Expression of novel potassium channels in the chick basilar papilla

Ionic currents are critical for the functioning of the inner ear auditory sensory epithelium. We set out to identify and molecularly clone the genes encoding the channels responsible for several currents in the chick basilar papilla. Here we describe an inward-rectifying K+ channel, cKir2.3, present in both hair cells and support cells in the apical end of the chick basilar papilla. The biophysical properties of the human ortholog, hKir2.3, are similar to those of an inward-rectifying channel found in the apical end of the chick basilar papilla, suggesting that this channel may contribute to the corresponding current. Additionally, we describe two new members of the Kv6 subfamily of putative regulatory voltage-gated K channels, cKv6.2 and cKv6.3. Both are expressed in hair cells in the apical end of the chick basilar papilla; cKv6.2 is also strongly expressed in support cells and in the brain.     

The cuticular plate of the hair cell in relation to morphological gradients of the chicken basilar papilla

The aim of the present study was to provide details on the diversity and morphological gradients in the anatomy of the cuticular plate of hair cells in the chicken basilar papilla. The structure of the cuticular plate, which is mainly made up of a network of actin filaments, may be related to differences in the mechanical demands on the anchorage of the stereovillar bundle. We describe the morphological gradients in the cuticular plates as seen in transverse section for four positions along the basilar papilla. Three different shapes of cuticular plate could be distinguished. In general, cuticular plates in neurally-lying hair cells have their main mass on the neural side of the cells; for abneural cells, the converse is true. The shape of the plates changes gradually across the papilla; symmetrical forms exist. The hair-cell bundle orientation (and thus the preferred direction of stimulation of the bundle), as measured using scanning EM preparations, does not correlate with the shape of the plate in transverse section. The present data confirm the notion developed from other studies that (1) there are no distinct populations of hair cells, (2) there are no linear or monotonic morphological gradients, and (3) the gradients on the papilla are species- and position-specific.     

Embryogenesis and functional development of the chick''s basilar papilla

BACKGROUND: After laparoscopy with carbon dioxide (CO2) insufflation early postoperative recovery is often complicated with drowsiness and postoperative nausea and vomiting (PONV). METHODS: 25 ASA I-II patients undergoing elective laparoscopic cholecystectomy under standardized anaesthesia were studied in a randomized, prospective study. The conventional CO2 pneumoperitoneum was compared with the mechanical abdominal wall lift (AWL) method with minimal CO2 insufflation with special reference to postoperative recovery. RESULTS: Postoperative drowsiness was of a significantly longer duration with the conventional method (p < 0.001) compared with the AWL technique. There was a positive correlation with the total amount of CO2 used and the duration of drowsiness (r = 0.75, p < 0.01). PONV was seen significantly more often in patients with CO2 insufflation of more than 121 (p < 0.05). CONCLUSIONS: Avoiding excessive CO2 is beneficial for smoother and more uneventful recovery after laparoscopic cholecystectomy.     

Genotoxicity studies on professional hair colorists exposed to oxidation hair dyes

The cytogenic repercussions of occupational exposure to oxidation hair dyes were assessed by using three assays in professional hair colorists. The assays were sister chromatid exchanges (SCE) in circulating lymphocytes to evaluate the interchange of DNA replication products at apparently homologous chromosomal loci, single cell gel electrophoretic (SCGE) assay to detect the presence of DNA strand breaks/alkali-labile damage, and the Ames assay using Salmonella typhimurium strain TA98 to detect the urine mutagenicity. The ability of these assays to detect genetic damage caused by oxidation hair dyes in man compared with closely matched controls produced the following findings. (i) The SCE assay could not detect the mutagenic effect in lymphocytes of exposed subjects from whom complete data were obtained. However, subjects (controls and exposed) with a history of smoking had slightly increased SCEs than the non-smokers in both groups. (ii) The extent of DNA migration (SCGE assay) did not distinguish between the samples in either the exposed or control subjects. Like the SCE results, the exposed and control smoker subjects showed a greater proportion of damaged lymphocytes with apparent migration of DNA. (iii) No clear differences in the mutagenic activity of the urine samples were observed between the exposed and control subjects. But, pooling exposed and controls together, a positive and significant variation in the urinary mutagenic effect was observed with the number of cigarettes smoked per day.     

Induction of hair growth in ear wounds by cultured dermal papilla cells

In the adult hair follicle the dermal papilla plays a crucial role in the dermal-epidermal interactions that control hair production and events of the growth cycle. It has previously been shown that cultured cells from rat vibrissa follicle dermal papillae can stimulate hair growth when implanted into amputated follicles. This study investigated the effects of implanting low-passage cultured papilla cells into small incisional wounds in the rat ear pinna. The groups of fibers that emerged from wound sites were much larger than local hairs, and often had vibrissa-type characteristics. Later-passage papilla cells or cultured skin fibroblasts failed to elicit the same response. Histology revealed that big follicles were formed when papilla cells were trapped between the cut edges of the epidermis. Abnormally large follicles were seen at wound sites many months post-operatively. Independent of epidermal influence, cultured papilla cells in the wound dermis formed rounded papilla-like aggregates that also persisted until biopsy. A previously described method of wrapping papilla cells in glabrous epidermis was less successful in percentage terms but resulted in the production of one very large vibrissa-type follicle and fiber. These results further illustrate that the inductive powers and developmental information retained by cultured dermal papilla cells parallel the properties of their embryonic precursors; the findings may have implications for human hair growth.     

Inhibitory effects of bFGF, VEGF and minoxidil on collagen synthesis by cultured hair dermal papilla cells

Dermal papilla cells of rat vibrissa follicles cultivated in monolayers and in three-dimensional collagen gels show a different morphology in these culture systems. Dermal papilla cells cultured in lattices tend to express morphological features resembling those seen in vivo. Quantification of total collagen by incorporation of 3H-proline in monolayer cultures and in collagen lattices show that the amount of collagen found in dermal papilla cells is higher than that secreted. Moreover, collagen synthesis measured in lattices is reduced to about 50% of that found in monolayer cultures. The influence of growth factors on collagen synthesis by hair dermal papilla cells was investigated. We studied the effects of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and minoxidil on collagen synthesis in monolayers and in lattices. VEGF, bFGF and minoxidil significantly decreased the total amount of collagen. In monolayer cultures, there was approximately a 30% inhibition of collagen production with 5 ng/ml bFGF, 0.1 ng/ml VEGF and 100 ng/ml minoxidil. However, in the lattices this inhibition was reduced to about half. These results suggest that both culture substrate and growth factors influence collagen production by rat hair dermal papilla cells.     

The tip link's role in asymmetric stereocilia motion of chick cochlear hair cells

The symmetry of chick cochlear hair bundle motion was examined in this study. Isolated segments from the basilar papilla were incubated in vitro in either normal or low calcium medium, which is known to disrupt tip links. Stereociliary bundles, stimulated with an oscillating water microjet, were oriented in profile and viewed in slow motion at high magnification with stroboscopic illumination. The displacement of the tallest hair in the bundle was fixed to 20 degrees peak-to-peak (P-P) motion. The angular deflections of the shortest and tallest hairs were then measured in both the positive (towards the tallest hair) and negative (towards the shortest) directions with respect to the non-stimulated position of the hair. The tallest hairs exhibited nearly symmetric motion in medium containing normal and low calcium. The shortest hairs, in normal calcium, displayed considerable asymmetry with angular deflections in the positive direction significantly larger than in the negative direction. This asymmetric motion disappeared after incubation in low calcium. The shortest hair angular displacement in the negative direction, however, was the same in both normal and low calcium conditions. These results indicated that the tallest and shortest hairs moved with equal angular deflection in the negative direction, while in the positive direction the shortest hair moved through a significantly greater angular deflection than the tallest hair. The implication of this finding is that the tip links contributed significantly to hair bundle motion in the positive direction only.     

The origin, migration and morphology of the primordial germ cells in the chick embryo

Chick primordial germ cells (PGCs) which separated from the "germinal crescent" entoderm in the period from stages 4 to 8 circulated mostly through the developing blood vessels from stage 10 onward and finally migrated into the gonad. The PGCs making their appearance up to this stage were generally spherical in profile, about 14 mum in diameter. Some of the PGCs in contrast, did not enter the blood vessels but remained in the tissue (mesenchyme) of the embryo proper (tissue PGCs) and possessed pseudopodial processes, suggesting their migration by means of amoeboid movements. The circulating PGCs emerged from blood vessels in the vicinity of developing gonads by three days (gonadal PGCs). The principal mechanism responsible for the subsequent migration of gonadal PGCs is assumed to be amoeboid movements as in the case of tissue PGCs. Notable amounts of PAS-positive glycogen were demonstrated in the cytoplasm of PGCs in all stages obsreved. They also contained yolk and lipids intracytoplasmically, the former dissipating in relatively early stages of development. Electron microscopic observation revealed the electron-opaque, "fragmented nucleolus" in the large nucleus (8 mum in diameter), which represented another prominent feature of chick PGCs. PGCs contained a well-developed Golgi complex and endoplasmic reticulum.     

Anatomical basis of a congenital hearing impairment: basilar papilla dysplasia in the Belgian Waterslager canary

We studied the role of proteases in apoptosis using a cell-free system prepared from a human leukemia cell line. HL60 cells are p53 null and extremely sensitive to a variety of apoptotic stimuli including DNA damage induced by the topoisomerase I inhibitor, camptothecin. We measured DNA fragmentation induced in isolated nuclei by cytosolic extracts using a filter elution assay. Cytosol from camptothecin-treated HL60 cells induced internucleosomal DNA fragmentation in nuclei from untreated cells. This fragmentation was suppressed by serine protease inhibitors. Serine proteases (trypsin, endoproteinase Glu-C, chymotrypsin A, and proteinase K) and papain by themselves induced DNA fragmentation in naive nuclei. This effect was enhanced in the presence of cytosol from untreated cells. Cysteine protease inhibitors (E-64, leupeptin, Ac-YVAD-CHO [ICE inhibitor]) did not affect camptothecin-induced DNA fragmentation. The apopain/Yama inhibitor, Ac-DEVD-CHO, and the proteasome inhibitor, MG-132, were also inactive both in the cell-free system and in whole cells. Interleukin-1 beta converting enzyme (ICE) or human immunodeficiency virus protease failed to induce DNA fragmentation in naive nuclei. Together, these results suggest that DNA damage activates serine protease(s) which in turn activate(s) nuclear endonuclease(s) during apoptosis in HL60 cells.     

Displacement-clamp measurement of the forces exerted by gating springs in the hair bundle

Mechanical stimuli applied to the hair bundle of a hair cell are communicated to the transduction channels by gating springs, elastic elements that are stretched when the bundle is displaced toward its tall edge. To quantify the magnitude and time dependence of the forces exerted by gating springs, we have developed a displacement-clamp system that constrains a bundle's motion while measuring the forces that the bundle produces during adaptation to mechanical stimuli, in response to channel blockage, and upon destruction of the gating springs. Our results suggest that each gating spring exerts a tension of approximately 8 pN in the resting bundle and can sustain at least 4-13 pN of additional tension. The experiments provide further evidence that the gating springs account for at least one-third of the hair bundle's dynamic stiffness and that a force of approximately 100 fN is sufficient to open a single transduction channel.     

Analysis of nicotine and cotinine in the hair of hospitality workers exposed to environmental tobacco smoke

The purpose of this study was to determine if hair nicotine and cotinine levels reflect relative exposure to environmental tobacco smoke (ETS) in subjects who worked in the hospitality industry, where public smoking was permitted. Hair samples from 26 subjects were analyzed by gas chromatograph/mass spectrometry techniques for nicotine and cotinine. An exposure gradient was shown for nicotine but not cotinine. Among nonsmokers, those working in bars where there are no public smoking restrictions had the highest hair nicotine levels, which were close to levels found in smokers. Nicotine measured in hair is useful as a biological marker for exposure to ETS from multiple sources. Bar workers in particular are exposed to high levels of ETS, which may adversely affect the health of nonsmokers.     

Hair cell mechano-transduction: its influence on the gross mechanical characteristics of a hair cell sense organ

Popular hair cosmetic treatments like bleaching or permanent waving were found to affect the stability of incorporated drugs and to cause alterations of the fibers at an ultrastructural level. This may result in a partial or complete loss of drug substances, depending on the particular drug molecule and on its concentration prior to the cosmetic treatment. Moreover, from literature, there is some evidence that drug molecules are not only incorporated into the growing fiber by passive diffusion from blood into the matrix cells and melanocytes, but that the substances enter the hair also via perspiration such as sweat and sebum. Since permed and bleached hair shows an enhanced sorption capacity, the risk of false positives or an unusually high drug concentration in cosmetically treated hair was under investigation. Virgin, permed, mildly as well as severely bleached tresses were exposed to artificial sweat or sebum containing cocaine, benzoylecgonine, 6-acetylmorphine, morphine and codeine (500 ng/g). Except codeine, the concentrations measured by GC/MS were very small and quite close to the detection limit indicating a minor importance of drug uptake into hair fiber from the endogenous-exogenous shunt via sebum or sweat. From the results it is concluded that an increased risk of false positive results in hair analysis on bleached and permanent waved hair fibers does exist, but is not particularly severe.     

Ontogeny of behavioral responsiveness to sound in the chick embryo as indicated by electrical recordings of motility.

Gathered normative behavioral data regarding the ontogeny of responsiveness to sound in the chicken embryo. As a prerequisite, a sensitive and accurate method for recording embryonic motility was developed (Exp I). By means of platinum electrodes inserted just beneath the shell membrane, potentials resulting from heartbeat and movement of 300 White Leghorn embryos were recorded on a polygraph. The technique was found to be effective when applied to chick embryos 6 days and older. Correlations between visual observations of activity and the records produced by the electronic technique substantiated its accuracy. Behavioral responses of 300 chick embryos (Stages 39–43) to acoustic stimulation (Exp II) were then recorded. High-intensity (115-db) tones of 400, 700, and 1,400 Hz were used as stimuli. The earliest consistent responses were recorded from Stage 40 (Days 14–25) Ss; the 700 and 1,400 Hz tones produced statistically reliable inhibition of movement during the stimulus period compared with the post-stimulus period. Reliable increases in movement during the stimulus period were first recorded at Stage 42 (Days 16–27) in response to 700 and 1,400 Hz and at Stage 43 (Days 17–28) in response to 400 Hz. (42 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Plasma protective effect on red blood cells exposed to mechanical stress

Hemodilution with plasma expanders is a widely applied practice during extracorporeal circulation and hemodialysis. Despite the immediate beneficial effects of hemodilution, such as reduction of blood viscosity and red blood cell (RBC) aggregation, elevation of blood flow in the microcirculation, etc., the dilution of plasma may cause some unfavorable effects on RBCs, amplifying the mechanical damage caused by circulatory assist devices. The authors investigated the effect of partial and total replacement of plasma on susceptibility of human and bovine RBCs to mechanical stress in vitro. Hemolysis was measured after the exposure of RBCs suspended in different media to similar mechanical stress. Experiments were performed at room temperature with control of osmolality and viscosity of the suspension media. The lowest hemolysis was obtained for RBCs suspended in serum, plasma, and albumin solutions. Hemolysis in PBS and Dextran suspensions was more than three times higher than that in plasma (p < 0.001). The protective effect depended upon protein concentration. Human RBCs were found to be significantly more sensitive to mechanical stress than bovine RBCs in all investigated suspension media (p < 0.005). Human RBCs from men suspended in plasma were found to be significantly (p < 0.05) more fragile than RBCs from women. The presence of even small amounts of plasma (such as 25%) in the suspension media significantly (p < 0.001) decreased hemolysis. However, a 30% replacement of plasma with PBS or Dextran solutions caused a statistically significant (p < 0.001) increase in mechanical hemolysis. This suggests that a decrease in the concentration of plasma proteins due to hemodilution may elevate blood damage during extracorporeal circulation and hemodialysis.     

Melatonin prevents death of neuroblastoma cells exposed to the Alzheimer amyloid peptide

Studies from several laboratories have generated evidence suggesting that oxidative stress is involved in the pathogenesis of Alzheimer's disease (AD). The finding that the amyloid beta protein (Abeta) has neurotoxic properties and that such effects are, in part, mediated by free radicals has provided insights into mechanisms of cell death in AD and an avenue to explore new therapeutic approaches. In this study we demonstrate that melatonin, a pineal hormone with recently established antioxidant properties, is remarkably effective in preventing death of cultured neuroblastoma cells as well as oxidative damage and intracellular Ca2+ increases induced by a cytotoxic fragment of Abeta. The effects of melatonin were extremely reproducible and corroborated by multiple quantitative methods, including cell viability studies by confocal laser microscopy, electron microscopy, and measurements of intracellular calcium levels. The importance of this finding is that, in contrast to conventional antioxidants, melatonin has a proposed physiological role in the aging process. Secretion levels of this hormone are decreased in aging and more severely reduced in AD. The reported phenomenon may be of therapeutic relevance in AD.     

Postnatal development of type I and type II hair cells in the mouse utricle: acquisition of voltage-gated conductances and differentiated morphology

The type I and type II hair cells of mature amniote vestibular organs have been classified according to their afferent nerve terminals: calyx and bouton, respectively. Mature type I and type II cells also have different complements of voltage-gated channels. Type I cells alone express a delayed rectifier, gK,L, that is activated at resting potential. We report that in mouse utricles this electrophysiological differentiation occurs during the first postnatal week. Whole-cell currents were recorded from hair cells in denervated organotypic cultures and in acutely excised epithelia. From postnatal day 1 (P1) to P3, most hair cells expressed a delayed rectifier that activated positive to resting potential and a fast inward rectifier, gK1. Between P4 and P8, many cells acquired the type I-specific conductance gK,L and/or a slow inward rectifier, gh. By P8, the percentages of cells expressing gK,L and gh were at mature levels. To investigate whether the electrophysiological differentiation correlated with morphological changes, we fixed utricles at different times between P0 and P28. Ultrastructural criteria were developed to classify cells when calyces were not present, as in cultures and neonatal organs. The morphological and electrophysiological differentiation followed different time courses, converging by P28. At P0, when no hair cells expressed gK,L, 33% were classified as type I by ultrastructural criteria. By P28, approximately 60% of hair cells in acute preparations received calyx terminals and expressed gK,L. Data from the denervated cultures showed that neither electrophysiological nor morphological differentiation depended on ongoing innervation.     

Protective effect of D-alpha-tocopherol on the function of human mesangial cells exposed to high glucose concentrations

Altered functions of mesangial cells (MCs) induced by high glucose levels are thought to play an important role in the pathogenesis of diabetic nephropathy. We investigate whether D-alpha-tocopherol (Toc), an antioxidant, can prevent malfunction of cultured human MCs induced by high-glucose media. Incubating MCs with 33 mmol/L glucose caused increased lipid peroxide (LPO) levels, disturbed cell replication, enhanced cytotoxicity, enhanced activity of the diacylglycerol (DAG)-protein kinase C (PKC) pathway, and overproduction of fibronectin and eicosanoids (6-keto prostaglandin F1 alpha [PGF1 alpha] and thromboxane B2 [TXB2]). The amount of LPO in MCs grown in 5 mmol/L glucose was reduced by the addition of Toc in a dose-dependent manner. Since the maximum effect of Toc on decreasing LPO was achieved at a concentration of 100 mumol/L, this dose was selected for the following experiments. Addition of Toc prevented increased LPO levels and [51Cr]-release from MCs induced by high-glucose media without affecting cell number. Toc decreased the total DAG level and PKC activity in membrane fractions in MCs cultured at both 5 and 33 mmol/L glucose. Furthermore, glucose-induced overproduction of fibronectin and eicosanoids from MCs was completely abolished by Toc. These results strongly suggest that Toc ameliorates glucose-induced malfunctions of MCs in vitro.     

Microsurgical pterional approach to aneurysms of the basilar bifurcation

The ultrastructure and component polysaccharides of the cell wall of Pythium debaryanum IFO-5919 were investigated. From results obtained by means of acid, alkali, Schweitzer reagent and beta-1, 3-glucanase treatments and electron microscopy, it was concluded that 1) the acid-extracted fraction was a 1,3-linked branched glucan, 2) the alkali-extranded fraction was a mixture of 1,3-, 1,6-, and 1,3,6-linked highly branched two glucans, 3) the Schweitzer reagent-extracted fraction was a beta-1, 4-linked glucan, 4) the cell wall was constructed from two types of cullulosic microfibrils, as a frame and as a finer network, and amorphous beta-1, 3-glucan including beta-1, 6-linkage, 5) cellulosic microfibrils were covered by matrix material consisting of a mixture of amorphous beta-1, 3-linked and beta-1, 6-linked branching glucans.