The tight regulation of Ca2+ is essential for inner ear function, and yet the role of Ca2+ binding proteins (CaBPs) remains elusive. Using immunofluorescence and RT-PCR, we investigated the expression of oncomodulin (Ocm), a member of the parvalbumin family, relative to other EF-hand CaBPs in cochlear and vestibular organs in the mouse. In the mouse cochlea, Ocm is found only in outer hair cells and is localized preferentially to the basolateral outer hair cell membrane and to the base of the hair bundle. Developmentally, Ocm immunoreactivity begins as early as postnatal day (P) 2 and shows preferential localization to the basolateral wall and hair bundle after P8. Unlike the cochlea, Ocm expression is substantially reduced in vestibular tissues at older adult ages. In vestibular organs, Ocm is found in type I striolar or central hair cells, and has a more diffuse subcellular localization throughout the hair cell body. Additionally, Ocm immunoreactivity in vestibular hair cells is present as early as E18 and is not obviously affected by mutations that cause a disruption of hair bundle polarity. We also find Ocm expression in striolar hair cells across mammalian species. These data suggest that Ocm may have distinct functional roles in cochlear and vestibular hair cells.

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The protein responsible for both nucleotide pyrophosphatase (EC 3.6

Here we describe preparatory techniques adapted for the study of proteins of inner ear tissues and fluids that have allowed us to apply state-of-the-art analytical techniques in spite of the minute size and anatomical complexities of this organ. Illustrative examples address unresolved issues of functional and clinical significance. First, we demonstrate how quick-freezing and freeze drying prevents artifacts that arise from sampling endolymphatic sac (ES) content in the liquid state. This set the stage for the generation of the first protein profile of the ES. Identification of crucial proteins will help elucidate mechanisms of endolymph volume regulation and pathogenesis of Meniere's disease. Second, we show how a unique situation allowed identification of otoconial proteins by mass spectrometric analysis without prior separation and we discuss possible roles for these minor otoconins in otoconial development and prevention of degenerative diseases that affect balance. Finally, we demonstrate techniques for the precise dissection of organ of Corti and its substructures, while preserving their near normal chemical state. We extended an earlier study in which we identified a novel calcium-binding protein by IEF, oncomodulin, localized in the outer hair cells and show here the applicability of prefractionation for the screening of calcium-binding proteins of organ of Corti. These studies demonstrate how advanced preparatory and analytical techniques can be applied to studies of the inner ear.

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Endothelial cells, which line the interior walls of blood vessels, proliferate at the site of blood vessel injury. Knowledge of the factors that control the proliferation of these cells would help elucidate the role of endothelial cells in wound healing, tumor growth, and arteriosclerosis. In vitro, endothelial cells organize into viable, three-dimensional tubular structures in environments that limit cell proliferation. The process of endothelial cell organization was found to result in decreased levels of the sis messenger RNA transcript and increased levels of the messenger RNA transcript for fibronectin. This situation was reversed on transition from the organized structure to a proliferative monolayer. These results suggest a reciprocity for two biological response modifiers involved in the regulation of endothelial cell proliferation and differentiation in vitro.

Human platelet-derived growth factor (PDGF) is a potent mitogenic polypeptide which is believed to be a heterodimer of A- and B-chains stabilized by interchain disulphide bonds. The B-chain of PDGF is encoded by the c-sis gene, the normal cellular homologue of the transforming gene of the simian sarcoma virus (SSV). cDNA clones of the B-chain from both normal and transformed cells have mutually consistent DNA sequences. Recently, an A-chain cDNA clone (D-1) was isolated from a transformed human glial cell cDNA library. We report the complete sequence of an A-chain cDNA clone (BT-1) isolated from a normal human umbilical vein endothelial (HUVE) cell cDNA library. BT-1 differs from the sequence of the D-1 clone by a 69 base pair deletion containing the predicted carboxy terminus of the protein. The mRNA levels of the A- and B-chains of PDGF in HUVE cells were analysed and shown to respond differently to the endothelial cell growth factor (ECGF).

Sequences are reported for portions of two mitochondrial genes from a domestic horse and a plains zebra and compared to those published for a quagga and a mountain zebra. The extinct quagga and plains zebra sequences are identical at all silent sites, whereas the horse sequence differs from both of them by 11 silent substitutions. Postmortem changes in quagga DNA may account for the two coding substitutions between the quagga and plains zebra sequences. The hypothesis that the closest relative of the quagga is the domestic horse receives no support from these data. From the extent of sequence divergence between horse and zebra mitochondrial DNAs (mtDNAs), as well as from information about the fossil record, we estimate that the mean rate of mtDNA divergence in Equus is similar to that in other mammals, i.e., roughly 2% per million years.