Encapsulation of Au Nanoparticles on a Silicon Wafer During Thermal Oxidation.

We report the behavior of Au nanoparticles anchored onto a Si(111) substrate and the evolution of the combined structure with annealing and oxidation. Au nanoparticles, formed by annealing a Au film, appear to "float" upon a growing layer of SiO2 during oxidation at high temperature, yet they also tend to become partially encapsulated by the growing silica layers. It is proposed that this occurs largely because of the differential growth rates of the silica layer on the silicon substrate between the particles and below the particles due to limited access of oxygen to the latter. This in turn is due to a combination of blockage of oxygen adsorption by the Au and limited oxygen diffusion under the gold. We think that such behavior is likely to be seen for other metal-semiconductor systems.

Na-K-Cl cotransporter expression in the developing and senescent gerbil cochlea.

Changes in the cellular expression pattern of the Na-K-Cl cotransporter (NKCC) were investigated during postnatal development and with advancing age in the gerbil cochlea. At birth, faint immunostaining for NKCC was discernable in the developing stria vascularis (StV), Reissner's membrane, interdental cells and some relatively undifferentiated cells lining the cochlear partition. Between 2 and 4 days after birth (DAB) immunostaining persisted and increased in the future interdental, inner and outer sulcus and claudius cells but then disappeared from these sites by 8 DAB. In contrast, NKCC immunoreactivity in the StV increased progressively during development and approached adult levels by 12 DAB. Immunostaining for NKCC in subpopulations of fibrocytes in the inferior portion of the spiral ligament, the suprastrial region and the spiral limbus was first detectable between 10 and 12 DAB and staining intensity reached adult levels around 16 DAB. Changes in NKCC expression with advancing age generally mimicked those previously observed for Na,K-ATPase in focal regions of atrophic lateral wall. Diminished immunostaining was first seen in the StV, presumably associated with the involution of the marginal cell's basolateral processes. Further atrophy culminated in complete loss of immunostaining in the StV and an associated down-regulation of NKCC expression in spiral ligament transport fibrocytes. The marked similarities in the developmental and age-related expression patterns of NKCC and Na,K-ATPase point to a high level of functional cooperativity between these two ion transport mediators, which together provide an efficient mechanism for generating and maintaining high K+ levels in endolymph and the endocochlear potential.

Immunohistochemical localization of the Na-K-Cl co-transporter (NKCC1) in the gerbil inner ear.

We mapped the cellular and subcellular distribution of the Na-K-Cl co-transporter (NKCC) in the adult gerbil inner ear by immunostaining with a monoclonal antibody (MAb T4) generated against human colon NKCC. Heavy immunolabeling was seen in the basolateral plasma membrane of marginal cells in the stria vascularis and dark cells in the vestibular system. Subpopulations of fibrocytes in the cochlear spiral ligament and limbus and underlying the vestibular neurosensory epithelium also stained with moderate to strong intensity, apparently along their entire plasmalemma. Because MAb T4 recognizes both the basolateral secretory (NKCC1) and the apical absorptive (NKCC2) isoforms of the co-transporter, we employed reverse transcription and the polymerase chain reaction (RT-PCR) to explore isoform diversity in inner ear tissues. Using NKCC1 and NKCC2 isoform-specific PCR primers based on mouse and human sequences, only transcripts for NKCC1 were detected in the gerbil inner ear. The presence of abundant NKCC1 in the basolateral plasmalemma of strial marginal and vestibular dark cells confirms conclusions drawn from pharmacological and physiological data. The co-expression of NKCC1 and Na,K-ATPase in highly specialized subpopulations of cochlear and vestibular fibrocytes provides further evidence for their role in recycling K+ leaked or effluxed through hair cells into perilymph back to endolymph, as postulated in current models of inner ear ion homeostasis.

Identification and cloning of site C splice variants of plasma membrane Ca-ATPase in the gerbil cochlea.

Plasma membrane Ca-ATPase (PMCA) gene products were identified in the gerbil cochlea by reverse-transcription polymerase chain reaction (RT-PCR). Cochlear cDNA was amplified using PMCA isoform-specific primers from splice site C, the calmodulin binding domain. PCR products were cloned and sequenced. The putative housekeeping PMCA genes, 1b and 4b, as expected, were present in the gerbil cochlea and shared 98.6 and 100% amino acid homology with published rat sequences, at splice site C, respectively. PMCA2b, 3a and 3b splice variants also were detected in cochlear cDNAs and shared 95, 94.3 and 98% amino acid homology with their rat counterparts. PMCA isoforms 2 and 3 have been shown to occur in highly specialized tissues, such as muscle and brain, that require finely tuned regulation of intracellular free Ca2+ levels. The presence of several isoforms and splice variants of PMCA in the cochlea most probably reflects their differential expression among the several cell types that have been shown to contain immunoreactive PMCA. This suggests that the cochlea has developed complex mechanisms that finely tune intracellular Ca2+ levels in different highly specialized cell types.

Expression of plasma membrane Ca-ATPase in the adult and developing gerbil cochlea.

The distribution of the plasma membrane Ca-ATPase (PMCA) was mapped in the adult and developing gerbil cochlea by immunostaining with a monoclonal antibody against the human erythrocyte PMCA. In the mature cochlea, intense immunoreactivity was present at the surface of stereocilia of both inner (IHC) and outer (OHC) hair cells. The basolateral plasma membrane of IHCs but not OHCs stained strongly whereas that of strial marginal cells and the epithelial cell layer of Reissner's membrane showed only weak reactivity. Nerve terminals underlying IHCs were also selectively stained. At birth, strong to moderate reactivity for PMCA was present in the basolateral plasma membrane of IHCs and OHCs, strial marginal cells, and epithelial cells lining the scala media surface of Reissner's membrane and in the neurolemma of spiral ganglion cells. Immunostaining in the basolateral plasmalemma of OHCs, strial marginal cells, and epithelial cells lining Reissner's membrane remained strong to moderate up to 14 days after birth when it diminished or disappeared entirely, suggesting a developmental role for PMCA activity in these sites. Expression of PMCA at the surface of IHC and OHC stereocilia was first observed at 10 days after birth and staining reached adult levels by 14 days after birth. The abundance of PMCA in the stereociliary plasma membrane of mature hair cells supports the suggested involvement of Ca2+ in regulating transduction and adaptation mechanisms.