Uptake of Gentamicin by Bullfrog Saccular Hair Cells in vitro

Vertebrate sensory hair cells in the inner ear are pharmacologically sensitive to aminoglycoside antibiotics. Although the ototoxicity of aminoglycosides is well known, the route of drug uptake by hair cells and mechanisms of cytotoxicity remain poorly understood. Previously published studies have documented the intracellular distribution of gentamicin using immunocytochemical, electron microscopic, and autoradiographic methods. In this article, we compare the subcellular distribution of fluorescently conjugated gentamicin (gentamicin–Texas Red, GTTR) with immunolabeled gentamicin using confocal or electron microscopy. Gentamicin (detected by postfixation immunocytochemistry) and GTTR were rapidly taken up by hair cells throughout the bullfrog saccular explant in vitro and preferentially in peripheral hair cells. Immunolabeled gentamicin and GTTR were observed at the apical membranes of hair cells, particularly in their hair bundles. GTTR was also identified within a variety of subcellular compartments within hair cells, including lysosomes, mitochondria, Golgi bodies, endoplasmic reticulum, and nuclei, and in similar structures by immunoelectron microscopy. The distributions of GTTR and immunolabeled gentamicin are largely identical and corroborate a variety of published immunocytochemical and autoradiography studies. Thus, GTTR is a valid fluorescent probe with which to investigate the pharmacokinetics and mechanisms of gentamicin accumulation

Noncoding Mutations of HGF Are Associated with Nonsyndromic Hearing Loss, DFNB39

A gene causing autosomal-recessive, nonsyndromic hearing loss, DFNB39, was previously mapped to an 18 Mb interval on chromosome 7q11.22-q21.12. We mapped an additional 40 consanguineous families segregating nonsyndromic hearing loss to the DFNB39 locus and refined the obligate interval to 1.2 Mb. The coding regions of all genes in this interval were sequenced, and no missense, nonsense, or frameshift mutations were found. We sequenced the noncoding sequences of genes, as well as noncoding genes, and found three mutations clustered in intron 4 and exon 5 in the hepatocyte growth factor gene (HGF). Two intron 4 deletions occur in a highly conserved sequence that is part of the 3′ untranslated region of a previously undescribed short isoform of HGF. The third mutation is a silent substitution, and we demonstrate that it affects splicing in vitro. HGF is involved in a wide variety of signaling pathways in many different tissues, yet these putative regulatory mutations cause a surprisingly specific phenotype, which is nonsydromic hearing loss. Two mouse models of Hgf dysregulation, one in which an Hgf transgene is ubiquitously overexpressed and the other a conditional knockout that deletes Hgf from a limited number of tissues, including the cochlea, result in deafness. Overexpression of HGF is associated with progressive degeneration of outer hair cells in the cochlea, whereas cochlear deletion of Hgf is associated with more general dysplasia