Identification of cisplatin-binding proteins using agarose conjugates of platinum compounds.

Cisplatin is widely used as an antineoplastic drug, but its ototoxic and nephrotoxic side-effects, as well as the inherent or acquired resistance of some cancers to cisplatin, remain significant clinical problems. Cisplatin's selectivity in killing rapidly proliferating cancer cells is largely dependent on covalent binding to DNA via cisplatin's chloride sites that had been aquated. We hypothesized that cisplatin's toxicity in slowly proliferating or terminally differentiated cells is primarily due to drug-protein interactions, instead of drug-DNA binding. To identify proteins that bind to cisplatin, we synthesized two different platinum-agarose conjugates, one with two amino groups and another with two chlorides attached to platinum that are available for protein binding, and conducted pull-down assays using cochlear and kidney cells. Mass spectrometric analysis on protein bands after gel electrophoresis and Coomassie blue staining identified several proteins, including myosin IIA, glucose-regulated protein 94 (GRP94), heat shock protein 90 (HSP90), calreticulin, valosin containing protein (VCP), and ribosomal protein L5, as cisplatin-binding proteins. Future studies on the interaction of these proteins with cisplatin will elucidate whether these drug-protein interactions are involved in ototoxicity and nephrotoxicity, or contribute to tumor sensitivity or resistance to cisplatin treatment.

MRNA expression of members of the IGF system in the organ of Corti, the modiolus and the stria vascularis of newborn rats.

We analyzed the mRNA expression of the insulin-like growth factor (IGF) family genes and of selected downstream pathway genes using the Affymetrix microarray system and confirmatory RT-PCR in the freshly prepared organ of Corti (OC), modiolus (MOD) and stria vascularis (SV) from neonatal rats (3-5 days old) and after 24h in culture. Among the seven members of the IGF family analyzed in this paper, IGF1, IGF2 and IGF-binding protein (IGFBP2) had the highest basal expression in all regions. Preparatory stress and culture increased the expression of IGF2, IGFBP2, IGFBP3, IGFBP5, glucose transporterl (GLUT1), signal transducer, and activator of transcription3 (STAT3), phosphoinositide-3-kinase regulatory subunit (Pik3r1), Jun oncogene (c-jun) and decreased that of mitogen-activated protein kinases MAPK3 and MAPK14 in all regions. Region-specific changes were observed in OC (GLUT1), MOD (IGFBP3 and c-jun) and SV (IGF2 and IGFBP2).

Synaptophysin immunohistochemistry in the human cochlea.

Light microscopy and immunohistochemical analyses of a freshly prepared human cochlea, removed at meningioma skull base surgery, were performed with particular emphasis on synaptophysin (SY) reactivity. Synaptophysin, a 38-kDa glycoprotein, is one of the most abundant integral membrane proteins of small presynaptic vesicles and is a useful marker for sites of synaptic transmission of the efferent olivocochlear system in the cochlea. Following fixation and decalcification, cryosections of 30 microm were prepared. To introduce immunostaining, free-floating sections were exposed to monoclonal SY antibody. Positive SY immunostaining was solely restricted to the neural and sensory structures and did not include supporting cells of the organ of Corti. Dense reaction products were noted around the hair cells, especially at the basal portion of the inner and outer hair cells and their neural poles, as well as around the inner spiral bundle, tunnel spiral bundle, outer spiral bundle and upper tunnel crossing fibers. The majority of spiral ganglion cells stained positively. An intermingling network of thin unmyelinated nerve fibers stained densely, especially at the basal portions of the cochlea. The spiral limbus, inner and outer sulcus cells, basilar membrane, myelinated nerve fibers, spiral ligament and the stria vascularis were unstained. Human cochlea obtained during surgery offers excellent conditions for immunohistochemical analysis. In the basal cochlea in the organ of Corti, outer hair cell area, there may be alterations due to noise trauma from the drilling procedure.

Localization of the P2Y4 receptor in the guinea pig organ of Corti.

Cochleae of guinea pigs were evaluated for the presence of the metabotropic receptor, P2Y4. Evidence is presented that P2Y4 protein is expressed in the guinea pig cochleae using Western blot analysis. A single protein band of 35 kDa was detected with P2Y4 receptor-specific antibody. The cellular distribution of P2Y4 purinoceptor protein was determined by immunohistochemistry of the whole organ of Corti. Immunoreactive staining for P2Y4 was seen in most cells of the organ of Corti. Staining of Hensen's cells and Deiters' cells, especially the outer Deiters' cells, was more intense than staining of the outer hair cells, inner hair cells, and pillar cells. Staining intensity was greatest at the basal turn and progressively decreased in the upper turns with the apex showing the weakest staining pattern. This is the first demonstration of a metabotropic P2Y receptor in the guinea pig organ of Corti.

Comparative expression patterns of T-, N-, E-cadherins, beta-catenin, and polysialic acid neural cell adhesion molecule in rat cochlea during development: implications for the nature of Kölliker's organ.

We investigated the expression patterns of several cell adhesion molecules (CAMs) during rat cochlea ontogeny, from embryo day 16 to adulthood, with the use of immunohistochemistry: neural cadherin (N-cad) and polysialic acid neural CAM (PSA-NCAM) as two different neural CAM paradigms; epithelial cadherin (E-cad), which was restricted to the epitheloid phenotype; and the cytoplasmic domain-free truncated-cadherin (T-cad). We made the following observations. (1) T-cad was present in all types of fibrocyte and in subdomains within the pillar cells. (2) E- and N-cad were expressed with mutually exclusive patterns and did not overlap with T-cad. All cochlear epithelial cells, including the sensory outer hair cells (OHCs), were E-cad-positive, except for the negative inner hair cells (IHCs) and the nonsensory Kölliker's organ domain close to the IHCs. N-cad expression appeared first in the developing IHCs and then in the neighboring Kölliker's organ in an increasingly mediolateral gradient in opposition to the E-cad gradient. The OHCs, which are never N-cad positive, intensively expressed E-cad, as did the Hensen cells at the beginning of their differentiation. (3) The cadherin-linked molecule beta-catenin, absent in fibrocytes, was detected in all epithelial cell membranes and was prominent in the E-cad-rich modiolar extremity of Kölliker's organ. (4) Gradual PSA-NCAM expression was observed in the lateral portion of Kölliker's organ, and the intense PSA-NCAM expression was seen surrounding the IHCs. As development proceeded, PSA-NCAM immunoreactivity progressively became restricted to the basal poles of the IHCs, where it remained in the adult rat cochlea, suggesting a synaptic plasticity. Synaptic plasticity in rat cochlea and hypotheses about T-cad functions and neosensory features of the Kölliker's organ are discussed.

Specific proteins of the organ of Corti.

The mammalian organ of Corti has achieved a degree of perfection unequaled in other hair cell systems. Although cellular metabolism requires the coordinated action of thousands of proteins, the physical processes underlying auditory transduction in the OC are undoubtedly mediated by a much smaller subset of these. OCP1, OCP2, and CBP-15-identified by 2D-PAGE-are apparently members of this elite class. OCP1 and OCP2 are restricted to the supporting cells of the organ of Corti and adjacent epithelia. Their distribution closely parallels the boundaries of the epithelial gap junction system, implying a role in cochlear potassium and pH homeostasis. CBP-15 was recently shown to be identical to oncomodulin, the mammalian beta-parvalbumin, heretofore documented only in the placenta and neoplasms. Expression of this small calcium-binding protein in the OC is restricted to the outer hair cells, where it may function as a calcium-dependent regulatory protein.

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.

Localization of bcl-2, bax, and bcl-x mRNAs in the developing inner ear of the mouse.

In situ hybridization was employed to study the expression of bcl-2 mRNA and its family members, bax and bcl-x mRNAs, in the developing inner ear. We found that in the cochlear structure, sensory epithelial cells, the spiral ganglion and stria vascularis expressed these mRNAs in postnatal period in a temporally similar manner, but in embryos, neither bax nor bcl-x mRNA were expressed in the sensory epithelium from embryonic day (E) 13 to 19. In contrast to these patterns, bcl-2 mRNA was expressed by E15 to E19, and the expression at E13 was below the lower limit of detection. Non-neuronal tissue (stria vascularis) also expressed these three transcripts during development. These results suggest that bcl-2 family members may be differentially involved in the differentiation of sensory epithelial cells, spiral ganglia, and stria vascularis. In particular, the differential expression patterns in the cochleovestibular neurons suggest that proliferating and differentiating neurons utilize distinct members of the bcl-2 family.

Expression of Na, K-ATPase alpha and beta isoforms in the neonatal rat cochlea.

The postnatal expression of five Na, K-ATPase alpha (alpha 1, alpha 2, alpha 3) and beta (beta 1, beta 2) subunit isoforms in the rat cochlea was investigated by immunocytochemistry. High levels of expression of the alpha 1 and beta 2 isoforms were observed in stria vascularis (SV) at all developmental stages. alpha 1 and beta 1 isoforms showed a distinct time-dependent developmental expression pattern in tissues of the spiral ligament (SL) and spiral limbus (SLi). Limited, temporary expression of alpha 2 and alpha 3 subunit isoforms were found in SV and SL. Expression of each isoform was also seen in organ of Corti (OC), spiral ganglion (SG), cochlear nerve (CN) and Kölliker's Organ (KO). These observations suggest that individual isoforms may exert specific actions postnatally during final cochlear maturation.

Gamma-aminobutyric acidA-receptor messenger ribonucleic acid (alpha-1 subunit) detection by in situ hybridization.

The inhibitory neurotransmitter gamma-aminobutyric acid (GABA) is most likely involved in the efferent cochlear neurotransmission. In situ hybridization (ISH) results in specific annealing of a labelled nucleic acid probe to complementary sequences in fixed tissue and allows subsequent visualization of the location of the probe. We used the ISH technique to localize messenger ribonucleic acid (mRNA) sequences of the alpha-1 subunit of the GABAA receptor with an S-35 labeled oligonucleotide probe. Experiments were performed in rat and guinea pig brain sections and surface tissue preparations of the guinea pig cochlea. Positive signals were obtained for the alpha-1 probe in cortical and hippocampal regions of the rat brain and had weaker expression in the guinea pig brain. Alpha-1 subunit mRNA was localized in Purkinje cells and in stellate and basket cells of the stratum moleculare in the rat and guinea pig cerebellum. In surface tissue preparations of the guinea pig cochlea mRNA sequences of the alpha-1 subunit were detectable with high signal expression. Positive signals were seen on both sides of the tunnel of Corti, predominantly in the region of the outer hair cells. The results indicate expression of GABAA-receptor mRNA in cochlear tissue, supporting the importance of GABAA receptors in cochlear neurotransmission.

The localization of synaptophysin in the organ of Corti of the human as shown by immunoelectron microscopy.

Synaptophysin, or p38, a polypeptide of molecular weight 38 kD, is a calcium-binding membrane protein found in synaptic vesicles of neurons and smooth surfaced vesicles of neuroendocrine cells. Six human neonatal and infant temporal bones were fixed in paraformaldehyde and glutaraldehyde, decalcified in EDTA and were than immunoreacted for synaptophysin (ICN Biomedicals) using the avidin-biotin reaction (ABC kit, Vector Labs). The tissue was then prepared for light microscopic surface preparation, radial sections of 5 microns, and serial section electron microscopy. At a light microscopic level, the inner spiral bundle, tunnel spiral bundle, upper tunnel crossing fibers and the base of outer hair cells were stained. At the base of outer hair cells, the immunoreactivity was seen to decrease from the base to the apex and from the first to third outer hair cells. At an electron microscopic level, immunoreactivity at the base of outer hair cells was limited to vesiculated efferent fibers. The degree of immunoreactivity between adjacent efferent fibers varied significantly. Immunoreactive vesiculated endings were also found in the supranuclear region of outer hair cells.

Hearing and glycoconjugates: localization of Le(y), Le(x) and sialosyl-Le(x) in guinea pig cochlea, particularly at the tectorial membrane and sensory epithelia of the organ of Corti.

Immunohistological examination of guinea pig cochleas was performed using a panel of 25 monoclonal antibodies directed to various lacto-, ganglio- and globo-series carbohydrate epitopes as well as mucin-type epitopes. Lacto-series structures were found to be localized at specific sites of the tectorial membrane (TM) and Corti's organ, i.e. alpha 1-->3 fucosyl type 2 chain (Le(x)) at Kimura's membrane, marginal band and covering net of TM; alpha 1-->2, alpha 1-->3 difucosyl type 2 chain (Le(y)) at covering net; and sialosyl-Le(x) and sialosyl-i at Kimura's membrane and sensory epithelia, particularly sensory tips of hair cells of Corti's organ. In striking contrast, ganglio-series structures (GM3, GD3, GD2, 9-O-Ac-GD3) were detected at spiral ganglion cells, neuronal fibres and stria vascularis, but were completely absent from Corti's organ and most of the TM. Other epitope structures defined by various antibodies were not detectable at any location. The functional roles of lacto-series carbohydrate epitopes expressed at TM and Corti's organ remain unknown. However, the expression of Le(y) (but not other structures) in association with developmental deficiency of TM induced by 6-N-propyl-2-thiouracil in rats suggests that Le(y) plays some role in normal TM development. The presence of Le(x) at Kimura's membrane and sialosyl-Le(x) at hair cell sensory tips of Corti's organ suggests the intriguing possibility that these fucosylated/sialosylated carbohydrate structures play some role in interactions (either attractive or repulsive) of these inner ear components, which have been implicated in the physiology of hearing, i.e. the conversion of sound waves to nerve impulses.

Construction of a cDNA library from microdissected guinea pig organ of Corti.

Poly (A) RNA was isolated from microdissected guinea pig organ of Corti and converted into cDNA with RNase H- murine leukemia virus reverse transcriptase. After size fractionation, the cDNA was directionally ligated into the vector pSPORT 1 and the plasmids were transformed into DH10B E. coli via electroporation. The library was found to have 3.35 x 10(6) independent colonies with ten percent of the colonies lacking an insert. After checking 33 randomly selected colonies for inserts, the average insert size was 1218 base pairs, ranging from 3300 base pairs to 400 base pairs. The library was screened with a beta-actin oligonucleotide probe and 1.4% of the colonies contained an insert hybridizing to the probe.

Expression of cytokeratin polypeptides during development of the rat inner ear.

The expression of cytokeratin polypeptides in the different epithelia of the developing inner ear of the rat from 12 days post conception to 20 days after birth was analysed immunohistochemically, using a panel of monoclonal antibodies. Throughout the development of the complex epithelial lining of the inner ear originating from the otocyst epithelium, only cytokeratins which are typical of simple epithelia were expressed. Cytokeratins 8, 18, and 19 were detectable shortly after the formation of the otocyst from the ectoderm (12 dpc), whereas cytokeratin 7 expression was delayed and first appeared in the vestibular portion and subsequently in the developing cochlear duct. During the development of the different types of specialized cells, differentiation-dependent modulation of the cytokeratin expression patterns was observed. In the mature inner ear, the specialized cell types displayed a function-related cytokeratin expression profile, both in the cochlear and vestibular portion. Cytokeratin expression in the flat epithelium of the vestibular portion suggests a more complex composition of this epithelium than has been established from routine morphology. Remarkably, the cochlear sensory cells were apparently devoid of cytokeratins, but no final conclusion could be drawn on the presence of cytokeratins in the sensory cells of the vestibular portion, because of the difficulty to delineate the cell borders between sensory cells and supporting cells.

Cytokeratin expression in the epithelia of the adult human cochlea.

Epithelia can be characterized by the specific expression pattern of their cytokeratin components. Therefore, we investigated the immunohistochemical expression of different cytokeratin subunits in frozen sections of chemically fixed, non-decalcified, adult human cochleas. The organ of Corti and the marginal cells of the stria vascularis showed reactivity for cytokeratin subunits 8, 18 and 19, whereas the other cochlear epithelia in addition expressed cytokeratin 7. The expression of cytokeratins 7, 8, 18 and 19 by the epithelia of the adult human cochlea is typical of "simple" epithelia. The deviant cytokeratin pattern of the organ of Corti and marginal cells of the stria vascularis may well reflect their differences in functional state and/or differentiation as compared to the other cochlear epithelia.

Partial amino acid sequence of organ of Corti protein OCP-II.

We have previously described two low-molecular-weight, highly acidic proteins which are present in the organ of Corti in extremely high concentrations. Since the function of these proteins is not known, they have been assigned the tentative names OCP-I and OCP-II. In the hope of obtaining information about their function through homology studies, we have initiated amino acid sequencing of these proteins. We have recently succeeded in obtaining a brief amino-terminal sequence of OCP-II. We now report on a significant extension of the amino-terminal sequence of OCP-II and our first results on sequences of peptide fragments obtained by limited digestion with V8 protease. Together, the sequenced segments account for about one-third of the total sequence. Comparisons with the sequences of known proteins suggest that OCP-II is not a structural protein, but that it may exhibit biologic activity.