A molecular mechanism for electrical tuning of cochlear hair cells

Cochlear frequency selectivity in lower vertebrates arises in part from electrical tuning intrinsic to the sensory hair cells. The resonant frequency is determined largely by the gating kinetics of calcium-activated potassium (BK) channels encoded by the slo gene. Alternative splicing of slo from chick cochlea generated kinetically distinct BK channels. Combination with accessory beta subunits slowed the gating kinetics of alpha splice variants but preserved relative differences between them. In situ hybridization showed that the beta subunit is preferentially expressed by low-frequency (apical) hair cells in the avian cochlea. Interaction of beta with alpha splice variants could provide the kinetic range needed for electrical tuning of cochlear hair cells.     

Maturation of postsynaptic acetylcholine receptors in cochlear outer hair cells

Sound transduction in the inner ear is controlled by olivocochlear efferents terminating predominantly at outer hair cells (OHC). Development of efferent fibers and thereby of postsynaptic OHC receptors was studied immunohistochemically in 13 cochleae from fetal guinea pigs. The gestational ages of the animals ranged from gestational day (GD) 35 to GD 56. To visualize nicotinic acetylcholine receptors (nAChR), sera were used from myasthenia gravis patients with confirmed nAChR antibodies. At GD 53 no staining was observed, whereas at GD 58 a striking nAChR-immunoreactivity was found. In cochleae from adult animals postsynaptic receptors were visualized at the bases of all three rows of OHCs. The region of the inner hair cells (IHC) was not stained. The present results indicate that nAChRs in guinea pig cochleae develop between GD 53 and GD 58. Maturation of the postsynaptic nAChRs coincides with development of OHC motile properties.     

Effects of efferent neurotransmitter acetylcholine and cochlear active substance ATP on cochlear outer hair cells

Toxoplasma antibody serological tests were carried out using the Dye test on sera of pregnant and postpartum Nigerian women to investigate whether there was any association between the levels of antibody titres and the occurrence of stillbirths and congenital malformations. There was a high prevalence of toxoplasma antibodies in the sera of both pregnant and postpartum women. The prevalence rates for the pregnant women ranged from 72.5% to 88.8% with an overall rate of 75.4%; whilst for the postpartum women, the prevalence rates ranged from 75.0% to 94.4% with an overall rate of 80.5%. The toxoplasma antibody titres of the sera from the live-born babies as well as stillbirths and congenitally malformed babies ranged from 1:16 to 1:1024. The exact role played by toxoplasma in the occurrence of stillbirths and congenital malformation in our area of study is, however, not clear. For future research, it is suggested that larger samples be studied in order to enhance the validity of the findings of the present study.     

Opposite phenotypes of hypomorphic and Y766 phosphorylation site mutations reveal a function for Fgfr1 in anteroposterior patterning of mouse embryos

Intercellular communication is needed for both the generation of the mesodermal germ layer and its division into distinct subpopulations. To dissect the functions of fibroblast growth factor receptor-1 (FGFR1) during mouse gastrulation as well as to gain insights into its possible roles during later embryonic development, we have introduced specific mutations into the Fgfr1 locus by gene targeting. Our results show functional dominance of one of the receptor isoforms and suggest a function for the autophosphorylation of site Y766 in the negative regulation of FGFR1 activity. Y766F and hypomorphic mutations in Fgfr1 generate opposite phenotypes in terms of homeotic vertebral transformations, suggesting a role for FGFR1 in patterning the embryonic anteriorposterior axis by way of regulation of Hox gene activity.     

Acute hyperpolarization and elongation of cochlear outer hair cells on superfusion with cis-platinum

The acute effects of cis-platinum on isolated cochlear outer hair cells (OHC) were investigated with whole-cell patch-clamps and measurements of cell length changes. Our findings demonstrated that cis-platinum reversibly induced a hyperpolarization and cellular elongation. These results suggest that the effects produced are the result of an interaction between cis-platinum and transduction channels in OHC. These acute effects are distinctly different from the chronic, irreversible ones that are followed by death of the OHC. The exact mechanism of these chronic effects remains unknown as yet.     

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.     

Cholinergic modulation of extracellular ATP-induced cytoplasmic calcium concentrations in cochlear outer hair cells

Outer hair cells (OHC) of the mammalian cochlea modulate the inner hair cell (IHC) mechanoelectrical transduction of sound. They are contacted by synapsing efferent neurons from the CNS, their main efferent neurotransmitter being acetylcholine (ACh). OHC function and in particular their control of [Ca2+]i is highly important and is modulated by ACh and also by other substances including extracellular (EC) ATP. OHC carry at their efferent synapse a not yet completely identified neuronal type of ionotropic ACh receptor (AChR), with an unusual pharmacology, which is, in vivo and in vitro, reversibly blocked by alpha-bungarotoxin (alpha-bgtx). The AChR mediates a fast influx of Ca2+ into OHC which, in turn, activates a closeby located outwardly-directed Ca(2+)-dependent K(+)-channel, thus shortly hyperpolarizing the cell. A cloned homomeric alpha 9 nAChR mimicks the function and pharmacology of this receptor. We here report results from a study designed to observe only slower effects triggered by EC ATP and the ACh-AChR system. EC presence of ATP at OHC increases [Ca2+]i by activating both P2x and P2y purinoceptors and also by indirect activation of OHC L-type Ca(2+)-channels. The L-type channel activation is responsible for a large part of the [Ca2+]i increase. Simultaneous EC presence of ACh and ATP at OHC was found to depress ATP-induced effects on OHC [Ca2+]i, an effect that is completely blocked in the presence of alpha-bgtx. Our observations suggest that the ACh-AChR system is involved in the modulation of the observed EC ATP-triggered events; possibly the OHC AChR is able to act both in its well known rapid ionotropic way, but also, perhaps after modification in a slower, metabotropic way interfering with the EC ATP-induced [Ca2+]i increase.     

Effects of mutations in the gamma-phosphate binding site of myosin on its motor function

The role of the highly conserved residues in the gamma-phosphate binding site of myosin upon myosin motor function was studied. Each of five residues (Ser181, Lys185, Asn235, Ser236, and Arg238) in smooth muscle myosin was mutated. K185Q has neither a steady state ATPase nor an initial Pi burst. Although ATP and actin bind to K185Q, it is not dissociated from actin by ATP. These results indicate that the hydrolysis of bound ATP by K185Q is inhibited. S236T has nearly normal basal Mg2+-ATPase activity, initial Pi burst, ATP-induced enhancement of intrinsic tryptophan fluorescence, and ATP-induced dissociation from actin. However, the actin activation of the Mg2+-ATPase activity and actin translocation of S236T were blocked. In contrast S236A has nearly normal enzymatic properties and actin-translocating activity. These results indicate that 1) the hydroxyl group of Ser236 is not critical as an intermediary of proton transfer during the ATP hydrolysis step, and 2) the bulk of the extra methyl group of the threonine residue in S236T blocks the acceleration of product release from the active site by actin. Arg238, which interacts with Glu459 at the Switch II region, was mutated to Lys and Ile, respectively. R238K has essentially normal enzymatic activity and motility. In contrast, R238I does not hydrolyze ATP or support motility, although it still binds ATP. These results indicate that the charge interaction between Glu459 and Arg238 is critical for ATP hydrolysis by myosin. Other mutants, S181A, S181T, and N235I, showed nearly normal enzymatic and motile activity.     

Protection of both auditory hair cells and auditory neurons from cisplatin induced damage

Knowledge about body composition is important in metabolic and nutritional studies. In this cross-sectional study the body composition of 403 healthy white Dutch children and adolescents was evaluated by using dual-energy X-ray absorptiometry (DXA). Possible determinants of body composition were analyzed. In 85 subjects the results of bioelectrical impedance analysis (BIA) were compared with DXA. Fat mass, lean tissue mass, and bone mineral content were greater in older boys and girls. Percentage body fat was greater in older girls but not in boys and it was higher in girls than in boys at all ages. From the age of 14 y boys had higher lean tissue mass and bone mineral content than girls. Tanner stage had a significant relation with body composition in both sexes. Percentage body fat was lower in boys in stage 4 than in stage 3 and was higher in consecutive Tanner stages in girls. After adjustment for age, Tanner stage was significantly positively related to lean tissue mass and bone mineral content in boys and girls and to percentage body fat and fat mass in girls. The profession of the parents and the education of the father had a significant negative correlation with percentage body fat and fat mass in girls (P < 0.01). Physical activity was related to lean tissue mass (P = 0.001) but not to fat mass in boys after adjustment for age. A high correlation and a small difference was found between lean body mass by BIA and lean tissue mass by DXA. Body composition in healthy Dutch children and adolescents is related to age, sex, Tanner stage, socioeconomic status, and physical activity.     

The in vitro development of innervated sensory hair cells of a mammal

Twelfth gestation day mouse embryo otocysts with their associated statoacoustic ganglions and otic mesenchyme were explanted to "in vitro". The otocyst explants developed for seven days in organ culture. They were then fixed and studied on a light microscopic and ultrastructural level for the development of synapses. Evidence is presented that strongly suggests that afferent synapses are developing "in vitro" in the vestibular sensory epithelium studied.     

Suppressors of cleavage-site mutations in the p62 envelope protein of Semliki Forest virus reveal dynamics in spike structure and function

The E2 spike glycoprotein of Semliki Forest virus is produced as a p62 precursor protein, which is cleaved by host proteases to its mature form, E2. Cleavage is not necessary for particle formation or release but is necessary for infectivity. Previous results had shown that phenotypic revertants of cleavage-deficient p62 mutants are generated, and here we show that these may contain second-site suppressor mutations in the vicinity of the cleavage site. These hot-spot sites were mutated to abolish the generation of such suppressor mutations; however, secondary mutations in another distant domain of the E2 protein appeared instead, all of which still caused cleavage-deficient mutations. Such mutants grew very poorly and were inefficient in virus entry and release. The mutated sites define domains of the spike protein which probably interact to regulate its structure and function. Because of their highly attenuated phenotype and the lower probability of reversion, the new mutations close to the cleavage site were used to make new helper vectors for packaging of recombinant RNA into infectious particles, thus increasing further the biosafety of the vector system based on the Semliki Forest virus replicon.     

Early development of the zebrafish pronephros and analysis of mutations affecting pronephric function

The zebrafish pronephric kidney provides a simplified model of nephron development and epithelial cell differentiation which is amenable to genetic analysis. The pronephros consists of two nephrons with fused glomeruli and paired pronephric tubules and ducts. Nephron formation occurs after the differentiation of the pronephric duct with both the glomeruli and tubules being derived from a nephron primordium. Fluorescent dextran injection experiments demonstrate that vascularization of the zebrafish pronephros and the onset of glomerular filtration occurs between 40 and 48 hpf. We isolated fifteen recessive mutations that affect development of the pronephros. All have visible cysts in place of the pronephric tubule at 2-2.5 days of development. Mutants were grouped in three classes: (1) a group of twelve mutants with defects in body axis curvature and manifesting the most rapid and severe cyst formation involving the glomerulus, tubule and duct, (2) the fleer mutation with distended glomerular capillary loops and cystic tubules, and (3) the mutation pao pao tang with a normal glomerulus and cysts limited to the pronephric tubules. double bubble was analyzed as a representative of mutations that perturb the entire length of the pronephros and body axis curvature. Cyst formation begins in the glomerulus at 40 hpf at the time when glomerular filtration is established suggesting a defect associated with the onset of pronephric function. Basolateral membrane protein targeting in the pronephric duct epithelial cells is also severely affected, suggesting a failure in terminal epithelial cell differentiation and alterations in electrolyte transport. These studies reveal the similarity of normal pronephric development to kidney organogenesis in all vertebrates and allow for a genetic dissection of genes needed to establish the earliest renal function.     

Global gene expression profiles during acute pathogen-induced pulmonary inflammation reveal divergent roles for Th1 and Th2 responses in tissue repair

T helper 1 responses are typically proinflammatory, while Th2 responses have been considered regulatory. Interestingly, Th2 responses characterize a number of pulmonary diseases, many of which terminate in tissue remodeling and fibrosis. We developed a mouse model using Schistosoma mansoni eggs and cytokine-deficient mice to induce highly polarized Th1- or Th2-type inflammation in the lung. In this study, we examined the pathology and cytokine profiles in Th1- and Th2-polarized environments and used oligonucleotide microarray analysis to decipher the genes responsible for these effects. We further elaborated on the results using IL-10- and IL-13-deficient mice because these cytokines are believed to be the central regulators of Th2-associated pathology. We found that the Th1-polarized mice developed small granulomas with less fibrosis while expressing genes characteristic of tissue damage. Th2-polarized mice, in contrast, formed large granulomas with massive collagen deposition and up-regulated genes associated with wound healing, specifically, arginase, collagens, matrix metalloproteinases (MMPs), and tissue inhibitors of MMP. In addition, several members of the chitinase-like family were up-regulated in the lung following egg challenge. We also developed a method of defining the net collagen deposition using the expression profiles of several collagen, MMP, and tissue inhibitors of MMP genes. We found that Th1-polarized mice did not elaborate collagens or MMPs and therefore did not have a significant capacity for repair in this model. Thus, Th1-mediated inflammation is characterized by tissue damage, while Th2 directs wound healing and fibrosis.     

Kinematic analysis of shear displacement as a means for operating mechanotransduction channels in the contact region between adjacent stereocilia of mammalian cochlear hair cells

In sensory hair cells of the cochlea, deflection of the stereociliary bundle results in direct mechanical gating of mechanoelectrical transduction channels, a function generally attributed to the tip link running between the tips of short stereocilia and the sides of adjacent taller ones. However, immunocytochemical experiments indicate that the channels may not be associated with the tip link but occur just below it in a region of contact between the stereocilia. To determine whether transduction channels in this location could be operated during physiologically appropriate deflections as effectively by shear displacement as if they were associated with the tip link, a two dimensional kinematic analysis of relative motion between stereocilia has been performed assuming contact between stereocilia is maintained during deflection. Bundle geometry and dimensions were determined from transmission electron micrographs of hair cells from several frequency locations between 0.27 and 13.00 kHz in the guinea-pig cochlea. The analysis indicates that for a 10 nm deflection of the tallest stereocilia of both inner and outer hair cells, i.e. within the range of the maximum sensitivity of mammalian hair bundles, the average shear displacement in the contact region would be 1.6 nm, but that it increases systematically towards higher frequency regions for outer hair cells. This displacement is comparable in magnitude to tip-link elongation for individual stereociliary pairs.     

TNF receptor-deficient mice reveal divergent roles for p55 and p75 in several models of inflammation

The pleiotropic activities of the potent proinflammatory cytokine TNF are mediated by two structurally related, but functionally distinct, receptors, p55 and p75, that are coexpressed on most cell types. The majority of biologic responses classically attributed to TNF are mediated by p55. In contrast, p75 has been proposed to function as both a TNF antagonist by neutralizing TNF and as a TNF agonist by facilitating the interaction between TNF and p55 at the cell surface. We have examined the roles of p55 and p75 in mediating and modulating the activity of TNF in vivo by generating and examining mice genetically deficient in these receptors. Selective deficits in several host defense and inflammatory responses are observed in mice lacking p55 or both p55 and p75, but not in mice lacking p75. In these models, the activity of p55 is not impaired by the absence of p75, arguing against a physiologic role for p75 as an essential element of p55-mediated signaling. In contrast, exacerbated pulmonary inflammation and dramatically increased endotoxin induced serum TNF levels in mice lacking p75 suggest a dominant role for p75 in suppressing TNF-mediated inflammatory responses. In summary, these data help clarify the biologic roles of p55 and p75 in mediating and modulating the biologic activity of TNF and provide genetic evidence for an antagonistic role of p75 in vivo.     

Temperature-sensitive mutations of the CKA1 gene reveal a role for casein kinase II in maintenance of cell polarity in Saccharomyces cerevisiae

Casein kinase II (CKII) of Saccharomyces cerevisiae contains two distinct catalytic subunits, alpha and alpha', that are encoded by the CKA1 and -2 genes, respectively. We have constructed conditional alleles of the CKA1 gene. In contrast to cka1 cka2(ts) strains, which exhibit a defect in both G1 and G2/M cell cycle progression, cka1(ts) cka2 strains continue to divide for three cell cycles after a shift to restrictive temperature and then arrest as a mixture of budded and unbudded cells with a spherical morphology. Arrested cells exhibit continued growth, a nonpolarized actin cytoskeleton, delocalized chitin deposition, and a significant fraction of multinucleate cell bodies, confirming the presence of a cell polarity defect in cka1(ts) strains. The presence of budded as well as unbudded cells in the arrested population suggests that CKII is required for maintenance rather than establishment of cell polarity, although a role in both processes is also possible. The terminal phenotype of cka1(ts) strains bears a strong resemblance to that of orb5 strains of Schizosaccharomyces pombe, which carry a temperature-sensitive CKII catalytic subunit mutation, but the underlying mechanism appears to be different in the two cases. These results establish a requirement for CKII in cell polarity in S. cerevisiae and provide the first evidence for functional specialization of CKA1 and -2.     

Expression of a potassium current in inner hair cells during development of hearing in mice

Excitable cells use ion channels to tailor their biophysical properties to the functional demands made upon them. During development, these demands may alter considerably, often associated with a change in the cells' complement of ion channels. Here we present evidence for such a change in inner hair cells, the primary sensory receptors in the mammalian cochlea. In mice, responses to sound can first be recorded from the auditory nerve and observed behaviourally from 10-12 days after birth; these responses mature rapidly over the next 4 days. Before this time, mouse inner hair cells have slow voltage responses and fire spontaneous and evoked action potentials. During development of auditory responsiveness a large, fast potassium conductance is expressed, greatly speeding up the membrane time constant and preventing action potentials. This change in potassium channel expression turns the inner hair cell from a regenerative, spiking pacemaker into a high-frequency signal transducer.     

Saccharomyces cerevisiae recA homologues RAD51 and DMC1 have both distinct and overlapping roles in meiotic recombination

BACKGROUND: Rad51 and Dmc1 are Saccharomyces cerevisiae homologues of the Escherichia coli recombination protein RecA. Mutant analysis has shown that both proteins are required for normal meiotic recombination, for timely and efficient formation of synaptonemal complex and for normal progression out from meiotic prophase. RESULTS: We have further characterized rad51 and dmc1 single mutants. A dmc1 mutation confers more severe defects in double strand break (DSB) resolution, crossover recombination and meiotic progression than does a rad51 mutant; in contrast, during return to growth, a rad51 mutation confers more severe defects in viability and intrachromosomal recombination than does a dmc1 mutation. Analysis of a rad51 dmc1 double mutant, in parallel with single mutants, shows that the double mutant is more defective with respect to the formation of crossovers during meiosis and, especially strikingly, with respect to interhomologue and intrachromosomal recombination during return to growth. Consistent with the observation of DMC1-dependent recombination in a rad51 mutant, subnuclear complexes of Dmc1 protein were detected for the first time in this mutant. In contrast to the effects on recombination, the effect of the double mutant on meiotic progression was similar to that of the rad51 single mutant. CONCLUSION: Rad51 and Dmc1 each make unique contributions to meiotic recombination. However, the two proteins are capable of substituting for one another under some circumstances, implying that they most likely share at least one recombination function. Recombination and cell cycle phenotypes are all consistent with the possibility that a dmc1 mutation causes an arrest of the post-DSB recombination complexes at a later, more stable stage than does a rad51 mutation.