Search

Adult-onset progressive hearing loss is a common, complex disease with a strong genetic component. Although to date over 150 genes have been identified as contributing to human hearing loss, many more remain to be discovered, as does most of the underlying genetic diversity. Many different variants have been found to underlie adult-onset hearing loss, but they tend to be rare variants with a high impact upon the gene product. It is likely that combinations of more common, lower impact variants also play a role in the prevalence of the disease.Here we present our exome study of hearing loss in a cohort of 532 older adult volunteers with extensive phenotypic data, including 99 older adults with normal hearing, an important control set. Firstly, we carried out an outlier analysis to identify genes with a high variant load in older adults with hearing loss compared to those with normal hearing. Secondly, we used audiometric threshold data to identify individual variants which appear to contribute to different threshold values. We followed up these analyses in a second cohort. Using these approaches, we identified genes and variants linked to better hearing as well as those linked to worse hearing.These analyses identified some known deafness genes, demonstrating proof of principle of our approach. However, most of the candidate genes are novel associations with hearing loss. While the results support the suggestion that genes responsible for severe deafness may also be involved in milder hearing loss, they also suggest that there are many more genes involved in hearing which remain to be identified. Our candidate gene lists may provide useful starting points for improved diagnosis and drug development.

Additional methods on drug screen, CSC sorting, real-time PCR, flow cytometric apoptosis assay and immunohistochemical (IHC) analysis are provided.

There is mounting evidence that cancer stem-like cells (CSC) are selectively enriched in residual tumors after anticancer therapies, which may account for tumor recurrence and metastasis by regenerating new tumors. Thus, there is a critical need to develop new therapeutic agents that can effectively eliminate drug-resistant CSCs and improve the efficacy of cancer therapy. Here, we report that Triptolide (C1572), a small-molecule natural product, selectively depletes CSCs in a dose-dependent fashion in human triple-negative breast cancer (TNBC) cell lines. Nanomolar concentrations of C1572 markedly reduced c-MYC (MYC) protein levels via a proteasome-dependent mechanism. Silencing MYC expression phenocopied the CSC depletion effects of C1572 and induced senescence in TNBC cells. Limited dilution assays revealed that ex vivo treatment of TNBC cells with C1572 reduced CSC levels by 28-fold. In mouse xenograft models of human TNBC, administration of C1572 suppressed tumor growth and depleted CSCs in a manner correlated with diminished MYC expression in residual tumor tissues. Together, these new findings provide a preclinical proof of concept defining C1572 as a promising therapeutic agent to eradicate CSCs for drug-resistant TNBC treatment. Cancer Res; 77(23); 6641–50. ©2017 AACR.

The Supplementary Figures and Legends provide additional information on Flow Cytometry gating strategy for TNBC CSC sorting, the effects of C1572 on additional TNBC cells, the evidence that C1572 reversibly inhibits MYC, comparing the effectiveness of C1572 and JQ1 at inhibiting MYC, and the impact of C1572 on body weight change in mice.

Radiation is widely used for cancer treatment but the radioresistance properties of cancer stem cells (CSCs) pose a significant challenge to the success of cancer therapy. Nuclear factor erythroid-2-related factor 2 (Nrf2) has emerged as a prominent regulator of cellular antioxidant responses and its over-activation is associated with drug resistant in cancer cells. However, the role of Nrf2 signaling in regulating the response of CSCs to irradiation has yet to be defined. Here, we show that exposure of triple-negative breast cancer (TNBC) cells to ionizing radiation (IR) upregulates Nrf2 expression and promotes its nuclear translocation in a reactive oxygen species (ROS)-dependent manner. Ectopic overexpression of Nrf2 attenuates, whereas knockdown of Nrf2 potentiates IR-induced killing of TNBC CSCs. Mechanistically, we found that Nrf2 knockdown increases IR-induced ROS production and impedes DNA repair at least in part via inhibition of DNA-PK. Furthermore, activation of Nrf2 by sulforaphane diminishes, whereas inhibition of Nrf2 by ML385 enhances IR-induced killing of TNBC CSCs. Collectively, these results demonstrate that IR-induced ROS production can activate Nrf2 signaling, which in turn counteracts the killing effect of irradiation. Therefore, pharmacological inhibition of IR-induced Nrf2 activation by ML385 could be a new therapeutic approach to sensitize therapy-resistant CSCs to radiotherapy.

Analysis approaches for single compositional data are well established; however, effective analysis strategies for paired compositional data remain to be investigated. The current project was motivated by studies of age-related hearing loss (presbyacusis), where subjects are classified into four audiometric phenotypes that need to be ranked within these phenotypes based on their paired compositional data. We address this challenge by formulating this problem as a classification problem and integrating a penalized multinomial logistic regression model with compositional data analysis approaches. We utilize Elastic Net for a penalty function, while considering average, absolute difference, and perturbation operators for compositional data. We applied the proposed approach to the presbyacusis study of 532 subjects with probabilities that each ear of a subject belongs to each of four presbyacusis subtypes. We further investigated the ranking of presbyacusis subjects using the proposed approach based on previous literature. The data analysis results indicate that the proposed approach is effective for ranking subjects based on paired compositional data.

Cancer stem cells (CSCs) have been shown to be resistant to current anticancer therapies and the induction of oxidative stress is an important mechanism of action for many anticancer agents. However, it is still largely unknown how CSCs respond to hydrogen peroxide (H(2)O(2))-induced oxidative stress. Here, we show that the levels of reactive oxygen species (ROS) are markedly lower in breast CSCs (BCSCs) than that in non-cancer stem cells (NCSCs). A transient exposure of breast cancer cells to sublethal doses of H(2)O(2) resulted in a dose-dependent increase of the epithelium-specific antigen (ESA)(+)/CD44(+)/CD24(−)subpopulations, a known phenotype for BCSCs. Although BCSCs survived sublethal doses of H(2)O(2) treatment, they lost the ability to form tumor spheres and failed to generate colonies as demonstrated by mammosphere-formation and clonogenic assays, respectively. Mechanistic studies revealed that H(2)O(2) treatment led to a marked increase of senescence-associated β-galactosidase activity but only minimal apoptotic cell death in BCSCs. Furthermore, H(2)O(2) triggers p53 activation and promotes p21 expression, indicating a role for the p53/p21 signaling pathway in oxidative stress-induced senescence in BCSCs. Taken together, these results demonstrate that the maintenance of a lower level of ROS is critical for CSCs to avoid oxidative stress and H(2)O(2)-induced BCSC loss of function is likely attributable to oxidative stress-triggered senescence induction, suggesting that ROS-generating drugs may have the therapeutic potential to eradicate drug-resistant CSCs via induction of premature senescence.

Noise exposure causes auditory nerve (AN) degeneration and hearing deficiency, though the proximal biological consequences are not entirely understood. Most AN fibers and spiral ganglion neurons are ensheathed by myelinating glia that provide insulation and ensure rapid transmission of nerve impulses from the cochlea to the brain. Here we show that noise exposure administered to mice of either sex rapidly affects myelinating glial cells, causing molecular and cellular consequences that precede nerve degeneration. This response is characterized by demyelination, inflammation, and widespread expression changes in myelin-related genes, including the RNA splicing regulatorQuaking(QKI) and numerous QKI target genes. Analysis of mice deficient in QKI revealed that QKI production in cochlear glial cells is essential for proper myelination of spiral ganglion neurons and AN fibers, and for normal hearing. Our findings implicate QKI dysregulation as a critical early component in the noise response, influencing cochlear glia function that leads to AN demyelination and, ultimately, to hearing deficiency.SIGNIFICANCE STATEMENTAuditory glia cells ensheath a majority of spiral ganglion neurons with myelin, protect auditory neurons, and allow for fast conduction of electrical impulses along the auditory nerve. Here we show that noise exposure causes glial dysfunction leading to myelin abnormality and altered expression of numerous genes in the auditory nerve, including QKI, a gene implicated in regulating myelination. Study of a conditional mouse model that specifically depleted QKI in glia showed that QKI deficiency alone was sufficient to elicit myelin-related abnormality and auditory functional declines. These results establish QKI as a key molecular target in the noise response and a causative agent in hearing loss.

There is mounting evidence that cancer stem-like cells (CSC) are selectively enriched in residual tumors after anticancer therapies, which may account for tumor recurrence and metastasis by regenerating new tumors. Thus, there is a critical need to develop new therapeutic agents that can effectively eliminate drug-resistant CSCs and improve the efficacy of cancer therapy. Here, we report that Triptolide (C1572), a small-molecule natural product, selectively depletes CSCs in a dose-dependent fashion in human triple-negative breast cancer (TNBC) cell lines. Nanomolar concentrations of C1572 markedly reduced c-MYC (MYC) protein levels via a proteasome-dependent mechanism. Silencing MYC expression phenocopied the CSC depletion effects of C1572 and induced senescence in TNBC cells. Limited dilution assays revealed that ex vivo treatment of TNBC cells with C1572 reduced CSC levels by 28-fold. In mouse xenograft models of human TNBC, administration of C1572 suppressed tumor growth and depleted CSCs in a manner correlated with diminished MYC expression in residual tumor tissues. Together, these new findings provide a preclinical proof of concept defining C1572 as a promising therapeutic agent to eradicate CSCs for drug-resistant TNBC treatment. Cancer Res; 77(23); 6641–50. ©2017 AACR.

Age-related hearing loss (or presbyacusis) is a progressive pathophysiological process. This study addressed the hypothesis that degeneration/dysfunction of multiple nonsensory cell types contributes to presbyacusis by evaluating tissues obtained from young and aged CBA/CaJ mouse ears and human temporal bones. Ultrastructural examination and transcriptomic analysis of mouse cochleas revealed age-dependent pathophysiological alterations in 3 types of neural crest–derived cells, namely intermediate cells in the stria vascularis, outer sulcus cells in the cochlear lateral wall, and satellite cells in the spiral ganglion. A significant decline in immunoreactivity for Kir4.1, an inwardly rectifying potassium channel, was seen in strial intermediate cells and outer sulcus cells in the ears of older mice. Age-dependent alterations in Kir4.1 immunostaining also were observed in satellite cells ensheathing spiral ganglion neurons. Expression alterations of Kir4.1 were observed in these same cell populations in the aged human cochlea. These results suggest that degeneration/dysfunction of neural crest–derived cells maybe an important contributing factor to both metabolic and neural forms of presbyacusis.

Highlights • Presbyacusis presents with metabolic, neural, and/or sensory phenotypes. • Reduced endocochlear potential is likely to be a key contributor to presbyacusis. • Genetic factors contribute to presbyacusis, including single-gene variants. • There appear to be downstream effects of presbyacusis on brain function and structure., There are multiple etiologies and phenotypes of age-related hearing loss or presbyacusis. In this review we summarize findings from animal and human studies of presbyacusis, including those that provide the theoretical framework for distinct metabolic, sensory, and neural presbyacusis phenotypes. A key finding in quiet-aged animals is a decline in the endocochlear potential (EP) that results in elevated pure-tone thresholds across frequencies with greater losses at higher frequencies. In contrast, sensory presbyacusis appears to derive, in part, from acute and cumulative effects on hair cells of a lifetime of environmental exposures (e.g., noise), which often result in pronounced high frequency hearing loss. These patterns of hearing loss in animals are recognizable in the human audiogram and can be classified into metabolic and sensory presbyacusis phenotypes, as well as a mixed metabolic+sensory phenotype. However, the audiogram does not fully characterize age-related changes in auditory function. Along with the effects of peripheral auditory system declines on the auditory nerve, primary degeneration in the spiral ganglion also appears to contribute to central auditory system aging. These inner ear alterations often correlate with structural and functional changes throughout the central nervous system and may explain suprathreshold speech communication difficulties in older adults with hearing loss. Throughout this review we highlight potential methods and research directions, with the goal of advancing our understanding, prevention, diagnosis, and treatment of presbyacusis.

The peripheral auditory nerve (AN) carries sound information from sensory hair cells to the brain. The present study investigated the contribution of mouse and human hematopoietic stem cells (HSCs) to cellular diversity in the AN following the destruction of neuron cell bodies, also known as spiral ganglion neurons (SGNs). Exposure of the adult mouse cochlea to ouabain selectively killed type I SGNs and disrupted the blood-labyrinth barrier. This procedure also resulted in the upregulation of genes associated with hematopoietic cell homing and differentiation, and provided an environment conducive to the tissue engraftment of circulating stem/progenitor cells into the AN. Experiments were performed using both a mouse-mouse bone marrow transplantation model and a severely immune-incompetent mouse model transplanted with human CD34+ cord blood cells. Quantitative immunohistochemical analysis of recipient mice demonstrated that ouabain injury promoted an increase in the number of both HSC-derived macrophages and HSC-derived nonmacrophages in the AN. Although rare, a few HSC-derived cells in the injured AN exhibited glial-like qualities. These results suggest that human hematopoietic cells participate in remodeling of the AN after neuron cell body loss and that hematopoietic cells can be an important resource for promoting AN repair/regeneration in the adult inner ear.

Cellular senescence is a form of permanent cell cycle arrest that can be triggered by a variety of cell-intrinsic and extrinsic stimuli, including telomere shortening, DNA damage, oxidative stress, and exposure to chemotherapeutic agents and ionizing radiation. Although the induction of apoptotic cell death is a desirable outcome in cancer therapy, mutations and/or deficiencies in the apoptotic signaling pathways have been frequently identified in many human cancer types, suggesting the importance of alternative apoptosis-independent therapeutic approaches for cancer treatment. A growing body of evidence has documented that senescence induction in tumor cells is a frequent response to many anticancer modalities including cyclin-dependent kinases 4/6 small molecule inhibitor-based targeted therapeutics and T helper-1 cytokine-mediated immunotherapy. This review discusses the recent advances and clinical relevance of therapy-induced senescence in cancer treatment.

Noise exposure causes auditory nerve (AN) degeneration and hearing deficiency, though the proximal biological consequences are not entirely understood. Most AN fibers and spiral ganglion neurons are ensheathed by myelinating glia that provide insulation and ensure rapid transmission of nerve impulses from the cochlea to the brain. Here we show that noise exposure administered to mice of either sex rapidly affects myelinating glial cells, causing molecular and cellular consequences that precede nerve degeneration. This response is characterized by demyelination, inflammation, and widespread expression changes in myelin-related genes, including the RNA splicing regulator Quaking (QKI) and numerous QKI target genes. Analysis of mice deficient in QKI revealed that QKI production in cochlear glial cells is essential for proper myelination of spiral ganglion neurons and AN fibers, and for normal hearing. Our findings implicate QKI dysregulation as a critical early component in the noise response, influencing cochlear glia function that leads to AN demyelination and, ultimately, to hearing deficiency.

// Xiaoyuan He 1, * , Aimin Yang 1, * , Daniel G. McDonald 2 , Ellen C. Riemer 1 , Kenneth N. Vanek 2 , Bradley A. Schulte 1 and Gavin Y. Wang 1, 3 1 Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC 29425, USA 2 Department of Radiation Oncology, Medical University of South Carolina, Charleston, SC 29425, USA 3 Cancer Genes and Molecular Regulation Program of Hollings Cancer Center, Medical University of South Carolina, Charleston, SC 29425, USA * These authors have contributed equally to this work Correspondence to: Gavin Y. Wang, email: wangy@musc.edu Keywords: microRNA-34a (miR-34a), ionizing radiation, non-small cell lung cancer, cellular senescence, c-Myc Received: February 28, 2017 Accepted: June 09, 2017 Published: July 15, 2017 ABSTRACT MicroRNAs (miRNAs) are a new class of gene expression regulators that have been implicated in tumorigenesis and modulation of the responses to cancer treatment including that of human non-small cell lung cancer (NSCLC). However, the role of miR-34a in ionizing radiation (IR)-induced senescence in NSCLC cells remains poorly understood. Here we report that IR-induced premature senescence correlates with upregulation of miR-34a expression in NSCLC cells. Ectopic overexpression of miR-34a by transfection with synthetic miR-34a mimics markedly enhances IR-induced senescence, whereas inhibition of miR-34a by transfection with a synthetic miR-34a inhibitor attenuates IR-induced senescence. Clonogenic assays reveal that treatment with miR-34a mimics augments IR-induced cell killing in human NSCLC cells. Mechanistically, we found that the senescence-promoting effect of miR-34a is associated with a dramatic down-regulation of c-Myc (Myc) expression, suggesting that miR-34a may promote IR-induced senescence via targeting Myc. In agreement with this suggestion, knockdown of Myc expression by RNAi recapitulates the senescence-promoting effect of miR-34a and enhances IR-induced cell killing in NSCLC cells. Collectively, these results demonstrate a previously unrecognized role for miR-34a in modulating IR-induced senescence in human NSCLC cells and suggest that pharmacological intervention of miR-34a expression may represent a new therapeutic strategy for improving the efficacy of lung cancer radiotherapy.

Objective: Health disparities exist between rural and urban areas. Rural physicians may lack sufficient medical knowledge, which may lead to poor quality of care. Therefore, we sought to determine whether medical knowledge differed between family physicians (FPs) practicing in rural areas compared with those practicing in metropolitan areas. Methods: We studied 8361 FPs who took the American Board of Family Medicine maintenance of certification (MOC) examination in 2009. Data sources were examination results and data from a demographic survey of practice structure and activities, completed as part of the examination application process. FPs9 location of practice was categorized as either rural or metropolitan using a moderate and conservative definition based on reported community size. Univariate statistics assessed differences in FP characteristics between rural and metropolitan areas. Logistic regression analyses determined the adjusted relationship between rural status and the odds of passing the MOC examination. Results: Metropolitan FPs were less likely than their rural counterparts to pass the MOC examination using both the moderate (odds ratio, 0.67; 95% confidence interval, 0.54–0.83) and conservative (odds ratio, 0.56; 95% confidence interval, 0.42–0.74) definitions. Physicians in solo practice were less likely to pass the examination than physicians in group practice. Conclusion: Rural physicians were more likely to pass the MOC examination, suggesting that rural health disparities do not result from a lack of provider knowledge.

Radiotherapy is used in >50% of patients during the course of cancer treatment both as a curative modality and for palliation. However, radioresistance is a major obstacle to the success of radiation therapy and contributes significantly to tumor recurrence and treatment failure, highlighting the need for the development of novel radiosensitizers that can be used to overcome tumor radioresistance and, thus, improve the efficacy of radiotherapy. Previous studies indicated that resveratrol (RV) may sensitize tumor cells to chemotherapy and ionizing radiation (IR). However, the mechanisms by which RV increases the radiation sensitivity of cancer cells have not been well characterized. Here, we show that RV treatment enhances IR-induced cell killing in non-small cell lung cancer (NSCLC) cells through an apoptosis-independent mechanism. Further studies revealed that the percentage of senescence-associated β-galactosidase (SA-β-gal)-positive senescent cells was markedly higher in cells treated with IR in combination with RV compared with cells treated either with IR or RV alone, suggesting that RV treatment enhances IR-induced premature senescence in lung cancer cells. Comet assays demonstrate that RV and IR combined treatment causes more DNA double-strand breaks (DSBs) than IR or RV treatment alone. DCF-DA staining and flow cytometric analyses demonstrate that RV and IR combined treatment leads to a significant increase in ROS production in irradiated NSCLC cells. Furthermore, our investigation show that inhibition of ROS production by N-acetyl-cysteine attenuates RV-induced radiosensitization in lung cancer cells. Collectively, these results demonstrate that RV-induced radiosensitization is associated with significant increase of ROS production, DNA-DSBs and senescence induction in irradiated NSCLC cells, suggesting that RV treatment may sensitize lung cancer cells to radiotherapy via enhancing IR-induced premature senescence.

With the exception of humans, the somata of type I spiral ganglion neurons (SGNs) of most mammalian species are heavily myelinated. In an earlier study, we used Ly5.1 congenic mice as transplant recipients to investigate the role of hematopoietic stem cells in the adult mouse inner ear. An unanticipated finding was that a large percentage of the SGNs in this strain were unmyelinated. Further characterization of the auditory phenotype of young adult Ly5.1 mice in the present study revealed several unusual characteristics including: 1) large aggregates of unmyelinated SGNs in the apical and middle turns; 2) symmetrical junction-like contacts between the unmyelinated neurons; 3) abnormal expression patterns for CNPase and connexin 29 in the SGN clusters; 4) reduced SGN density in the basal cochlea without a corresponding loss of sensory hair cells; 5) significantly delayed auditory brainstem response (ABR) wave I latencies at low and middle frequencies as compared to control mice with similar ABR threshold and 6) elevated ABR thresholds and deceased wave I amplitudes at high frequencies. Taken together, these data suggest a defect in Schwann cells that leads to incomplete myelinization of SGNs during cochlear development. The Ly5.1 mouse strain appears to be the only rodent model so far identified with a high degree of the “human-like” feature of unmyelinated SGNs that aggregate into neural clusters. Thus, this strain may provide a suitable animal platform for modeling human auditory information processing such as synchronous neural activity and other auditory response properties.

Gerbils aged in quiet show a decline of the endocochlear potential (EP) and elevated auditory nerve compound action potential (CAP) thresholds. However, establishing a direct relationship between an age-related reduction in the EP and changes in the activities of primary auditory neurons is difficult owing to the complexity of age-related histological changes in the cochlea. To address this issue, we developed a young gerbil model of "metabolic" presbyacusis that uses an osmotic pump to deliver furosemide into the round window niche for 7 days, resulting in a chronically reduced EP. In this model, the only major histopathologic changes were restricted to the hook region of the cochlea and consisted of loss of strial intermediate cells and massive edema in the lateral wall. The morphological and physiological evidence suggests that the cochlea can adapt to furosemide application over time. The morphology of spiral ganglion cells and hair cells appeared normal throughout the cochlea. CAP responses and EP values in this model are similar to those of quiet-aged ears. The spontaneous activity of single auditory fibers (n = 188) was assessed in 15 young gerbils treated with furosemide for 7 days. The percentage of recorded low-spontaneous rate (SR) fibers at characteristic frequencies (CFs)or = 6 kHz was significantly lower in furosemide-treated than in control ears. Recovery function tests of CAP responses after prior stimulation also showed a decline in activity of the low-SR population with CFsor = 6 kHz in the treated cochleas. A similar loss in the activity of low-SR fiber has been previously shown in quiet-aged gerbils. These results suggest that dysfunction of the cochlear lateral wall and subsequent chronic reduction in the EP can directly affect the activity patterns of primary auditory neurons in a manner similar to that seen in aged gerbils.

A dense population of vesicles largely fills the infranuclear compartment of gerbil inner hair cells (IHCs). Although the nature of the cargo in these vesicles has not been determined, the absence of a Golgi apparatus from the IHC’s basal compartment suggests that the vesicles lack the glycosylated protein that Golgi cisternae would provide. Instead, they likely possess neurotransmitter and function as synaptic vesicles. The morphologic mechanism for generating the vesicles also remains unexplained. Ultrastructural examination revealed a few discrete clusters of mitochondria in the IHC’s basal compartment. The clustered mitochondria made contact either with intermingling single cisternae or with one end of an unique set of polarized parallel cisternae. Both of these cisternal forms belong to a novel, mitochondria-activated category of cisternae which transforms into aligned segments where contacting mitochondria. Mitochondria-activated cisternae also envelope the vesicles in Hensen bodies of outer hair cells (OHCs). Coexistence of the mitochondria-activated cisternae with a specialized population of cytoplasmic vesicles in both IHCs and OHCs implicated this type of cisterna in synthesis of the cell specific vesicles. Assumedly, the mitochondria-activated cisternae possess an ATPase of the Class IV type. This class of enzymes, also designated flippases, translocates aminophospholipid from the outer to inner leaflet of the lipid bilayer and appears thereby to induce a lipid asymmetry which leads to cisternal segmentation and then vesiculation. In support of such an interpretation, RT-PCR analysis demonstrated the presence of Class IV ATPase in the Organ of Corti.

Voltage-gated chloride channels (ClCs) are important mediators of cellular ion homeostasis and volume regulation. In an earlier study, we used immunohistochemical, Western blot, and reverse transcriptase PCR (RT-PCR) approaches to identify ClC-K variants in types II, IV, and V fibrocytes of the rodent spiral ligament. We have now confirmed the expression of ClC-K2 in these cells by in situ hybridization. All three of these fibrocyte subtypes are thought to be involved in cochlear K(+) recycling; thus, it is important to understand the precise mechanisms regulating their membrane conductance and the role played by ClCs in this process. In this study, we report the characterization of a secondary cell line derived from explants from the region of the rat spiral ligament underlying and inferior to the spiral prominence. The cultured cells were immunopositive for vimentin, Na,K/ATPase, Na,K,Cl-cotransporter, carbonic anhydrase isozyme II, and creatine kinase isozyme BB, but not for cytokeratins or Ca/ATPase, an immunostaining profile indicative of the type IV subtype. Evaluation of the cultures by RT-PCR and Western blot analysis confirmed the presence of both ClC-2 and -K2. Whole-cell patch clamp recordings identified two biophysically distinct Cl(-) currents in the cultured cells. One, an inwardly rectifying Cl(-) current activated by hyperpolarization or decreasing extracellular pH corresponded with the properties of ClC-2. The other, a weak outwardly rectifying Cl(-) current regulated by extracellular pH, Cl(-), and Ca(2+) resembled the channel characteristics of ClC-K2 when expressed in Xenopus oocytes. These findings suggest that at least two functionally different chloride channels are involved in regulating membrane anion conductance in cultured type IV spiral ligament fibrocytes.

Degeneration of the spiral ganglion neurons (SGNs) of the auditory nerve occurs with age and in response to acoustic injury. Histopathological observations suggest that the neural degeneration often begins with an excitotoxic process affecting the afferent dendrites under the inner hair cells (IHCs), however, little is known about the sequence of cellular or molecular events mediating this excitotoxicity. Nuclear factor κB (NFκB) is a transcription factor involved in regulating inflammatory responses and apoptosis in many cell types. NFκB is also associated with intracellular calcium regulation, an important factor in neuronal excitotoxicity. Here, we provide evidence that NFκB can play a central role in the degeneration of SGNs. Mice lacking the p50 subunit of NFκB (p50−/−mice) showed an accelerated hearing loss with age that was highly associated with an exacerbated excitotoxic-like damage in afferent dendrites under IHCs and an accelerated loss of SGNs. Also, as evidenced by immunostaining intensity, calcium-buffering proteins were significantly elevated in SGNs of the p50−/−mice. Finally, the knock-out mice exhibited an increased sensitivity to low-level noise exposure. The accelerated hearing loss and neural degeneration with age in the p50−/−mice occurred in the absence of concomitant hair cell loss and decline of the endocochlear potential. These results indicate that NFκB activity plays an important role in protecting the primary auditory neurons from excitotoxic damage and age-related degeneration. A possible mechanism underlying this protection is that the NFκB activity may help to maintain calcium homeostasis in SGNs.

Current models of the lateral K + recycling pathway in the mammalian cochlea include two multicellular transport networks separated from one another by three interstitial gaps. The first gap is between outer hair cells and Deiters cells, the second is between outer sulcus cells and type II spiral ligament fibrocytes and the third is between intermediate and marginal cells in the stria vascularis. K + taken up by cells bordering these interstitial spaces is accompanied by Cl − . Maintaining appropriate electrolyte balance and membrane potentials in these cells requires a mechanism for exit of the resorbed Cl − . One possible candidate for regulating this Cl − efflux is ClC-K, a chloride channel previously thought to be kidney specific. Here, we demonstrate the expression of both known isoforms of ClC-K in the organ of Corti, spiral ligament and stria vascularis of the rat cochlea by immunohistochemical, Western blot and RT-PCR analysis. These results indicate a role for ClC-K in mediating Cl − recycling in the cochlea. The widespread expression of both ClC-K isoforms in the cochlea may help to explain the symptoms of Bartter’s syndrome Type III, a mutation in the hClC-Kb gene (human homologue of ClC-K2), which results in renal salt wasting without deafness. These data support the hypothesis that both isoforms of ClC-K are co-expressed in some cell membranes and account for the preservation of hearing in the presence of a mutation in only one channel isoform.

Bone marrow (BM)-derived stem cells have shown plasticity with a capacity to differentiate into a variety of specialized cells. To test the hypothesis that some cells in the inner ear are derived from BM, we transplanted either isolated whole BM cells or clonally expanded hematopoietic stem cells (HSCs) prepared from transgenic mice expressing enhanced green fluorescent protein (EGFP) into irradiated adult mice. Isolated GFP(+) BM cells were also transplanted into conditioned newborn mice derived from pregnant mice injected with busulfan (which ablates HSCs in the newborns). Quantification of GFP(+) cells was performed 3-20 months after transplant. GFP(+) cells were found in the inner ear with all transplant conditions. They were most abundant within the spiral ligament but were also found in other locations normally occupied by fibrocytes and mesenchymal cells. No GFP(+) neurons or hair cells were observed in inner ears of transplanted mice. Dual immunofluorescence assays demonstrated that most of the GFP(+) cells were negative for CD45, a macrophage and hematopoietic cell marker. A portion of the GFP(+) cells in the spiral ligament expressed immunoreactive Na, K-ATPase, or the Na-K-Cl transporter (NKCC), proteins used as markers for specialized ion transport fibrocytes. Phenotypic studies indicated that the GFP(+) cells did not arise from fusion of donor cells with endogenous cells. This study provides the first evidence for the origin of inner ear cells from BM and more specifically from HSCs. The results suggest that mesenchymal cells, including fibrocytes in the adult inner ear, may be derived continuously from HSCs.

Radiotherapy and chemotherapy are commonly used or treatment of cancer. Unfortunately, these treatments frequently cause acute and/or long-term bone marrow (BM) injury that can adversely affect the quality of life and the course of treatment. Our recent studies suggest that induction of hematopoietic stem cell (HSC) senescence by ionizing radiation (IR) and certain chemotherapeutic agents may contribute to long-term BM injury by impairing the ability of HSCs to self-renew. This suggestion is in agreement with a growing body of evidence demonstrating that HSCs from Bmi-1(-/-) and ATM(-/-) mice can lose their ability to self-renew by undergoing premature senescence. Interestingly, IR and different chemotherapeutic agents may induce HSC senescence and long-term BM injury in an agent-specific manner by activation of the p53-p21 and/or p16-Rb pathways. It will be of a great interest to determine if inhibition of these pathways can ameliorate radiotherapy and chemotherapy induced long-term BM injury.

Strial atrophy underlying age-related hearing loss was investigated by ultrastructural comparisons in young and senescent gerbils. In young animals strial marginal cells (MCs) projected primary processes which gave rise to and were connected by numerous ultrathin secondary processes. In 30-36-month-old gerbils, the MC secondary processes degenerated into lamellar or amorphous profiles as the first manifestation of strial atrophy. Some short primary processes shorn of projecting and connecting secondaries coalesced to form mitochondria-filled lobules. Strial involution appeared to progress with transformation of the degenerating processes and lobules into permanent residues of laminated amorphous substance. A second apparently unique form of degeneration was observed in which areas filled with homogeneous granular material replaced the processes that comprise the basal half of the normal MC. An abrupt line of transition separated this structureless degradation product below from the viable upper half of the MC. The terminally involuted stria consisted of MC bodies lining scala media, along with vestigial remnants of MC processes, nearby normal appearing intermediate cells (ICs) and unaltered basal cells. The only age-related change in ICs involved incorporation of melanosomes into very large, matrix-filled lysosomes. A profile of one MC in apparent necrosis provided evidence for an infrequent occurrence of MC death. These data support a progression of pathologic changes beginning with the demise of MC secondary processes and ending with ablation of secondary and primary processes. The initial injury apparently occurs as a result of oxidative self-damage to mitochondria in the MCs primary processes, leading to insufficient ATP for the Na,K-ATPase of the secondary processes. The reduced ATP level may cause cytotoxic alteration of the cytosolic Na(+)/K(+) ratio first in MC secondary processes and later in the primaries, with consequent degeneration of these structures.

Intracellular free Ca 2+ levels are critical to the activity of BK channels in inner ear type I spiral ligament fibrocytes. However, the mechanisms for regulating intracellular Ca 2+ levels in these cells are currently poorly understood. Using patch-clamp technique, we have identified a voltage-dependent L-type Ca 2+ channel in type I spiral ligament fibrocytes cultured from gerbil inner ear. With 10 mM Ba 2+ as the conductive cation, an inwardly rectifying current was elicited with little inactivation by membrane depolarization. The voltage activation threshold and the half-maximal voltage activation were −40 and −6 mV, respectively. This inward whole-cell current reached its peak at around 10 mV of membrane potential. The amplitude of the peak current varied among cells ranging from 50 to 274 pA with an average of 132.4±76.2 pA ( n =19); 10 −6 M nifedipine significantly inhibited the inward currents by 90.3±1.2% ( n =11). RT-PCR analysis revealed that cultured type I spiral ligament fibrocytes express the α 1C isoform of the L-type Ca 2+ channels encoded by the Ca v 1.2 gene. The expression of this channel in gerbil inner ear was confirmed by RT-PCR analysis using freshly isolated spiral ligament tissues. The Ca v 1.2 channel may function in conjunction with a previously identified intracellular Ca-ATPase (SERCA) to regulate intracellular free Ca 2+ levels in type I spiral ligament fibrocytes, and thus modulate BK channel activity in these cells.

Recent experimental and clinical studies have provided considerable evidence to support the phenomenon of K(+) recycling in the mammalian cochlea. However, the precise cellular and molecular mechanisms underlying and regulating this process remain only partially understood. Here, we report that cultured type I spiral ligament fibrocytes (SLFs), a major component of the K(+) recycling pathway, have a dominant K(+) membrane conductance that is mediated by BK channels. The averaged half-maximal voltage-dependent membrane potential for the whole-cell currents was 70+/-1.2 mV at 1 nM intracellular free Ca(2+) and shifted to 38+/-0.2 mV at 20 microM intracellular free Ca(2+) (n=4-6). The reversal potential of whole-cell tail currents against different bath K(+) concentrations was 52 mV per decade (n=3-6). The sequence of relative ion permeability of the whole-cell conductance was K(+)Rb(+)z.Gt;Cs(+)Na(+) (n=5-17). The whole-cell currents were inhibited by extracellular tetraethylammonium and iberiotoxin (IbTx) with IC(50) values of 0.07 mM and 0.013 microM, respectively (n=3-7). The membrane potentials of type I SLFs measured with conventional zero-current whole-cell configuration were highly K(+)-selective and sensitive to IbTx (n=4-9). In addition, the BK channels in these cells exhibited voltage-dependent and incomplete inactivation properties and the recovery time was estimated to be approximately 6 s with repetitive voltage pulses from -70 to 80 mV (n=3). These data suggest that BK channels in type I SLFs play a major role in regulating the intracellular electrochemical gradient in the lateral wall syncytium responsible for facilitating the K(+) movement from perilymph to the stria vascularis.

Ultrastructural examination revealed an epithelium of about five tectal cells (TCs) roofing the outer tunnel (OT) in the mid to upper, but not the basal, region of gerbil and chinchilla cochlea. Structures in TCs that are apparently specialized for retrieval of K(+) released into tunnel fluid from outer hair cells (OHCs) include surface fimbriae in the gerbil and canalicular reticulum in the chinchilla. A tunnel roof of organelle-rich TCs appeared to be better equipped for ion resorption than a roof composed of organelle-poor Hensen cells (HCs). Fimbriae, filopodia, and the cell body of TCs descended to contact the third Deiters cell (DC3) in the gerbil, and the hypertrophied DC3 phalanx rose to contact TCs in the chinchilla, which suggests a solute exchange between TCs and DCs. Previously unrecognized structures that are speculated to provide ATP ligand for cochlear purinoreceptors occurred in the chinchilla DC and gerbil TC. The observation of a microtubule stalk in DCs indicated that they also function in cochlear mechanics. A newly delineated lateral tunnel cell (LTC) intervened between the DC3 and HC in both species. The apicomedial plasmalemma of all DCs fitted closely to the base of OHCs and enveloped afferent nerves. The morphologic specializations reported here provide further support for the proposed transcellular lateral flow route for K(+) currents generated by sound exposure and neural activity. The previously demonstrated expansion of Boettcher cells, outer sulcus cell roots, type Il and IV fibrocytes, and apical microvilli on HCs and Claudius cells (CCs) in the base of the cochlea is postulated here to mediate a basal parallel current that could supply the increased K(+) transport required for the basally elevated electric potential (EP).

Hematologic toxicity is a major cause of mortality in radiation emergency scenarios and a primary side effect concern in patients undergoing chemo-radiotherapy. Therefore, there is a critical need for the development of novel and more effective approaches to manage this side effect. Catalase is a potent antioxidant enzyme that coverts hydrogen peroxide into hydrogen and water. In this study, we evaluated the efficacy of catalase as a protectant against ionizing radiation (IR)-induced toxicity in hematopoietic stem and progenitor cells (HSPCs). The results revealed that catalase treatment markedly inhibits IR-induced apoptosis in murine hematopoietic stem cells and hematopoietic progenitor cells. Subsequent colony-forming cell and cobble-stone area-forming cell assays showed that catalase-treated HSPCs can not only survive irradiation-induced apoptosis but also have higher clonogenic capacity, compared with vehicle-treated cells. Moreover, transplantation of catalase-treated irradiated HSPCs results in high levels of multi-lineage and long-term engraftments, whereas vehicle-treated irradiated HSPCs exhibit very limited hematopoiesis reconstituting capacity. Mechanistically, catalase treatment attenuates IR-induced DNA double-strand breaks and inhibits reactive oxygen species. Unexpectedly, we found that the radioprotective effect of catalase is associated with activation of the signal transducer and activator of transcription 3 (STAT3) signaling pathway and pharmacological inhibition of STAT3 abolishes the protective activity of catalase, suggesting that catalase may protect HSPCs against IR-induced toxicity via promoting STAT3 activation. Collectively, these results demonstrate a previously unrecognized mechanism by which catalase inhibits IR-induced DNA damage and apoptosis in HSPCs.

Abnormal activation of the mammalian target of rapamycin (mTOR) signaling pathway has been observed in a variety of human cancers. Therefore, targeting of the mTOR pathway is an attractive strategy for cancer treatment and several mTOR inhibitors, including AZD8055 (AZD), a novel dual mTORC1/2 inhibitor, are currently in clinical trials. Although bone marrow (BM) suppression is one of the primary side effects of anticancer drugs, it is not known if pharmacological inhibition of dual mTORC1/2 affects BM hematopoietic stem and progenitor cells (HSPCs) function and plasticity. Here we report that dual inhibition of mTORC1/2 by AZD or its analogue (KU-63794) depletes mouse BM Lin−Sca-1+c-Kit+cells in cultures via the induction of apoptotic cell death. Subsequent colony-forming unit (CFU) assays revealed that inhibition of mTORC1/2 suppresses the clonogenic function of hematopoietic progenitor cells (HPCs) in a dose-dependent manner. Surprisingly, we found that dual inhibition of mTORC1/2 markedly inhibits the growth of day-14 cobblestone area-forming cells (CAFCs) but enhances the generation of day-35 CAFCs. Given the fact that day-14 and day-35 CAFCs are functional surrogates of HPCs and hematopoietic stem cells (HSCs), respectively, these results suggest that dual inhibition of mTORC1/2 may have distinct effects on HPCs versus HSCs.

Dystroglycan (DG) forms part of a cell surface laminin receptor complex and is believed to play a critical role in the assembly and homeostasis of basement membranes (BM). The receptor complex is made up of alpha- and beta-DG subunits and is found in muscle, epithelial and nerve tissue. In the cochlea, DG may be involved in the abnormal accumulation of laminin seen in the thickened BM of strial capillaries with age. This excess deposition of laminin is thought to lead to capillary necrosis and contribute to degeneration of the stria vascularis (SV). Here we assessed the presence and distribution of DG in the developing, mature and senescent gerbil cochlea in order to ascertain whether altered patterns of expression are a factor in age-related pathology. Western blots of proteins isolated from the entire cochlea demonstrated the presence of the alpha-DG subunit. mRNA encoding DG was identified in microdissected specimens of the lateral wall and the combined organ of Corti/modiolus by RT-PCR analysis. Immunohistochemical experiments localized alpha-DG in epithelial BMs and regions of epithelial cell-cell contact with no intervening BM in the developing and mature cochlea. Immunoreactive alpha-DG was present in the BM underlying strial capillaries and in vessels of the central portion of the auditory nerve, but was not detected in any other vessels in the cochlea. Age-related changes in alpha-DG expression were observed only in the SV where a marked decrease in alpha-DG immunoreactivity was seen in the BM of strial capillaries as well as throughout the SV. The results demonstrate the selective expression of alpha-DG in both BM and non-BM sites in the mature cochlea and suggests its involvement in both developmental and aging processes.

The C57BL/6J mouse suffers from cochlear degeneration beginning at an early age and has been used as a model of age-related hearing loss (presbyacusis). Here, the endocochlear potential (EP) and compound action potential (CAP) responses were determined in one-, four-, 12- and 24-month-old C57BL/6J mice. CAP measures included thresholds to tone pips, input/output (I/O) functions, and recovery functions to conditioning tones. EP values among the four age groups did not differ significantly (P>0.05) in either the basal or apical turns. CAP thresholds were increased significantly by 10 to 30 dB in the four-month group compared to the one-month controls at 11.3, 16, 20, and 22.6 kHz. CAP I/O functions were shallower in the four-month group compared to controls at all frequencies. In the 12- and 24-month-old mice, CAP responses were absent, despite normal EP values in these animals. Recovery functions after conditioning tones were obtained at 8, 16, 20 and 22.6 kHz; the functions had fast and slow components at all frequencies tested in both the one- and four-month-old groups. The corresponding recovery curves were identical for both age groups, even with significant threshold shifts in the older group. The two component recovery curves provide the first physiological evidence that different spontaneous rate (SR) classes of auditory neurons exist in the C57BL/6J mouse. Moreover, the unchanged recovery functions in the older group suggest that there was no loss of activity of the low-SR fiber population with age under conditions where the EP remains stable, in contrast to the gerbil model of presbyacusis where there is a loss of low-SR fiber activity and EP does decline with age.

Triple-negative breast cancer (TNBC), the most aggressive subtype of breast cancers, is highly refractory to chemotherapy and has a high incidence of metastasis and tumor relapse. Targeted therapies for TNBC do not exist because this subtype lacks druggable hormone receptors and HER2 amplification or overexpression. Although Taxane-based chemotherapy is a standard of care for TNBC treatment, paclitaxel (Taxol)-induced cancer stem cell (CSC) enrichment presents a significant challenge to the success of breast cancer treatment. Thus, there is a critical need to understand the mechanisms by which chemotherapy induces CSC enrichment in residual tumors following anticancer therapies. Here we report that Taxol-induced enrichment for CSCs correlates well with a marked upregulation of Twist1 expression in human TNBC cells. Knockdown of Twist1 inhibits the clonogenic capacity of TNBC cells in vitro and TNBC tumorigenesis in vivo. Mammosphere formation assays indicate that silencing of Twist1 greatly diminishes the tumor sphere-forming potential of breast CSCs. Furthermore, limited dilution assays and stem cell xenotransplantation reveal that knockdown of Twist1 expression decreases the frequency of CSCs by 72-fold in human TNBC cells. More importantly, we show that silencing of Twist1 blocks Taxol-induced CSC enrichment in residual tumor cells that have survived drug treatment. Together, these results demonstrate a previously unrecognized role for Twist1 in mediating Taxol-induced CSC enrichment in residual tumors of human TNBC and suggest that targeted inhibition of the Twist1 signaling pathway may represent a novel therapeutic strategy to tackle therapy-induced CSC enrichment in breast cancer treatment. Citation Format: Aimin Yang, Xiaoyuan He, Bradley A. Schulte, Steven L. Carroll, Stephen P. Ethier, Gavin Y. Wang. Twist1 modulates paclitaxel-induced cancer stem cell enrichment in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1919. doi:10.1158/1538-7445.AM2017-1919

Objectives:Our study aims to determine the immunostaining pattern of Kir4.1 channels in human cochlear tissues. Potassium recycling pathways critical for maintaining cochlear ion homeostasis and the endocochlear potential have been defined largely in animal models. Potassium effluxed from sensory hair cells is taken up by supporting cells and returned to the stria vascularis via two distinct transcellular syncytial networks consisting of epithelial cells and spiral ligament fibrocytes. The inward rectifying potassium channel Kir4.1 appears to be an essential component of this process, as evidenced by murine knockout models lacking an endocochlear potential. Animal models have demonstrated robust Kir4.1 expression in several cell types along the cochlear potassium recycling pathway. However, Kir4.1 immunostaining patterns in the human cochlea remain undefined.Methods:Postmortem human temporal bones were collected through the Hearing Research Program at the Medical University of South Carolina. Temporal bon...

The number, size and distribution of myelinated nerve fibers were analyzed in the osseous spiral lamina (OSL) of young and old gerbils raised in a quiet environment. Because decreased endocochlear potentials (EPs) play a significant role in age-related hearing loss in the gerbil, we correlated morphometric and topographical data for nerve fibers with EP measurements in the same ear. Fibers were analyzed at the 2 and 10 kHz locations. The number of fibers at the 2 kHz location ranged from 12 to 47% greater than at the 10 kHz place in both young and aged specimens. No significant correlation was found between the number of fibers and the EP. Nerve fibers in gerbil tend to be distributed vertically by size within the OSL [Slepecky et al. (2000) Hear. Res. 144, 124-134], a result also found in cats and guinea pigs. Smaller fibers are more often found towards the scala vestibuli side of the OSL, whereas larger fibers are concentrated towards the scala tympani. The present data confirmed this distribution in young gerbils; however, in aged ears the distribution often became more uniform. Moreover, fiber distribution and ganglion cell size were highly correlated with EP. As EP declined, the fiber size distribution in the OSL became more uniform and the mean cross-sectional area of spiral ganglion cells and fiber diameter decreased. Thus, for whatever reason, certain indices of auditory nerve fiber morphometrics appear to be associated with the EP.

Objectives/Hypothesis Morphological and immunohistochemical studies of human temporal bones are challenging as a result of difficulties in obtaining reliably fi-ed specimens and the lengthy time required for decalcification, typically 4 to 7 months. A novel method of processing human temporal bones using a microwave oven to accelerate decalcification is described. This procedure provides a rapid means of decalcifying temporal bones with good preservation of tissue morphology and antigenicity. Methods Human temporal bone specimens obtained at autopsy (n = 12, from specimens aged 43–91 y) were fixed within 6.5 hours of death by transtympanic perilymphatic perfusion of the inner ear. Decalcification was carried out using ethylenediaminetetra-acetic acid (EDTA) in a microwave oven and required only 3 to 6 weeks. Specimens were then dehydrated, embedded in paraffin, sectioned, and mounted on slides for morphological and immunohistochemical evaluation. Results Microscopic e-amination revealed no obvious artifacts attributable to the microwave decalcification process. The quality of morphological preservation was largely dependent on the postmortem fi-ation interval and adequacy of perilymphatic perfusion. Immunohistochemical analysis demonstrated strong positive staining for the enzyme Na,K-ATPase, an integral membrane protein. Conclusions This study demonstrates that microwave decalcification provides an efficient and reliable means of processing human temporal bones for histological and histochemical e-amination. Decalcification time is significantly reduced with no apparent adverse effects on structural preservation or antigenicity.

The possibility that phospholipase C contributes to intracellular signaling in the cochlea was investigated by immunostaining for eight different isoforms of the enzyme. In the mature gerbil cochlea, expression of the isozymes varied widely among different cell types. The phospholipase C-beta1 isoform was detected in inner and outer hair cells, and spiral ganglion neurons where it may participate in regulating Ca(2+) flux. The beta3 isozyme was expressed in epithelial cells thought to mediate lateral and medial circulation of potassium. The beta2 isozyme was present in border, inner phalangeal and Hensen cells, the stria vascularis, and suprastrial and supralimbal fibrocytes where it also may be involved in regulating ion transport activities. The phospholipase C-gamma isozymes were expressed in supporting cells, the stria vascularis, and certain fibrocytes where they possibly participate in activating tyrosine kinase and modulating ion conductances. The delta2 isoform was found in pillar, outer sulcus and strial marginal cells as well as spiral ganglion neurons and their radial processes. Documentation of changes in the expression pattern of phospholipase C isoforms during postnatal development and knowledge of their distribution in several positive control tissues provided further data for speculation about the biologic significance of the cochlear reactivity. The results demonstrate a wide diversity of isozyme distribution in the cochlea and suggest that the enzymes affect activities of various cochlear cell types in different ways.

The NF-kappaB/IkappaB complex is a major transcription regulator of inflammatory and immune responses. Helicobacter pylori infection causes chronic inflammation in gastric mucosa by inducing dissociation of the inhibitory IkappaB protein from the complex with a resulting increased expression of interleukin (IL)-8. To clarify which of several known IkappaB proteins could be involved in this inflammatory response, we undertook immunohistochemical examination of normal mouse stomach as well as other murine tissues for comparison, using polyclonal antibodies specific for alpha-, beta-, gamma-, and in-isoforms of IkappaB. The results showed strong immunoreactivity for the alpha-isoform in parietal cells and for the beta-isoform in pit cells of the stomach, along with the presence of these proteins in various other sites. Comparative staining revealed a similar but not identical distribution of IkappaB proteins in the Mongolian gerbil, a rodent model for H. pylori infection. The findings suggest that the alpha- and beta-isoforms are dominant IkappaB proteins in gastric parietal and foveolar cells, respectively, and point to a role for these transcription regulators in modulating pathological responses in stomach and other organs. (J Histochem Cytochem 48:191-199, 2000)

Postfixation with a ferrocyanide-osmium tetroxide solution preserved a dense network of canaliculi extending from the apical to the upper lateral plasma membrane in cochlear inner hair cells (IHCs). Numerous Golgi bodies intermingled with this apical canalicular reticulum (CR). Osmium-ferrocyanide treatment also disclosed several previously unreported structures below the IHC nucleus. The first consisted of stacks of six or eight and sets of three parallel cisternae of rough endoplasmic reticulum spanning between clustered mitochondria. Some parallel cisternae ended with segmentation where they contacted mitochondria, and others terminated by transforming into blebs or continuing into canaliculi. A second feature was comprised of a complex of segmented cisternae and branching canaliculi with clustered mitochondria. Branching minicanaliculi with associated vesicles neighbored the complexes. A fourth entity consisted of synaptic-like vesicles that largely filled the subnuclear cytosol and congregated at synapses. An additional infranuclear structure was composed of slender canaliculi that collected near or streamed to plasmalemma, often next to a synapse. A paradoxical absence of rough endoplasmic reticulum above and Golgi zones below the nucleus provided evidence of atypical mechanisms for generating the membrane in CR and forming synaptic vesicles. The observations offer the view that IHCs are compartmentalized into an apical mechanoreceptor half and a basal half that affects neurotransmission. The apical CR provided a possible structural basis for sequestering the K+ known to influx apically and for directing its diffusion to the site of known efflux across the lateral plasmalemma. The codistribution of parallel cisternae, canalicular-mitochondrial complexes, and synaptic-like vesicles, all of which are unique to IHCs, implicated the cisternae and complexes in the genesis of the vesicles.

SUMMARY Members of the glutathione S-transferase (GST) family of detoxification enzymes play a role in chemotherapy resistance in certain cancers but have not been directly implicated as agents whose absence may predispose tissues to hormonally induced tumorigenesis. Here we report the development of a polyclonal antiserum to a hamster mu class GST, and immunohistochemical analysis of alpha, mu, and pi class GSTs to study the effects of hormone treatment on their expression in reproductive tract tissues of male golden Syrian hamsters. These animals develop leiomyosarcomas in the vas deferens after treatment with testosterone propionate (TP) and 17 b -estradiol (E2). High levels of all three GST classes were detected throughout the reproductive tract epithelium of control animals. In 100% of the experimental animals, 4 weeks of treatment either with E2 alone, or with E2 plus TP promoted a complete loss of immunostaining for alpha and mu class GSTs, but not for pi class GSTs, only in the epithelial lining of the vas deferens. In contrast, treatment with TP alone resulted in moderate hyperplasia of smooth muscle in the proximal vas deferens, with no cellular atypia and no changes in immunoreactivity of any of the GST classes. The consistent and site-specific nature of these results strongly suggests a functional role for GSTs in hormonally induced carcinogenic process. (J Histochem Cytochem 47:91‐98, 1999)

A smooth membrane system consisting of subsurface cisternae (SSC) underlies the lateral plasmalemma of auditory outer hair cells (OHCs). The SSC contain Ca-ATPase and are regarded as an intracellular Ca2+ reservoir like the sarcoplasmic reticulum of myocytes. Recently, it has been demonstrated that Ca-ATPase activity in sarcoplasmic reticulum is regulated by Ca2+/calmodulin-dependent protein kinases (CaM kinases). Here we investigated the presence of CaM kinases in OHCs and their possible association with the SSC. Inner ears collected from adult gerbils and from neonates at 2-day intervals between 0 and 20 days after birth were immunostained with antibodies specific for different CaM kinases. A polyclonal antiserum against CaM kinase IV yielded a strong immunostaining reaction along the lateral wall of OHCs. The staining appeared after the tenth postnatal day and continued into adulthood. No other site in the inner ear, including cochlear inner hair cells and vestibular hair cells, was reactive. The kinase's apparent association with the SSC strongly supports its involvement in intracellular Ca2+ homeostasis and suggests a role in regulating the OHCs' slow motile responses.