2'-hydroxy C16-ceramide induces apoptosis-associated proteomic changes in C6 glioma cells.

Ceramide is a bioactive sphingolipid involved in regulation of numerous cell signaling pathways. Evidence is accumulating that differences in ceramide structure, such as N-acyl chain length and desaturation of sphingoid base, determine the biological activities of ceramide. Using synthetic (R)-2'-hydroxy-C16-ceramide, which is the naturally occurring stereoisomer, we demonstrate that this ceramide has more potent pro-apoptotic activity compared to its (S) isomer or non-hydroxylated C16-ceramide. Upon exposure to (R)-2'-hydroxy-ceramide, C6 glioma cells rapidly underwent apoptosis as indicated by caspase-3 activation, PARP cleavage, chromatin condensation, and annexin V stain. A 2D gel proteomics analysis identified 28 proteins whose levels were altered during the initial 3 h of exposure. Using the list of 28 proteins, we performed a software-assisted pathway analysis to identify possible signaling events that would result in the observed changes. The result indicated that Akt and MAP kinase pathways are among the possible pathways regulated by (R)-2'-hydroxy-ceramide. Experimental validation confirmed that 2'-hydroxy-ceramide significantly altered phosphorylation status of Akt and its downstream effector GSK3ß, as well as p38, ERK1/2, and JNK1/2 MAP kinases. Unexpectedly, robust phosphorylation of Akt was observed within 1 h of exposure to 2'-hydroxy-ceramide, followed by dephosphorylation. Phosphorylation status of MAPKs showed a complex pattern, in which rapid phosphorylation of ERK1/2 was followed by dephosphorylation of p38 and ERK1/2 and phosphorylation of the 46 kDa isoform of JNK1/2. These data indicate that (R)-2'-hydroxy-ceramide regulates multiple signaling pathways by affecting protein kinases and phosphatases with kinetics distinct from that of the extensively studied non-hydroxy-ceramide or its unnatural stereoisomer.

Unmyelinated auditory type I spiral ganglion neurons in congenic Ly5.1 mice.

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 compared with 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.

Mitochondria-activated cisternae generate the cell specific vesicles in auditory hair cells.

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.

Topical application of mitomycin C to the middle ear is ototoxic in the gerbil.

HYPOTHESIS: Mitomycin C is ototoxic when applied topically to the structures of the middle ear. BACKGROUND: Mitomycin C is a topically applied medication widely used in a variety of surgical procedures to prevent excessive scar tissue formation. Its safety for use during otologic procedures has not been fully evaluated. METHODS: A laboratory study was undertaken using the Mongolian gerbil as an animal model. Both acute and chronic effects on cochlear function of mitomycin C were assessed with measurements of compound action potential (CAP) thresholds of the auditory nerve, CAP input/output functions, distortion product otoacoustic emissions, and endocochlear potentials. Morphologic changes were assessed with light microscopy using hematoxylin-eosin staining as well as transmission electron microscopy. RESULTS: Five-minute applications of mitomycin C (0.5 mg/ml) to the entire surface of the middle ear adversely affected CAP thresholds, input/output functions, distortion product otoacoustic emissions, and the endocochlear potential. Ninety-minute exposures of mitomycin C solely to the round window produced similar changes. Histologic evaluation of animals 1 week after treatment showed damage to cochlear hair cells, the stria vascularis, and spiral ganglion neurons when compared with controls. CONCLUSION: Mitomycin C can produce substantial sensorineural hearing loss when applied topically to the gerbil middle ear for even brief periods. Consequently, its safety for topical use in the human middle ear is highly questionable.

Contribution of bone marrow hematopoietic stem cells to adult mouse inner ear: mesenchymal cells and fibrocytes.

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.

Presence of surfactant lamellar bodies in normal and diseased sinus mucosa.

BACKGROUND: Pulmonary surfactant originates from phospholipid lamellar bodies secreted from the type II epithelial cell of the alveolus. In the lower airway, surfactant optimizes surface tension and oxygen exchange, decreases mucus viscosity and aids in mechanical elimination of inhaled pathogens. In addition to the lung, lamellar bodies have been identified in many other cell types throughout the human body. However, no prior studies have identified lamellar bodies in human sinus mucosa. OBJECTIVES: We performed ultrastructural studies to assess whether lamellar bodies are present in the human sinus in a variety of diseased and normal epithelium. METHODS: We biopsied sinus mucosa from 5 subjects, 1 each with allergic fungal sinusitis, eosinophilic mucin rhinosinusitis, cystic fibrosis, frontal sinus mucocele, and cerebrospinal fluid leak (healthy control). Mouse lung served as a positive control. Specimens were prepared using ferrocyanide-reduced osmium tetroxide and thiocarbohydrazide for fixation (R-OTO method) to avoid extraction of phospholipids during dehydration and were viewed with transmission electron microscopy. RESULTS: We identified lamellar bodies in the sinus mucosa of all patients. Additionally, preservation of mouse lung lamellar bodies confirms that the R-OTO method is a valid technique to preserve these structures. CONCLUSIONS: We describe a simpler, faster technique for identification of cellular phospholipid components than those used previously. Definitive identification of these lamellar bodies within ciliated pseudostratified epithelium of the upper airway indicates that surfactant may have a role in sinus function and pathophysiology.