Tetrabromobisphenol-A induces apoptotic death of auditory cells and hearing loss.

Phenolic tetrabromobisphenol-A (TBBPA) and its derivatives are commonly used flame-retardants, in spite of reported toxic effects including neurotoxicity, immunotoxicity, nephrotoxicity, and hepatotoxicity. However, the effects of TBBPA on ototoxicity have not yet been reported. In this study, we investigated the effect of TBBPA on hearing function in vivo and in vitro. Auditory Brainstem Response (ABR) threshold was markedly increased in mice after oral administration of TBBPA, indicating that TBBPA causes hearing loss. In addition, TBBPA induced the loss of both zebrafish neuromasts and hair cells in the rat cochlea in a dose-dependent manner. Mechanistically, hearing loss is largely attributed to apoptotic cell death, as TBBPA increased the expression of pro-apoptotic genes but decreased the expression of anti-apoptotic genes. We also found that TBBPA induced oxidative stress, and importantly, pretreatment with NAC, an anti-oxidant reagent, reduced TBBPA-induced reactive oxygen species (ROS) generation and partially prevented cell death. Our results show that TBBPA-mediated ROS generation induces ototoxicity and hearing loss. These findings implicate TBBPA as a potential environmental ototoxin by exerting its hazardous effects on the auditory system.

Panel 4: Recent advances in otitis media in molecular biology, biochemistry, genetics, and animal models.

BACKGROUND: Otitis media (OM) is the most common childhood bacterial infection and also the leading cause of conductive hearing loss in children. Currently, there is an urgent need for developing novel therapeutic agents for treating OM based on full understanding of molecular pathogenesis in the areas of molecular biology, biochemistry, genetics, and animal model studies in OM. OBJECTIVE: To provide a state-of-the-art review concerning recent advances in OM in the areas of molecular biology, biochemistry, genetics, and animal model studies and to discuss the future directions of OM studies in these areas. DATA SOURCES AND REVIEW METHODS: A structured search of the current literature (since June 2007). The authors searched PubMed for published literature in the areas of molecular biology, biochemistry, genetics, and animal model studies in OM. RESULTS: Over the past 4 years, significant progress has been made in the areas of molecular biology, biochemistry, genetics, and animal model studies in OM. These studies brought new insights into our understanding of the molecular and biochemical mechanisms underlying the molecular pathogenesis of OM and helped identify novel therapeutic targets for OM. CONCLUSIONS AND IMPLICATIONS FOR PRACTICE: Our understanding of the molecular pathogenesis of OM has been significantly advanced, particularly in the areas of inflammation, innate immunity, mucus overproduction, mucosal hyperplasia, middle ear and inner ear interaction, genetics, genome sequencing, and animal model studies. Although these studies are still in their experimental stages, they help identify new potential therapeutic targets. Future preclinical and clinical studies will help to translate these exciting experimental research findings into clinical applications.

Cisplatin ototoxicity involves cytokines and STAT6 signaling network.

We herein investigated the role of the STAT signaling cascade in the production of pro-inflammatory cytokines and cisplatin ototoxicity. A significant hearing impairment caused by cisplatin injection was observed in Balb/c (wild type, WT) and STAT4(-/-), but not in STAT6(-/-) mice. Moreover, the expression levels of the protein and mRNA of pro-inflammatory cytokines, including TNF-α, IL-1ß, and IL-6, were markedly increased in the serum and cochlea of WT and STAT4(-/-), but not STAT6(-/-) mice. Organotypic culture revealed that the shape of stereocilia bundles and arrays of sensory hair cell layers in the organ of Corti from STAT6(-/-) mice were intact after treatment with cisplatin, whereas those from WT and STAT4(-/-) mice were highly distorted and disarrayed after the treatment. Cisplatin induced the phosphorylation of STAT6 in HEI-OC1 auditory cells, and the knockdown of STAT6 by STAT6-specific siRNA significantly protected HEI-OC1 auditory cells from cisplatin-induced cell death and inhibited pro-inflammatory cytokine production. We further demonstrated that IL-4 and IL-13 induced by cisplatin modulated the phosphorylation of STAT6 by binding with IL-4 receptor alpha and IL-13Rα1. These findings suggest that STAT6 signaling plays a pivotal role in cisplatin-mediated pro-inflammatory cytokine production and ototoxicity.

Evidence that cisplatin-induced auditory damage is attenuated by downregulation of pro-inflammatory cytokines via Nrf2/HO-1.

Recently, we demonstrated that pro-inflammatory cytokines such as TNF-alpha, IL-1beta, and IL-6 played a critical role in cisplatin-induced cochlear injury and that flunarizine, known as a T-type Ca(2+) channel antagonist, induced a cytoprotective effect against cisplatin cytotoxicity in HEI-OC1 cells by the activation of NF-E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) cascade through PI3K-Akt signaling but calcium-independent pathway. We report here that flunarizine markedly attenuates cisplatin-induced pro-inflammatory cytokine secretion and their messenger RNA transcription as well as cisplatin cytotoxicity through the activation of Nrf2/HO-1 and downregulation of NF-kappaB. In HEI-OC1 cells, overexpression of Nrf2/HO-1 by gene transfer or pharmacological approaches attenuated cisplatin-induced cytotoxicity and pro-inflammatory cytokine production. On the contrary, inhibition of Nrf2/HO-1 signaling by pharmacological inhibitors or specific small interfering RNAs significantly abolished the beneficial effects of flunarizine. Flunarizine also attenuated cisplatin-mediated MAPK activation and pharmacological inhibition of MAPKs, especially MEK1/ERK, blocked cisplatin-induced NF-kappaB activation in HEI-OC1 cells. Furthermore, WT-Nrf2 overexpression effectively blocked MAPK activation after cisplatin exposure. Finally, orally administrated Sibelium, the trade name of flunarizine, suppressed the increase of pro-inflammatory cytokines by cisplatin in both serum and cochleas of mice, whereas it increased HO-1 expression in cochleas. These results indicate that flunarizine induces a protective effect against cisplatin ototoxicity through the downregulation of NF-kappaB by Nrf2/HO-1 activation and the resulting inhibition of pro-inflammatory cytokine production in vitro and in vivo.

Expression of water channel proteins (aquaporins) in the rat Eustachian tube and middle ear mucosa.

CONCLUSION: Diverse expression of the different subtypes of aquaporins in different parts of the Eustachian tube and middle ear suggests region-specific functions of the aquaporins in the normal physiology of the tubotympanum and also suggests that they may play roles in the pathophysiology of otitis media. OBJECTIVES: The epithelial cells of the middle ear and Eustachian tube must maintain adequate water balance for normal function of the mucociliary system. Since aquaporins (AQPs) are known to play critical roles in water homeostasis, we investigated their expression in the tubotympanum of the rat. METHODS: The expression of AQP subtypes 1, 2, 4, 5, and 7 were examined in the rat Eustachian tube and middle ear using RT-PCR, Western blotting, and immunohistochemistry. RESULTS: Transcripts for AQP 1, 4, and 5 were detected in the Eustachian tube and middle ear. Expression of these molecules at the protein level was confirmed by Western blot analysis. Immunohistochemical analysis demonstrated that AQP 4 was localized to the basolateral membranes of ciliated epithelial cells while AQP 5 was localized to the apical surface of serous gland cells, but not goblet cells, in the rat Eustachian tube. AQP 1 was found to be expressed by the subepithelial fibroblasts.

Cisplatin cytotoxicity of auditory cells requires secretions of proinflammatory cytokines via activation of ERK and NF-kappaB.

The ototoxicity of cisplatin, a widely used chemotherapeutic agent, involves a number of mechanisms, including perturbation of redox status, increase in lipid peroxidation, and formation of DNA adducts. In this study, we demonstrate that cisplatin increased the early immediate release and de novo synthesis of proinflammatory cytokines, including TNF-alpha, IL-1beta, and IL-6, through the activation of ERK and NF-kappaB in HEI-OC1 cells, which are conditionally immortalized cochlear cells that express hair cell markers. Both neutralization of proinflammatory cytokines and pharmacologic inhibition of ERK significantly attenuated the death of HEI-OC1 auditory cells caused by cisplatin and proinflammatory cytokines. We also observed a significant increase in the protein and mRNA levels of proinflammatory cytokines in both serum and cochleae of cisplatin-injected rats, which was suppressed by intraperitoneal injection of etanercept, an inhibitor of TNF-alpha. Immunohistochemical studies revealed that TNF-alpha expression was mainly located in the spiral ligament, spiral limbus, and the organ of Corti in the cochleae of cisplatin-injected rats. NF-kappaB protein expression, which overlapped with terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling-positive signal, was very strong in specific regions of the cochleae, including the organ of Corti, spiral ligament, and stria vascularis. These results indicate that proinflammatory cytokines, especially TNF-alpha, play a central role in the pathophysiology of sensory hair cell damage caused by cisplatin.

Spiral ligament fibrocytes release chemokines in response to otitis media pathogens.

CONCLUSION: Spiral ligament fibrocytes (SLFs) may be involved in the innate immune response of the inner ear by producing chemoattractants for recruiting inflammatory cells such as neutrophils and monocytes. OBJECTIVE: The purpose of this study was to investigate the cellular responses of SLFs when challenged by inflammatory stimuli such as components of otitis media pathogens or proinflammatory cytokines. MATERIALS AND METHODS: To detect released inflammatory cytokines and chemokines, cells were treated for 48 h with whole lysates of nontypable Haemophilus influenzae (NTHi), Streptococcus pneumoniae, or with interleukin 1 alpha (IL-1alpha). The culture medium was then collected and applied to protein arrays. To compare mRNA levels of chemokines, total RNA was extracted after 3 h of treatment with the above agents, and quantitative real-time PCR was performed. RESULTS: Protein array analysis showed that in response to NTHi or S. pneumoniae, rat SLFs released monocyte chemotactic protein 1, macrophage inflammatory protein 3 alpha, TNF-alpha, and cytokine-induced neutrophil chemoattractant 2 and 3. Treatment with IL-1alpha, on the other hand, resulted in release of MCP-1 but not the other molecules. Tissue inhibitor of metalloproteinase 1 and vascular endothelial growth factor were released regardless of the inflammatory stimulus used.

Synergistic effect of interleukin 1 alpha on nontypeable Haemophilus influenzae-induced up-regulation of human beta-defensin 2 in middle ear epithelial cells.

BACKGROUND: We recently showed that beta-defensins have antimicrobial activity against nontypeable Haemophilus influenzae (NTHi) and that interleukin 1 alpha (IL-1 alpha) up-regulates the transcription of beta-defensin 2 (DEFB4 according to new nomenclature of the Human Genome Organization) in human middle ear epithelial cells via a Src-dependent Raf-MEK1/2-ERK signaling pathway. Based on these observations, we investigated if human middle ear epithelial cells could release IL-1 alpha upon exposure to a lysate of NTHi and if this cytokine could have a synergistic effect on beta-defensin 2 up-regulation by the bacterial components. METHODS: The studies described herein were carried out using epithelial cell lines as well as a murine model of acute otitis media (OM). Human cytokine macroarray analysis was performed to detect the released cytokines in response to NTHi exposure. Real time quantitative PCR was done to compare the induction of IL-1 alpha or beta-defensin 2 mRNAs and to identify the signaling pathways involved. Direct activation of the beta-defensin 2 promoter was monitored using a beta-defensin 2 promoter-Luciferase construct. An IL-1 alpha blocking antibody was used to demonstrate the direct involvement of this cytokine on DEFB4 induction. RESULTS: Middle ear epithelial cells released IL-1 alpha when stimulated by NTHi components and this cytokine acted in an autocrine/paracrine synergistic manner with NTHi to up-regulate beta-defensin 2. This synergistic effect of IL-1 alpha on NTHi-induced beta-defensin 2 up-regulation appeared to be mediated by the p38 MAP kinase pathway. CONCLUSION: We demonstrate that IL-1 alpha is secreted by middle ear epithelial cells upon exposure to NTHi components and that it can synergistically act with certain of these molecules to up-regulate beta-defensin 2 via the p38 MAP kinase pathway.

Expression of hepatocyte growth factor and c-Met in hypopharyngeal squamous cell carcinoma.

CONCLUSION: Our results suggest that hepatocyte growth factor (HGF) may play an important role in the progression of hypopharyngeal cancer. OBJECTIVE: HGF, a potent stimulator of hepatocyte growth, stimulates the motility, invasiveness, proliferation and morphogenesis of epithelium and may be involved in physiologic and pathologic processes such as embryogenesis, wound healing, organ regeneration, inflammation and tumor invasion. We therefore examined the role of HGF and c-Met in the invasion and metastasis of hypopharyngeal squamous cell carcinoma (SCC). MATERIAL AND METHODS: We performed immunohistochemical staining of 40 specimens each of normal mucosa and SCC of the hypopharynx with HGF and c-Met antibodies. For reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting, fresh normal and cancer tissue from the hypopharynx obtained from five patients were used. RESULTS: Positive rates of HGF and c-Met expression in hypopharyngeal SCC were 77.5% and 70%, respectively. HGF staining was significantly correlated with lymph node metastasis and pathologic stage (p < 0.05). c-Met staining was only significantly correlated with lymph node metastasis (p < 0.05). Increased expression of c-Met mRNA (RT-PCR) and protein (Western blotting) was detected in hypopharyngeal cancer tissue.

Expression of female sex hormone receptors in human middle-ear cholesteatomas.

The purpose of this study was to establish the eventual presence of progesterone receptor (PGR) and oestrogen receptor (EGR) in human middle-ear cholesteatoma (MECh) tissues and to compare their expression between male and female patients. An immunohistochemical technique was employed for detection of PGR- and EGR-specific immunoreactivity in MECh samples using formalin-fixed paraffin-embedded tissue sections. The positive results were verified with reverse transcriptase polymerase chain reaction (RT-PCR). The morphological study revealed stable expression of PGR in suprabasal layers of all cholesteatoma samples. Weaker immunoreactivity for PGR was demonstrated in external auditory canal skin (EACS) samples in comparison with MECh, while PGR-specific staining was not observed in retroauricular skin (RAS) samples. EGR was detected only at mRNA levels. Stronger expression of EGR PCR products was disclosed in female cholesteatoma samples, while PGR mRNA was predominantly detected in male cholesteatoma specimens. Our preliminary experimental results give us ground to assume that female sex hormones may stimulate proliferation and affect differentiation of MECh keratinocytes.

Nucleoplasm staining patterns and cell cycle-associated expression of Ki-67 in middle ear cholesteatoma.

OBJECTIVES: To compare Ki-67 expression patterns in middle ear cholesteatoma with the corresponding retroauricular and external auditory canal skins, and to determine the cell cycle-dependent localization of Ki-67. MATERIAL AND METHODS: MIB-1 monoclonal antibody was used for comparative assessment of proliferative activity of middle ear cholesteatoma, external auditory canal skin, and retroauricular skin samples on formalin-fixed paraffin-embedded tissue sections. Primary keratinocytes from cholesteatoma tissue were isolated and subjected to kinetic analysis of the cell cycle. RESULTS: Higher proliferative activity was established in cholesteatoma in comparison with retroauricular and external auditory canal skins. Three different staining patterns have been described. Kinetic analysis revealed continuous expression of Ki-67 during all active phases of the cell cycle and remained "silent" in resting cells. CONCLUSION: The established correlation between the staining patterns and cell cycle-associated expression of Ki-67 specifies Ki-67 as a reliable and stable marker of proliferation for middle ear cholesteatoma.

3D micro-CT images of ossicles destroyed by middle ear cholesteatoma.

OBJECTIVES: To reconstruct the 3D structures of normal ossicles and ossicles destroyed by different cholesteatomas, and to compare the volume fractions of the two types of ossicles. MATERIALS AND METHOD: Thirteen pathological ossicles from four kinds of middle ear cholesteatoma obtained during surgery were used. Normal ossicles harvested from cadaveric temporal bone specimens were used as controls. The structures of the ossicles were analyzed using micro-CT by filming 2D cross-sectional images perpendicular to the long axis, from which 3D images were reconstructed. The volume fraction was measured using a CT-Analyzer and Ant. RESULTS: The volume fraction, indicating the relative strength of normal ossicles, was lower in the portion close to the articular surface. As the four types of cholesteatoma were found to express their patterns of destruction at various sites in the ossicles, they revealed a different volume fraction in each ossicle. CONCLUSION: Our preliminary data on ossicular structure obtained using micro-CT will be helpful for elucidating the normal and pathological structure of ossicles and for the further development of artificial prostheses.

Antimicrobial activity of innate immune molecules against Streptococcus pneumoniae, Moraxella catarrhalis and nontypeable Haemophilus influenzae.

BACKGROUND: Despite its direct connection to the nasopharynx which harbors otitis media pathogens as part of its normal flora, the middle ear cavity is kept free of these bacteria by as yet unknown mechanisms. Respiratory mucosal epithelia, including those of the middle ear and eustachian tube, secrete antimicrobial effectors including lysozyme, lactoferrin and beta defensins-1 and -2. To elucidate the role of these innate immune molecules in the normal defense and maintenance of sterility of respiratory mucosa such as that of the middle ear, we assessed their effect on the respiratory pathogens nontypeable Haemophilus influenzae (NTHi) 12, Moraxella catarrhalis 035E, and Streptococcus pneumoniae 3, and 6B. METHODS: Two assay methods, the radial assay and the liquid broth assay, were employed for testing the antimicrobial activity of the molecules. This was done in order to minimize the possibility that the observed effects were artifacts of any single assay system employed. Also, transmission electron microscopy (TEM) was employed to evaluate the effect of antimicrobial innate immune molecules on OM pathogens. For the statistical analysis of the data, Student's t-test was performed. RESULTS: Results of the radial diffusion assay showed that beta defensin-2 was active against all four OM pathogens tested, while treatment with beta defensin-1 appeared to only affect M. catarrhalis. The radial assay results also showed that lysozyme was quite effective against S. pneumoniae 3 and 6B and was partially bacteriostatic/bactericidal against M. catarrhalis. Lysozyme however, appeared not to affect the growth of NTHi. Thus, lysozyme seems to have a more pronounced impact on the growth of the Gram-positive S. pneumoniae as compared to that of Gram-negative pathogens. Lactoferrin on the other hand, enhanced the growth of the bacteria tested. The results of the radial assays were confirmed using liquid broth assays for antimicrobial activity, and showed that lysozyme and beta defensin-2 could act synergistically against S. pneumoniae 6B. Moreover, in the liquid broth assay, beta defensin-1 showed a modest inhibitory effect on the growth of S. pneumoniae 6B. As assessed by ultrastructural analysis, lysozyme and beta defensin-2, and to a much lesser extent, beta defensin-1, appeared to be able to cause damage to the bacterial membranes. CONCLUSIONS: Here we report that lysozyme and the beta defensins can inhibit the growth of clinical isolates of otitis media pathogens - namely NTHi strain 12, S. pneumoniae strains 3 and 6B and M. catarrhalis strain 035E - and cause ultrastructural damage to these pathogens. Moreover, we demonstrate that lysozyme and beta defensin-2 can act synergistically against S. pneumoniae. These findings are consistent with the concept that secreted antimicrobial peptides and other components of innate immunity constitute the first line of defense protecting host mucosal surfaces, including the tubotympanal (eustachian tube and middle ear cavity) mucosa, against pathogens.

Three cases of Waardenburg syndrome type 2 in a Korean family.

Waardenburg syndrome (WS) is a rare, autosomal dominant disorder characterized by sensorineural hearing loss, pigmentary disturbances of the skin, hair, and iris, and other developmental defects such as lateral displacement of both medial canthi and lacrimal puncta called dystopia canthorum. While mutations of the PAX3 (paired box) gene have been identified in about 99% of WS type 1 cases, WS type 2 is a heterogeneous group, with about 15% of cases caused by mutations in microphthalmia associated transcription factor (MITF). We have experienced three cases of typical WS type 2 in a Korean family, for whom full ocular examination and genetic studies were performed. The genetic studies revealed no mutation in either PAX3 or MITF genes. The genetic basis, as yet unknown for most cases of WS type 2, might be found with further investigation.

Various causes and clinical characteristics in vertigo in children with normal eardrums.

OBJECTIVE: The differential diagnosis of vertigo in children is extensive. Otitis media and middle ear effusion could be the most common causes of vertigo in children, but there are some problems in detecting the other causes for vertigo because they are one of most frequent diseases of childhood. The purpose of this study is to review the clinical characteristics and both the audiological and vestibular findings of vertigo in children with normal eardrums, who do not show otitis media or middle ear effusion, and to assist in making a differential diagnosis of vertigo. METHODS: The fifty five children (< 16 years old) with vertigo, who visited the Department of Otolaryngology, Ajou University Hospital, Suwon, South Korea between January 1995 and December 2001 were selected for this study. These excluded the patients with abnormal eardrums/tympanograms or those that did not perform questionnaires, audiological, or vestibular evaluations. They were retrospectively analyzed for clinical symptoms, vestibular functions, and differential diagnosis. RESULTS: The most common causes for vertigo in children were migraine in 17 (30.9%) and benign paroxysmal vertigo of childhood (BPVC) in 14 (25.5%). Other less frequent causes included four cases of trauma, two cases each of Meniere's disease, delayed endolymphatic hydrops, benign positional vertigo, and one case only for cerebellopontine angle tumor, seizure, acute vestibular neuritis, juvenile rheumatoid arthritis, leaving ten cases (18.2%) as unclassified. Abnormal findings were noted in 13 (23.6%) in pure tone audiogram, 3 (5.5%) in positioning test, 6 (10.9%) in bithermal caloric test, and 36 (65.5%) in rotation chair test. CONCLUSIONS: The vertigo in children with normal eardrums, who did not show otitis media or middle ear effusion, was most commonly caused by migraine and BPVC. These findings have shown to be very different from those with adult vertigo. The evaluation of vertigo in children requires a questionnaire for extensive and complete history taking, audiograms and vestibular function tests. And in selected cases, electroencephalography, hematological evaluation, imaging of the brain or temporal bone should be performed.

Expression of beta-defensins in human middle ear cholesteatoma.

OBJECTIVE: To establish the eventual presence of human beta-defensins (hBD) in middle ea r cholesteatoma and paired retroauricular skin samples obtained during surgical treatment. MATERIAL AND METHODS: hBD were detected using immunohistochemical techniques and reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: It is hypothesized that hBD-1 and -2 are upregulated in cholesteatoma in comparison with retroauricular skin. The immunohistochemical study demonstrated stronger hBD-2-positive staining in middle ear cholesteatoma in comparison with retroauricular skin. No significant differences in hBD-1 expression were detected between cholesteatoma and skin samples. RT-PCR established elevated expression of hBD-2 mRNA in middle ear cholesteatomas. HBD-1 mRNA was constitutively expressed in cholesteatoma and retroauricular skin specimens. CONCLUSION: These preliminary experimental results lead us to believe that hBD may play an important role in the chronic inflammatory state of middle ear cholesteatoma.

Immortalization of rat eustachian tube epithelial cells by adenovirus 12-simian virus 40 hybrid virus.

The eustachian tube epithelial cells play an important role in the initial pathogenesis of otitis media. In order to study the role of the eustachian tube epithelial cells in the pathogenesis of otitis media, we have established a rat eustachian tube epithelial cell line. The cell line was derived by infecting primary cultures of eustachian tube epithelial cells with the adenovirus 12-simian virus 40 (Adl2-SV40) hybrid virus. The immortalized cells have retained the morphological characteristics of the parental cells and show positive staining with anti-cytokeratin antibodies (a marker for epithelial cells), but not with anti-vimentin antibodies (a fibroblast marker). The cells have been in continuous culture for more than 10 months and have undergone 38 passages. Western blotting and cell staining have confirmed the expression of the SV40 T antigen and p53. Chromosomal analysis indicates that the cell line is aneuploid and derived from male rat epithelial cells. Together, our results suggest that the cell line originated from eustachian tube epithelial cells from a male rat and was successfully immortalized by the Ad12-SV40 virus.

Functional study of GJB2 in hereditary hearing loss.

OBJECTIVES/HYPOTHESIS: The gene of the gap junction protein connexin 26 (Cx26) was found to be the main causative gene of autosomal recessive nonsyndromic hearing loss (DFNB1). Although 35delG has been known as the major mutation in Western countries, 235delC was reported to be a specific form of mutation in Asian populations. The objective of the study was to identify how 235delC and E114G changes found in the Korean population affected the function of using molecular biological techniques. METHODS: Genes containing 235delC and E114G were cloned into the pcDNA3 vector, and HeLa cells were transfected with the recombinant DNA samples by the liposome complex method. The expression and subcellular localization of Cx26 were determined, using antibodies against amino acid sequences in the intracellular loop (IL) and N-terminal (NT) portions of Cx26. To analyze functions of the as a gap junction channel, we examined Lucifer yellow dye transfer between cells with a scrape-loaded technique. Wild-type (WT) with normal hearing was used as a positive control, and mock transfected cells were used as a negative control. RESULTS: Immunocytochemical analysis showed that cells transfected with E114G and WT gave characteristic punctate patterns of reaction in the cell membrane with both antibodies. However, 235delC cells were not stained with anti-IL antibody but stained slightly just around the nucleus only with anti-NT antibody. In a functional study of, transfer of Lucifer yellow into contiguous cells was detected in both WT and E114G, but no transfer activity was observed in 235delC. CONCLUSIONS: The 235delC mutation showed a loss of targeting activity to the cell membrane and severe deterioration of gap junction activity. For the E114G, we did not find any difference from WT transfected cells.