the direct conversion of cells into otic stem/progenitor cells.

In addition to well-known curcuminoids, three colored metabolites were isolated from cultured cell clumps that had been induced from buds on turmeric rhizomes. The isolated compounds were identified as dihydro derivatives of curcuminoids, dihydrocurcumin (dihydroCurc), dihydrodesmethoxycurcumin-a (dihydroDMC-a), and dihydrobisdesmethoxycurcumin (dihydroBDMC). The cell clumps did not contain dihydroDMC-b, an isomer of dihydroDMC-a. A comparison of the distribution profiles of curcuminoids and dihydrocurcuminoids in the cell clumps with those in the rhizomes, leaves, and roots revealed the following differences: Unlike rhizomes, the cell clumps, leaves, and roots contained dihydrocurcuminoids as the major colored constituents. Whereas dimethoxy compounds, curcumin and dihydrocurcumin, respectively, were most abundant in the rhizomes and leaves, one of the monomethoxy derivatives, dihydroDMC-a, was found most abundantly in the cell clumps and roots. While both dihydroDMC-a and b were detected in the rhizomes, dihydroDMC-b was not detectable in the cell clumps, leaves, or roots. The occurrence of only one of the two possible isomers of dihydroDMC suggests biosynthetic formation of dihydrocurcuminoids in turmeric.

Curcuminoids found in the rhizome of turmeric, Curcuma longa, possess various biological activities. Despite much attention regarding the biosynthesis of curcuminoids because of their pharmaceutically important properties and biosynthetically intriguing structures, no enzyme systems have been elucidated. Here we propose a pathway for curcuminoid biosynthesis in the herb C. longa, which includes two novel type III polyketide synthases. One of the type III polyketide synthases, named diketide-CoA synthase (DCS), catalyzed the formation of feruloyldiketide-CoA by condensing feruloyl-CoA and malonyl-CoA. The other, named curcumin synthase (CURS), catalyzed the in vitro formation of curcuminoids from cinnamoyldiketide-N-acetylcysteamine (a mimic of the CoA ester) and feruloyl-CoA. Co-incubation of DCS and CURS in the presence of feruloyl-CoA and malonyl-CoA yielded curcumin at high efficiency, although CURS itself possessed low activity for the synthesis of curcumin from feruloyl-CoA and malonyl-CoA. These findings thus revealed the curcumin biosynthetic route in turmeric, in which DCS synthesizes feruloyldiketide-CoA, and CURS then converts the diketide-CoA esters into a curcuminoid scaffold.

In order to investigate the biosynthesis of curcuminoid in rhizomes of turmeric (Curcuma longa), we established an in vitro culture system of turmeric plants for feeding 13C-labeled precursors. Analyses of labeled desmethoxycurcumin (DMC), an unsymmetrical curcuminoid, by 13C-NMR, revealed that one molecule of acetic acid or malonic acid and two molecules of phenylalanine or phenylpropanoids, but not tyrosine, were incorporated into DMC. The incorporation efficiencies of the same precursors into DMC and curcumin were similar, and were in the order malonic acid > acetic acid, and cinnamic acid > p-coumaric acid >> ferulic acid. These results suggest the possibility that the pathway to curcuminoids utilized two cinnamoyl CoAs and one malonyl CoA, and that hydroxy- and methoxy-functional groups on the aromatic rings were introduced after the formation of the curcuminoid skeleton.

We investigate the thermopower due to the orbital Kondo effect in a single quantum dot system by means of the noncrossing approximation. It is elucidated how the asymmetry of tunneling resonance due to the orbital Kondo effect affects the thermopower under gate-voltage and magnetic-field control., 4 pages, 4 figures, proceeding of Second International Symposium on Nanometer-Scale Quantum Physics

This study investigated the potential of bone-marrow stromal cell transplantation for cell replacement therapy in the cochlea. Bone-marrow stromal cells labeled with enhanced green fluorescent protein were injected into the perilymphatic space of normal cochleae in mice. Histological analysis 2 weeks after transplantation demonstrated that transplanted cells settled within the cochlear tissues, especially in the spiral ligament and the spiral limbus, although most transplants were located in the perilymphatic space. Some of the transplanted cells expressed the cochlear gap-junction protein connexin 26. These findings indicate the potential of bone-marrow stromal cells for delivering therapeutic molecules and for the restoration of cochlear cells, particularly in the spiral ligament and the spiral limbus.

We study transport properties of the orbital Kondo effect via single quantum dot systems with plural electrons by applying the noncrossing approximation to the Anderson impurity model with orbitals. Investigating the case for the half filling of the quantum dot with three orbitals, we discuss the effects of the orbital level splitting on the conductance and the thermopower.

Our results indicate that myosin VIIa-positive cells are generated from embryonic stem cells (ESCs) co-cultured with PA6 cells; however, bone morphogenetic protein 4 (BMP4) may not be a key molecule for induction of myosin VIIa-positive cells from the ESCs.ESCs have been considered as a basis for cell therapy in a range of organs, because of their potential for self-renewal and pluripotency. Co-culture with PA6 stromal cells can induce differentiation of ESCs into various types of ectodermal cells including sensory progenitors. BMP4 plays an essential role in the development of sensory hair cells in the inner ear.We examined effects of BMP4 on differentiation of ESCs into the hair cell immunophenotype. BMP4 was supplemented at different time points to ESCs co-cultured on PA6 stromal cells. The ESCs were then collected and examined for the expression of myosin VIIa, a hair cell marker, and betaIII-tubulin, a neural marker. The expression of myosin VIIa and betaIII-tubulin was identified.Quantitative assessments revealed that exogenous BMP4 has significant effects on the expression of betaIII-tubulin, but not of myosin VIIa.

Various Curcuma rhizomes have been used as medicines or spices in Asia since ancient times. It is very difficult to distinguish them morphologically, especially when they are boiled and dried, which causes misidentification leading to a loss of efficacy. We developed a method for discriminating Curcuma species by intron length polymorphism markers in genes encoding diketide-CoA synthase and curcumin synthase. This method could apply to identification of not only fresh plants but also samples of crude drugs or edible spices. By applying this method to Curcuma specimens and samples, and constructing a dendrogram based on these markers, seven Curcuma species were clearly distinguishable. Moreover, Curcuma longa specimens were geographically distinguishable. On the other hand, Curcuma kwangsiensis (gl type) specimens also showed intraspecies polymorphism, which may have occurred as a result of hybridization with other Curcuma species. The molecular method we developed is a potential tool for global classification of the genus Curcuma.

We study the orbital Kondo effect due to two electrons occupying three orbital levels in quantum dot (QD) systems. By applying the non-crossing approximation to the three-orbital Anderson impurity model, we calculate the conductance and the thermopower. We consider the realistic situation in QD systems, where the correlation effect induces the orbital-level splitting even at zero field. It is found that the level splitting at zero field larger than temperature revives the maximum structure in the conductance clearly, which characterizes the singlet-triplet Kondo effect. We also show that the enhancement of the Kondo temperature due to the singlet-triplet mechanism gives rise to the characteristic behavior in the thermopower as well. This is in contrast with the two-orbital case, where the thermopower is zero irrespective of the level splitting. (© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Sensorineural hearing loss is a common disability, but treatment options are currently limited to cochlear implants and hearing aids. Studies are therefore being conducted to provide alternative means of biological therapy, including gene therapy. Safe and effective methods of gene delivery to the cochlea need to be developed to facilitate the clinical application of these therapeutic treatments for hearing loss. In this study, we examined the potential of cell-gene therapy with nonviral vectors for delivery of therapeutic molecules into the cochlea. NIH3T3 cells were transfected with the brain-derived neurotrophic factor (Bdnf) gene using lipofection and then transplanted into the mouse inner ear. Immunohistochemistry and Western blotting demonstrated the survival of grafted cells in the cochlea for up to 4 weeks after transplantation. No significant hearing loss was induced by the transplantation procedure. A Bdnf-specific enzyme-linked immunosorbent assay revealed a significant increase in Bdnf production in the inner ear following transplantation of engineered cells. These findings indicate that cell-gene delivery with nonviral vectors may be applicable for the local, sustained delivery of therapeutic molecules into the cochlea.

Objective: The aim of this experimental study was to examine the potential of local recombinant human insulin-like growth factor-1 (rhIGF-1) application through a biodegradable hydrogel for the treatment of cochleae. Methods: A hydrogel immersed with rhIGF-1 was placed on the round window membrane of Sprague-Dawley rats while a hydrogel immersed with physiological saline was applied to control animals. On day 3 after drug application, the animals were exposed to white noise at 120 dB sound pressure level (SPL) for 2 hours. Cochlear function was monitored using measurements of auditory brain stem responses (ABRs) at frequencies of 8, 16, and 32 kHz. The temporal bones were collected 7 or 30 days after noise exposure and the loss of hair cells was quantitatively analyzed. Results: Local rhIGF-1 treatment significantly reduced the elevation of ABR thresholds on days 7 and 30 after noise exposure. Histologic analysis revealed that local rhIGF-1 treatment significantly prohibited the loss of outer hair cells. Conclusions: These findings demonstrate that local IGF-1 application through the biodegradable hydrogel has the potential for protection of cochleae from noise trauma.

This study aimed to evaluate the potential of embryonic stem cell-derived neural progenitors for use as transplants for the replacement of the auditory primary neurons, spiral ganglion neurons. Mouse embryonic stem cell-derived neural progenitors were implanted into the base of the cochlear modiolus of normal or deafened guinea pigs, which contains spiral ganglion neurons and cochlear nerve fibers. Histological analysis demonstrated the survival and neural differentiation of transplants in the cochlear modiolus and active neurite outgrowth of transplants toward host peripheral or central auditory systems. Functional assessments indicated the potential of transplanted embryonic stem cell-derived neural progenitors to elicit the functional recovery of damaged cochleae. These findings support the hypothesis that transplantation of embryonic stem cell-derived neural progenitors can contribute to the functional restoration of spiral ganglion neurons.

Objectives: This study aimed to investigate the efficacy of encapsulating therapeutic molecules in poly lactic/glycolic acid (PLGA) nanoparticles for drug delivery to the cochlea. Study Design: An experimental study. Methods: We examined the distribution of rhodamine, a fluorescent dye, in the cochlea, liver, and kidney of guinea pigs. Intravenous injection of rhodamine or rhodamine-encapsulated PLGA nanoparticles was used to target the fluorescent dye systemically to the liver, kidney, and cochlea, and these molecules were applied locally to the round window membrane (RWM) of the cochlea. The localization of rhodamine fluorescence in each region was quantitatively analyzed. Results: After systemic application of rhodamine nanoparticles, fluorescence was identified in the liver, kidney, and cochlea. The systemic application of nanoparticles had a significant effect on targeted and sustained delivery of rhodamine to the liver but not the kidney or cochlea. Rhodamine nanoparticles placed on the RWM were identified in the scala tympani as nanoparticles, indicating that the PLGA nanoparticles can permeate through the RWM. Furthermore, the local application of rhodamine nanoparticles to the RWM was more effective in targeted delivery to the cochlea than systemic application. Conclusions: These findings indicate that PLGA nanoparticles can be an useful drug carrier to the cochlea via local application.

Objective: The present study aimed to evaluate the efficacy of a biodegradable hydrogel as a drug-delivery medium for the inner ear. Brain-derived neurotrophic factor (BDNF) was chosen as the agent to be administered. Method: First, we used an enzyme-linked immunosorbent assay to measure BDNF concentrations in the cochlear fluid after placing a hydrogel containing this agent onto the round-window membrane of the ear. Second, the functional and histologic protection of the auditory primary neurons (spiral ganglion neurons [SGNs]) by BDNF applied through the hydrogel was examined using an animal model of SGN degeneration. Results: The results revealed sustained delivery of BDNF into the cochlear fluid by way of the hydrogel. Second, the functional and histologic protection of the auditory primary neurons (SGNs) by BDNF applied through the hydrogel was examined using an animal model of SGN degeneration. The measurement of electrically evoked auditory-brainstem responses demonstrated that BDNF delivered by way of the hydrogel significantly reduced the threshold elevation. Immunohistochemistry for neurofilament 200 kD demonstrated increased survival of SGNs because of BDNF application through the hydrogel. Conclusion: These findings indicate that biodegradable hydrogels can be used for drug delivery to the inner ear.

This study aimed to examine the potential of embryonic stem cell (ESC)-derived neural progenitors for restoration of the neural network in the peripheral vestibular system. Mouse ESC-derived neural progenitors were co-cultured with explants of vestibular sensory epithelia from neonatal mice. Histological analyses demonstrated that ESC-derived neurons substantially elongated their neurites towards vestibular hair cells, and attached to hair cells at the regions corresponding to the location of nerve endings in normal vestibular epithelia. Immunoreactivity for synaptophysin, a marker for synaptic vesicles, was present only in the cytoplasm of hair cells in sensory epithelia cultured alone, while the nerve endings of ESC-derived neurons attached to hair cells exhibited intense immunoreactivity for synaptophysin and some hair cells were moderately reactive in co-cultured specimens. The pattern of synaptophysin expression in co-cultured specimens was very similar to that observed in developing sensory epithelia, in which synaptic connections between hair cells and nerve endings are actively formed. These findings indicate that ESC-derived neurons have the potential to restore neural connections in the peripheral vestibular system.

The intracellular junction complexes, which consist of tight junctions (TJ), adherens junctions (AJ), and desmosomes, mediate cell-cell adhesion in epithelial cells. E-cadherin, which is a major component of AJ, plays a role not only in the maintenance of cell-cell junctions, but also in repressing cell proliferation. In this study, we examined changes of E-cadherin expression in mouse vestibular epithelia following local application of neomycin using immunohistochemistry and western blotting, and morphology of cell-cell junctions by transmission electron microscopy (TEM). Immunohistochemistry and western blotting revealed down-expression of E-cadherin and its consecutive recovery. TEM demonstrated temporal disruption of cell-cell junctions. Morphology of cell-cell junctions was more rapidly restored than recovery of E-cadherin expression. Transient disruption of cell-cell junctions and down-expression of E-cadherin is a rational response for the deletion of dying hair cells, and may be associated with a limited capacity for cell proliferations in mammalian vestibular epithelia following their rapid restoration.

This study investigated surgical procedures for cell transplantation into the mouse inner ear. Female C57BL/6 mice were used as recipient animals. Fetal mouse neural stem cells expressing green fluorescence were used as donor cells. Two methods, an injection of transplants from the lateral semicircular canal (LSCC) and from the cochlear lateral wall (CLW), were examined. Two weeks after transplantation, the distribution of transplant-derived cells in the cochlea was examined. Effects on auditory function were assessed by measurement of auditory brain stem responses (ABRs). Cochleae receiving cell transplantation from the LSCC exhibited robust survival of transplant-derived cells mainly in the scala vestibuli and scala tympani. Transplantation from the LSCC caused elevation of ABR thresholds by less than 10 dB SPL. However, transplantation from the CLW resulted in considerable hearing loss, even though transplant-derived cells settled in the scala media. These findings demonstrate that an approach from the LSCC can be utilized for cell transplantation into the perilymph without causing apparent auditory disorder, while an approach from the CLW delivers cells to the endolymph but appears to cause auditory dysfunction.

This study investigated the role of beta-catenin in the development of mouse auditory epithelia. Inner ears obtained from embryonic and newborn mice were used. Expression of beta-catenin was examined together with the expression of Ki-67, a marker for proliferating cells, or myosin VIIa, a marker for differentiated hair cells. In the early phase of development, intense expression of beta-catenin was found in auditory epithelia in which a number of Ki-67-positive cells were identified. Together with a decrease in proliferating cells, the intensity and area of beta-catenin expression were reduced. In addition, during differentiation and maturation of hair cells, the area of beta-catenin expression was further limited. These findings suggest that patterns of expression of beta-catenin are closely linked with the status of auditory epithelia development.

The reversing effects of carvedilol and other beta-adrenoceptor antagonists on multidrug resistance (MDR) were assessed in HeLa cells and the MDR1-overexpressing derivative Hvr100-6 cells, established by stepwise increases of vinblastine concentration in the culture medium. The inhibitory effects on the transcellular transport and intracellular accumulation of [3H]vinblastine and [3H]daunorubicin were also assessed using LLC-GA5-COL150 cell monolayers, established by transfection of human MDR1 cDNA into porcine kidney epithelial LLC-PK1 cells. The cytotoxic effects of vinblastine, paclitaxel, doxorubicin and daunorubicin in Hvr100-6 were reversed 1.4- to 7.1-fold by carvedilol at the realistic clinical concentration of 1 microM, whereas other beta-adrenoceptor antagonists had weaker or no such effects. Transport experiments using LLC-GA5-COL150 cell monolayers demonstrated that this effect of carvedilol was due to the inhibition of MDR1-mediated transport of vinblastine, paclitaxel, doxorubicin and daunorubicin. These MDR1-mediated reversing effects of carvedilol were similar to those of 1 microM verapamil, suggesting that carvedilol could be a candidate modulator of MDR in clinical use. Since other beta-adrenoceptor antagonists had no inhibitory effect on transport, the effects of carvedilol were not related to beta-adrenoceptors and might have been due to antioxidant activity.

The multidrug transporter, MDR1-mediated interaction of digoxin with antiarrhythmic or antianginal drugs was examined in vitro by using the MDR1-overexpressing LLC-GA5-COL150 cells, which were established by transfection with human MDR1 cDNA into porcine kidney epithelial LLC-PK(1) cells. Amiodarone, its active metabolite monodesethyl-amiodarone (DEA), and quinidine markedly inhibited the basal-to-apical transport (renal secretion) of [(3)H]digoxin and increased the apical-to-basal transport (reabsorption), but cibenzoline and lidocaine showed slight inhibition of the transport, and disopyramide and mexiletin had no such effects. The IC(50) values for amiodarone, DEA and quinidine on [(3)H]digoxin transport in LLC-GA5-COL150 cells were 5.48 microM, 1.27 microM and 9.52 microM, respectively. These were comparable to, or only several times the achievable concentration in clinical use, suggesting that MDR1 could be responsible for the drug interaction between digoxin and amiodarone found in clinical reports and that DEA contributes the elevation of digoxin serum concentration. Similarly, dipyridamole altered the transport, but isosorbide showed only slight modification of the transport. The IC(50) value for dipyridamole was 40.0 microM, also only several times the achievable concentration in clinical use, indicating a risk of interaction.

In anti-Helicobacter pylori therapy using omeprazole and antimicrobials, the efficacy can be related to the CYP2C19 genotype groups; the eradication rates were 83% in extensive metabolizers and 100% in poor metabolizers. The present study was undertaken to help predict the optimal dosage of omeprazole for extensive metabolizers in this therapy. Seven healthy Japanese subjects, classified based on the CYP2C19 genotype into extensive metabolizers (n=4) and poor metabolizers (n=3), participated in this study. Each subject received a single oral dose of omeprazole 20, 40, and 80 mg, with at least a 1-week washout period between each dose. Plasma concentrations of omeprazole and its two metabolites were monitored for 12 h after each dose of medication. After each dose was administered, the pharmacokinetic profiles of omeprazole and its two metabolites were significantly different between extensive metabolizers and poor metabolizers. The area under the plasma concentration-time curve (AUC) of omeprazole in extensive metabolizers was disproportionally increased 3.2- or 19.2-fold with dose escalation from 20 to 40 or 80 mg omeprazole, respectively. In contrast, the AUC of omeprazole was proportionally increased with the higher dose in poor metabolizers. The AUC of omeprazole after 20 mg administration to poor metabolizers was almost equal to the AUC in extensive metabolizers after 80 mg administration. In anti-H. pylori therapy, the recommended dose of omeprazole for extensive metabolizers is suggested to be a maximum of 80 mg x 2/d based on pharmacokinetic considerations.

When hair cells are once damaged by disease, noise trauma, or aging, hearing loss occurs in a person. Although fish, amphibians, and birds exhibit the ability to grow new hair cells to replace damaged ones, mammals cannot regenerate hair cells by themselves. In this chapter, the regenerative mechanisms in avian inner ear are reviewed comparing with mammals.

Genotyping of polymorphic drug metabolizing enzymes may be useful to estimate the blood concentration, efficacy, and toxicity of drugs before administration. Blood samples are most generally used for genotyping; however, sampling is invasive and complicated by handling and transport. Therefore, the authors developed genotyping methods using nonblood specimens, and then each genotype was compared with that from blood. Healthy Japanese volunteers provided hairs (n = 50), buccal cell swabs (n = 50), and fingernails (n = 30) for N-acetyltransferase 2 and CYP2C19 genotyping. Recovery of genomic DNA from each nonblood specimen was lower than that from 0.5 mL blood. Using a modification of the DNA extraction and polymerase chain reaction amplification method, genotypes were diagnosed without failure, even for those with very low levels of DNA. Both genotypes from these specimens completely matched the genotypes from the blood of the same subject. These nonblood specimens can be convenient, accessible, and economical alternatives to blood as a source of DNA for genotyping.

Purpose. To evaluate the β2-adrenergic receptor (β2AR) genotype frequency in the Japanese population and the relationship between β2AR genotype at amino acid position 16 (β2AR-16) and desensitization to β2-agonist ex vivo.

Purpose. To predict the CYP2C19 genotype-dependence in anti-Helicobacter pylori(H. pylori) therapy when lansoprazole or rabeprazole was used instead of omeprazole as a proton pump inhibitor (PPI).

Sulfapyridine (SP) is metabolized by polymorphic N-acetyltransferase 2 (NAT2) [EC 2.3.1.5]. In this study, the correlation between the NAT2 genotype and the pharmacokinetics of SP after multiple oral dosing of sulfasalazine (SASP) was examined to elucidate the effect of multiple dosing on the predictability of the phenotype by NAT2 genotyping. Seven healthy subjects were classified into two groups; the homozygotes for the wild-type allele, NAT2*4/*4 (Group I) and the compound heterozygotes for the mutant allele (NAT2*4/*6A or NAT2*4/*7B) (Group II). All received once-daily 1 g of SASP (Salazopyrin®) orally for 8 d. Plasma concentrations and urinary recoveries of SASP, SP and N-acetylsulfapyridine (AcSP) were monitored for 8 d. At 24 h on Day 1, the plasma concentration of SASP was lower and those of SP and AcSP were higher in Group II compared with Group I, but there was no significant difference. The plasma concentration ratio of AcSP to SP (AcSP/SP) tended to be lower in Group II. Urinary recoveries of SP and AcSP were increased in Group II, and their ratio was slightly reduced in Group II. Multiple dosing for 8 d resulted in an increase in the plasma concentrations of SASP, SP and AcSP. The difference between Group I and II was marked compared with single dosing, resulting in a significant difference in the plasma concentration of SP and the ratio of AcSP/SP. The simple input-output pharmacokinetic model applied for the analysis of plasma concentrations and urinary recoveries of SP and AcSP suggested the acetylation of SP into AcSP was 2.7-fold reduced in Group II (p=0.064).

Purpose. A combination of proton pump inhibitors and antimicrobials has been applied as an anti–Helicobacter pylori (H. pylori) therapy. Omeprazole, one of the proton pump inhibitors, is metabolized by CYP2C19, which exhibits genetic polymorphism. It was reported previously that the overall anti–H. pylori efficacy can be related to the CYP2C19 genotype. The main aim of the present study was to obtain a rational explanation for the relationship between the overall anti–H. pylori efficacy and the CYP2C19 genotype.

Isoniazid (INH) is metabolized by polymorphic N-acetyltransferase2 (NAT2). In the present study, the relationship between the NAT2 genotype and the INH acetylator phenotype was examined in Japanese tuberculous patients and compared with healthy subjects. Subjects were classified according to the genotyping into NAT2*5B (allele4), NAT2*6A (allele3) and NAT2*7B (allele2), using the PCR-RFLP method. Twelve healthy subjects and 7 tuberculous patients participated in the INH acetylator phenotyping study, in which each subject was administered an oral dose of INH, followed by urine sampling for 24 h. Urinary concentrations of INH and N-acetylisoniazid (AcINH) were measured by the HPLC method. The urinary recoveries of INH (% of dose) in healthy subjects in relation to NAT2 genotyping were as follows: 6.4+/-2.2 in the homozygotes for the wild-type allele, 10.7+/-2.2 in the compound heterozygotes for the mutant allele, and 38.6+/-6.4 in the homozygotes for the mutant allele. In the patients study, the findings in the corresponding three groups were 4.0+/-1.7, 8.8 and 18.3+/-9.3. Although no significant difference was found because of the lower systemic exposure of INH in patients compared with healthy subjects, there were differences in the disposition kinetics of INH between subjects with and without mutations in the NAT2 gene, and these findings were observed not only in healthy subjects but also in patients who had comedicated drugs and hepatic dysfunctions. The findings indicated that the metabolism of INH by NAT2 is clearly impaired in subjects with mutations in the NAT2 gene, and thus genotyping for three NAT2 point mutations was adequate to predict the metabolism of INH in Japanese tuberculous patients as well as healthy subjects. This NAT2 genotyping could become a useful alternative to TDM for INH.