Sacculatane diterpenoids from the Chinese liverwort Pellia epiphylla with protection against H2O2-induced apoptosis of PC12 cells.

Eight previously undescribed sacculatane diterpenoids, epiphyllins A-H, and one unknown bibenzyl-based isopentene along with seven known compounds were isolated from the Chinese liverwort Pellia epiphylla (L.) Corda. Their structures were established unequivocally on the basis of spectroscopic data and CD measurement. The quinine reductase-inducing activity evaluation demonstrated that epiphyllins A-D, 1ß-hydroxysacculatanolide and pellianolactone B displayed moderate antioxidant effect. Further investigation of pellianolactone B revealed its protective effects on H2O2-induced oxidative insults and apoptosis in PC12 cells.

Oncomodulin Expression Reveals New Insights into the Cellular Organization of the Murine Utricle Striola.

Oncomodulin (OCM, aka ß-parvalbumin) is an EF-hand calcium binding protein that is expressed in a restricted set of hair cells in the peristriolar region of the mammalian utricle. In the present study, we determined the topologic distribution of OCM among hair cell phenotypes to advance our understanding of the cellular organization of the striola and the relationship of these phenotypes with characteristics of tissue polarity. The distributions of OCM-positive (OCM+) hair cells were quantified in utricles of mature C57Bl/6 mice. Immunohistochemistry was conducted using antibodies to OCM, calretinin, and ß3-tubulin. Fluorophore-conjugated phalloidin was used to label hair cell stereocilia, which provided the basis for determining hair cell counts and morphologic polarizations. We found OCM expression in striolar types I and II hair cells, though the distributions were dissimilar to the native striolar type I and II distributions, favoring type I hair cells. The distribution of OCM immunoreactivity among striolar type I hair cells also reflected nonrandom distribution among type Ic and Id phenotypes (i.e., those receiving calretinin-positive and calretinin-negative calyces, respectively). However, many OCM+ hair cells were found lateral to the striola, and within the epithelial region encompassing OCM+ hair cells, the distributions of OCM+ types Ic and Id hair cells were similar to the native distributions of Ic and Id in this region. Summarily, these data provide a quantitative perspective supporting the existence of different underlying factors driving the topologic expression of OCM in hair cells than those responsible for tissue polarity characteristics associated within the utricular striola, including calretinin expression in afferent calyces.

Reactions of all Escherichia coli lytic transglycosylases with bacterial cell wall.

The reactions of all seven Escherichia coli lytic transglycosylases with purified bacterial sacculus are characterized in a quantitative manner. These reactions, which initiate recycling of the bacterial cell wall, exhibit significant redundancy in the activities of these enzymes along with some complementarity. These discoveries underscore the importance of the functions of these enzymes for recycling of the cell wall.

Biomechanics of hair cell kinocilia: experimental measurement of kinocilium shaft stiffness and base rotational stiffness with Euler-Bernoulli and Timoshenko beam analysis.

Vestibular hair cell bundles in the inner ear contain a single kinocilium composed of a 9+2 microtubule structure. Kinocilia play a crucial role in transmitting movement of the overlying mass, otoconial membrane or cupula to the mechanotransducing portion of the hair cell bundle. Little is known regarding the mechanical deformation properties of the kinocilium. Using a force-deflection technique, we measured two important mechanical properties of kinocilia in the utricle of a turtle, Trachemys (Pseudemys) scripta elegans. First, we measured the stiffness of kinocilia with different heights. These kinocilia were assumed to be homogenous cylindrical rods and were modeled as both isotropic Euler-Bernoulli beams and transversely isotropic Timoshenko beams. Two mechanical properties of the kinocilia were derived from the beam analysis: flexural rigidity (EI) and shear rigidity (kGA). The Timoshenko model produced a better fit to the experimental data, predicting EI=10,400 pN µm(2) and kGA=247 pN. Assuming a homogenous rod, the shear modulus (G=1.9 kPa) was four orders of magnitude less than Young's modulus (E=14.1 MPa), indicating that significant shear deformation occurs within deflected kinocilia. When analyzed as an Euler-Bernoulli beam, which neglects translational shear, EI increased linearly with kinocilium height, giving underestimates of EI for shorter kinocilia. Second, we measured the rotational stiffness of the kinocilium insertion (κ) into the hair cell's apical surface. Following BAPTA treatment to break the kinocilial links, the kinocilia remained upright, and κ was measured as 177±47 pN µm rad(-1). The mechanical parameters we quantified are important for understanding how forces arising from head movement are transduced and encoded by hair cells.

Zebrafish pax5 regulates development of the utricular macula and vestibular function.

The zebrafish otic vesicle initially forms with only two sensory epithelia, the utricular and saccular maculae, which primarily mediate vestibular and auditory function, respectively. Here, we test the role of pax5, which is preferentially expressed in the utricular macula. Morpholino knockdown of pax5 disrupts vestibular function but not hearing. Neurons of the statoacoustic ganglion (SAG) develop normally. Utricular hair cells appear to form normally but a variable number subsequently undergo apoptosis and are extruded from the otic vesicle. Dendrites of the SAG persist in the utricle but become disorganized after hair cell loss. Hair cells in the saccule develop and survive normally. Otic expression of pax5 requires pax2a and fgf3, mutations in which cause vestibular defects, albeit by distinct mechanisms. Thus, pax5 works in conjunction with fgf3 and pax2a to establish and/or maintain the utricular macula and is essential for vestibular function.

Observation of the morphology and calcium content of vestibular otoconia in rats after simulated weightlessness.

Reduction in bone formation may have been the main reason for the lower calcium content of the otoconia after simulated weightlessness in rats. The head-ward distribution of blood volume may explain the morphological changes observed in the middle and inner ears. To observe morphological changes in the vestibular organs and measure the calcium content of otoconia in rats after simulated weightlessness. We used a tail suspension model of simulated weightlessness and then investigated changes in the vestibular organs using scanning electron microscopy and X-ray microanalysis. In comparison to untreated rats, the vestibular otoconia of the rats subjected to simulated weightlessness were small, irregularly shaped or fissured, and were arranged loosely and out of order. In addition, the calcium content of the otoconia was markedly decreased.

Myo3A, one of two class III myosin genes expressed in vertebrate retina, is localized to the calycal processes of rod and cone photoreceptors and is expressed in the sacculus.

The striped bass has two retina-expressed class III myosin genes, each composed of a kinase, motor, and tail domain. We report the cloning, sequence analysis, and expression patterns of the long (Myo3A) and short (Myo3B) class III myosins, as well as cellular localization and biochemical characterization of the long isoform, Myo3A. Myo3A (209 kDa) is expressed in the retina, brain, testis, and sacculus, and Myo3B (155 kDa) is expressed in the retina, intestine, and testis. The tails of these two isoforms contain two highly conserved domains, 3THDI and 3THDII. Whereas Myo3B has three IQ motifs, Myo3A has nine IQ motifs, four in its neck and five in its tail domain. Myo3A localizes to actin filament bundles of photoreceptors and is concentrated in the calycal processes. An anti-Myo3A antibody decorates the actin cytoskeleton of rod inner/outer segments, and this labeling is reduced by the presence of ATP. The ATP-sensitive actin association is a feature characteristic of myosin motors. The numerous IQ motifs may play a structural or signaling role in the Myo3A, and its localization to calycal processes indicates that this myosin mediates a local function at this site in vertebrate photoreceptors.

Localization of glucocorticoid receptors in the murine inner ear.

We performed an immunohistochemical investigation of the distribution of glucocorticoid receptors (GRs) in the murine inner ear and found that GRs were expressed extensively, but with various degrees of immunoreactivity in different regions. We observed the strongest GR expression in the type III fibrocytes of the spiral ligament. Although the immunoreactivity of the cochlear hair cells and of the vestibular sensory epithelia was weak, the neighboring cochlear supporting cells and the subepithelial regions of the vestibular sensory epithelia were immunostained. Staining for GRs was also positive in the spiral ganglia and vestibular ganglia, as well as in the endolymphatic sac. The role of GRs in the inner ear is discussed.

Weightlessness affects cytoskeleton of rat utricular hair cells during maturation in vitro.

The aim of this study was to investigate whether an altered gravitational environment affected the phenotype of vestibular hair cells during maturation. We developed, using an automated incubator, a 3D culture of utricles from newborn rats. These cultures were subjected to weightlessness for 1 or 3 days, and then compared with control cultures developed in natural and induced 1G gravity. Immunocytochemistry for alpha-tubulin and calretinin revealed disorganisation of the microtubules and a loss of hair cell shape in cells subjected to weightlessness during maturation. We conclude that the lack of gravitational strain affected cytoskeletal dynamics, resulting in loss of the specific morphological phenotype of the cells.

Alteration of E-cadherin and beta-catenin in mouse vestibular epithelia during induction of apoptosis.

The aim of this study was to examine if adhesion molecules had relation with degeneration and regeneration processes of mammalian vestibular epithelia. The distribution of E-cadherin and beta-catenin was immunohistochemically examined in normal and aminoglycoside-treated utricles of mice. E-cadherin and beta-catenin linearly expressed between epithelial cells in normal specimens. Aminoglycoside injury resulted in temporal alteration in distribution of these molecules with induction of apoptosis in hair cells. Degradation of both molecules was widely observed in vestibular epithelia, while some supporting cells exhibited accumulation of beta-catenin. After completion of induction of apoptosis, expression of these adhesion molecules was normal in distribution. These findings suggest that the E-cadherin-beta-catenin complex plays roles in degeneration and subsequent repair processes in vestibular epithelia affected by aminoglycosides.

Functional expression of exogenous proteins in mammalian sensory hair cells infected with adenoviral vectors.

To understand the function of specific proteins in sensory hair cells, it is necessary to add or inactivate those proteins in a system where their physiological effects can be studied. Unfortunately, the usefulness of heterologous expression systems for the study of many hair cell proteins is limited by the inherent difficulty of reconstituting the hair cell's exquisite cytoarchitecture. Expression of exogenous proteins within hair cells themselves may provide an alternative approach. Because recombinant viruses were efficient vectors for gene delivery in other systems, we screened three viral vectors for their ability to express exogenous genes in hair cells of organotypic cultures from mouse auditory and vestibular organs. We observed no expression of the genes for beta-galactosidase or green fluorescent protein (GFP) with either herpes simplex virus or adeno-associated virus. On the other hand, we found robust expression of GFP in hair cells exposed to a recombinant, replication-deficient adenovirus that carried the gene for GFP driven by a cytomegalovirus promoter. Titers of 4 x 10(7) pfu/ml were sufficient for expression in 50% of the approximately 1,000 hair cells in the utricular epithelium; < 1% of the nonhair cells in the epithelium were GFP positive. Expression of GFP was evident as early as 12 h postinfection, was maximal at 4 days, and continued for at least 10 days. Over the first 36 h there was no evidence of toxicity. We recorded normal voltage-dependent and transduction currents from infected cells identified by GFP fluorescence. At longer times hair bundle integrity was compromised despite a cell body that appeared healthy. To assess the ability of adenovirus-mediated gene transfer to alter hair cell function we introduced the gene for the ion channel Kir2.1. We used an adenovirus vector encoding Kir2.1 fused to GFP under the control of an ecdysone promoter. Unlike the diffuse distribution within the cell body we observed with GFP, the ion channel-GFP fusion showed a pattern of fluorescence that was restricted to the cell membrane and a few extranuclear punctate regions. Patch-clamp recordings confirmed the expression of an inward rectifier with a conductance of 43 nS, over an order of magnitude larger than the endogenous inward rectifier. The zero-current potential in infected cells was shifted by -17 mV. These results demonstrate an efficient method for gene transfer into both vestibular and auditory hair cells in culture, which can be used to study the effects of gene products on hair cell function.

Localization of myosin-Ibeta near both ends of tip links in frog saccular hair cells.

Current evidence suggests that the adaptation motor of mechanoelectrical transduction in vertebrate hair cells is myosin-Ibeta. Previously, confocal and electron microscopy of bullfrog saccular hair cells using an anti-myosin-Ibeta antibody labeled the tips of stereocilia. We have now done quantitative immunoelectron microscopy to test whether myosin-Ibeta is enriched at or near the side plaques of tip links, the proposed sites of adaptation, using hair bundles that were serially sectioned parallel to the macular surface. The highest particle density occurred at stereocilia bases, close to the cuticular plate. Also, stereocilia of differing lengths had approximately the same number of total particles, suggesting equal targeting of myosin-Ibeta to all stereocilia. Finally, particles tended to clump in clusters of two to five particles in the distal two-thirds of stereocilia, suggesting a tendency for self-assembly of myosin-Ibeta. As expected from fluorescence microscopy, particle density was high in the distal 1 micrometer of stereocilia. If myosin-Ibeta is the adaptation motor, a difference should exist in particle density between regions containing the side plaque and those excluding it. Averaging of particle distributions revealed two regions with approximately twice the average density: at the upper ends of tip links in a 700-nm-long region centered approximately 100 nm above the side plaque, and at the lower ends of tip links within the tip plaques. Controls demonstrated no such increase. The shortest stereocilia, which lack side plaques, showed no concentration rise on their sides. Thus, the specific localization of myosin-Ibeta at both ends of tip links supports its role as the adaptation motor.

Postnatal development of type I and type II hair cells in the mouse utricle: acquisition of voltage-gated conductances and differentiated morphology.

The type I and type II hair cells of mature amniote vestibular organs have been classified according to their afferent nerve terminals: calyx and bouton, respectively. Mature type I and type II cells also have different complements of voltage-gated channels. Type I cells alone express a delayed rectifier, gK,L, that is activated at resting potential. We report that in mouse utricles this electrophysiological differentiation occurs during the first postnatal week. Whole-cell currents were recorded from hair cells in denervated organotypic cultures and in acutely excised epithelia. From postnatal day 1 (P1) to P3, most hair cells expressed a delayed rectifier that activated positive to resting potential and a fast inward rectifier, gK1. Between P4 and P8, many cells acquired the type I-specific conductance gK,L and/or a slow inward rectifier, gh. By P8, the percentages of cells expressing gK,L and gh were at mature levels. To investigate whether the electrophysiological differentiation correlated with morphological changes, we fixed utricles at different times between P0 and P28. Ultrastructural criteria were developed to classify cells when calyces were not present, as in cultures and neonatal organs. The morphological and electrophysiological differentiation followed different time courses, converging by P28. At P0, when no hair cells expressed gK,L, 33% were classified as type I by ultrastructural criteria. By P28, approximately 60% of hair cells in acute preparations received calyx terminals and expressed gK,L. Data from the denervated cultures showed that neither electrophysiological nor morphological differentiation depended on ongoing innervation.

Calcitonin gene-related peptide immunoreactivity in the rat efferent vestibular system during development.

The organization of the efferent fiber network during postnatal development was investigated by immunocytochemical detection of the calcitonin gene-related peptide (CGRP) in rat vestibular receptors from postnatal day 0 (PD 0) to adulthood. CGRP was detected at birth in a few efferent fibers below the sensory epithelia of cristae and maculae. Thereafter, the nerve fibers in the cristae progressively invaded the epithelia with an apex to base gradient from PD 2 to PD 4. There was also a rearrangement of the fibers during maturation of the efferent innervation, such that after reaching the surface of the epithelium, they turned back and moved towards the base of the sensory cells, producing numerous synaptic contacts. Analysis of surface preparations of utricules showed the irregular and asymmetric topographic organization of the efferent fiber network and the extensive, complex distribution of this innervation. The presence and broad distribution of CGRP in the epithelium at critical stages of development and synaptogenesis suggests that it is involved in the maturation of vestibular receptors.

Immunocytochemical localization of calmodulin in the vestibular end-organs of the gerbil.

This study demonstrates the presence of calmodulin in the vestibular end-organs of the gerbil by use of immunocytochemistry. Using fluorescence microscopy, calmodulin was localized to the cytoplasm, cuticular plate, and stereocilia of both type I and type II hair cells in the sensory epithelia of the utricle and cristae ampullaris; no label was found in the supporting cells, the dark cells, or the nerve fibers. There was no immunoreactive distinction between the labeling of type I and type II hair cells in the striolar or extrastriolar regions. Thus, immunocytochemical labeling for calmodulin provides a good marker for hair cells in gerbil vestibular epithelium. The presence of calmodulin in the stereocilia was confirmed by immunoelectron microscopy using secondary antibodies coupled to colloidal gold.

The combined effects of trkB and trkC mutations on the innervation of the inner ear.

Previous research has demonstrated that only the two neurotrophins and their cognate receptors are necessary for the support of the inner ear innervation. However, detailed analyses of patterns of innervation in various combinations of neurotrophin receptor mutants are lacking. We provide here such an analysis of the distribution of afferent and efferent fibers to the ear in various combinations of neurotrophin receptor mutants using the lipophilic tracer Dil. In the vestibular system, trkC+/- heterozygosity aggravates the trkB-/- mutation effect and causes almost complete loss of vestibular neurons. In the cochlea innervation, various mutations are each characterized by specific topological absence of spiral neurons in Rosenthal's canal of the cochlea. trkC-/- mutation alone or in combination with trkB+/- heterozygosity causes absence of all basal turn spiral neurons and afferent fibers extend from the middle turn to the basal turn along inner hair cells with little or no contribution to outer hair cells. Both types of basal turn spiral neurons appear to develop and project via radial fibers to inner and, more sparingly, outer hair cells. Simple trkB-/- mutations show a reduction of fibers to outer hair cells in the apex and, less obvious, in the basal turn. Basal turn spiral neurons may be the only neurons present at birth in the cochlea of a trkB-/- mutant mouse combined with trkC+/- heterozygosity. In addition, the trkB-/- mutation combined with trkC+/- heterozygosity has a patchy and variable loss of middle turn spiral neurons in mice of different litters. Comparisons of patterns of innervation of afferent and efferent fibers show a striking similarity of absence of fibers to topologically corresponding areas. For example, in trkC-/- mutants afferents reach the basal turn, spiraling along the cochlea, rather than through radial fibers and efferent fibers follow the same pathway rather than emanating from intraganglionic spiral fibers. The data presented suggest that there are regional specific effects with some bias towards a specific spiral ganglion type: trkC is essential for support of basal turn spiral neurons whereas trkB appears to be more important for middle and apical turn spiral neurons.

Distribution of ionocytes in the saccular epithelium of the inner ear of two teleosts (Oncorhynchus mykiss and Scophthalmus maximus).

The saccular membranes of trout (Oncorhynchus mykiss) and turbot (Scophthalmus maximus) were examined to characterize specialized epithelial cells that might be responsible for ion exchange. The approach for localizing cell types was new for this tissue, as observations were made with a stereomicroscope and a light microscope in order to have a general view of the epithelium. No important differences between the two species were seen. The saccular tissue is a monolayer epithelium (except for the macula neural zone) surrounded by a layer of connective tissue invaded by many blood vessels. The use of the fluorescent probe DAPSMI and zinc iodide/osmium fixation-coloration defined two areas in which ionocytes were present. In the first, large ionocytes were grouped into a nearly complete, crowned meshwork around, but separated from, the macula. In the second area, opposite the macula, the ionocytes were smaller, cubical, and grouped in patches. Cells rich in Na+, K+-ATPase and carbonic anhydrase II were present in both areas. Contrary to previous studies in mammals and fish, ionocytes were also found in the epithelium of the saccule.

Identification of a structural constituent and one possible site of postembryonic formation of a teleost otolithic membrane.

A gelatinous otolithic membrane (OM) couples a single calcified otolith to the sensory epithelium in the bluegill sunfish (Lepomis macrochirus) saccule, one of the otolithic organs in the inner ear. Though the OM is an integral part of the anatomic network of endorgan structures that result in vestibular function in the inner ear, the identity of the proteins that make up this sensory accessory membrane in teleosts, or in any vertebrate, is not fully known. Previously, we identified a cDNA from the sunfish saccular otolithic organ that encoded a new member of the collagen family of structural proteins. In this study, we examined biochemical features and the localization of the saccular collagen (SC) protein in vivo using polyclonal antisera that recognize the noncollagenous domains of the SC protein. The SC protein, in vivo, was identified as a 95-kDa glycoprotein in sunfish whole-saccule lysate and in homogenates of microdissected saccular OMs. Immunohistochemical analyses demonstrated that the SC protein was localized within one of the two distinct layers of the sunfish saccular OM. The SC protein was also detected within the cytoplasm of supporting cells at the edges of the saccular sensory epithelium, indicating that these cells are a primary site for the synthesis of this structural protein. Further studies of the organization of this matrix molecule in the OM may help clarify the role of this sensory accessory membrane in vestibular sensory function.

Receptors for glucocorticoids in the human inner ear.

Glucocorticoid receptors were detected in the human inner ear. The highest concentration of glucocorticoid receptor protein was measured by enzyme-linked immunosorbent assay in the spiral ligament tissues; the lowest concentration of glucocorticoid receptors was measured in the macula of the saccule. The demonstration of the presence of glucocorticoid receptors in human Inner ear tissues provides a basis to consider the direct effects of glucocorticoid action on select inner ear cells, rather than assuming a systemic antiinflammatory or immunosuppressive effect during the therapeutic treatment of patients with given inner ear disorders.