Nerve growth factor regulates Na+ transport in human airway epithelial cells

Nerve growth factor (NGF) was discovered for its ability to enhance nerve growth, but recent evidence suggests there is a correlation between elevated NGF levels in the lung and airway diseases, including lung inflammatory diseases and respiratory virus infections. NGF can be produced and act upon both structural and non-structural cells of the airways, and overexpression of NGF causes morphological and physiological changes in the airways, such as an increased innervation resulting in a neuronal remodeling of the lung, airway smooth muscle thickening, increased vascularization, airway hyperreactivity to capsaicin, and subepithelial thickening. Although work has been conducted to investigate NGFs effects on ion transport in non-airway cells, such as PC12, MTAL, and HEK-293 cells, no information is available regarding the effect NGF has on ion transport of airway epithelial cells. To investigate whether NGF can affect epithelial ion transport, a well-differentiated human primary cultured epithelial cell model was developed. The ability for these cells to differentiate into epithelial cells, which represent in situ epithelial morphology, was confirmed using several imaging techniques. Cells were placed in Ussing chambers to obtain transepithelial voltage (Vt, -7.1 +/- 3.4 mV), short-circuit current ( Isc, 5.9 +/- 1.0 muA), and transepithelial resistance (Rt, 750 Ohm x cm2), and to measure responses to ion transport inhibitors. Apical and basolateral NGF concentration-response curves were generated, but NGF only evoked bioelectric responses apically with the maximum response occurring at 1 ng/ml. To investigate the ionic basis for the bioelectric responses to NGF, responses to known ion transport inhibitors were generated in the absence or presence of 1 ng/ml NGF. The addition of 1 ng/ml of NGF to the apical membrane decreased Isc by 5.3 %. Amiloride (apical, 3.5x10-5 M), which inhibits Na+ transport, decreased Isc by 55.3 % in the absence of NGF, but this response was reduced (41.6 %; p = 0.0127) in the presence of 1 ng/ml NGF, which indicated NGF was affecting amiloride-sensitive Na + transport. There were no differences in response to NPPB or ouabain, indicating NGF did not have an affect on Cl- transport or the Na+/K+-ATPase. To investigate if the trkA receptor was responsible for mediating the NGF-induced reduction in Na+ transport, the non-specific tyrosine kinase inhibitor, K-252a (10 nM, apical), was used. K-252a reduced the NGF bioelectric response as well as attenuated the NGF-induced reduction in Na+ transport. The trkA receptor activates the Erk 1/2 signaling pathway, which has been shown to phosphorylate ENaC and reduce Na+ transport by channel degradation through a NEDD4-mediated ubiquitin pathway. To investigate if NGF is activating the Erk 1/2 signaling pathway downstream of trkA, the specific Erk 1/2 inhibitor, PD-98059 (30 microM, apical and basolateral), was used. PD-98059 reduced the NGF-induced bioelectric response as well as attenuated the NGF-induced reduction in Na+ transport. Protein analysis using western blot techniques confirmed NGF-mediated reduction in Na+ transport was a result of Erk 1/2 activation and ENaC phosphorylation.;To investigate if incubation with NGF can elicit changes in ion transport, cells were incubated with NGF for 24 or 48 h prior to placing to cells into Ussing chambers. Cells exposed to NGF for either 24 or 48 h did not demonstrate changes in ion transport as compared to control cells, indicating NGF did not have a genomic effect on ion transporter subunit expression. These results also suggest that the rapid reduction in amiloride-sensitive Na+ transport is a transient reduction. To determine if this lack of response was a result of a decreased concentration of NGF during the incubation period, a NGF-specific ELISA assay was used. Cells internalized or metabolized 94% of initial concentration of NGF applied within 5 min, as inserts without cells did not demonstrate a reduction in NGF concentration.;The findings discussed in this dissertation indicate that NGF causes a transient and non-genomic reduction in Na+ transport in epithelium through a trkA-Erk1/2-mediated signaling pathway, resulting in the internalization and degradation of ENaC. This reduction in Na+ transport would result in the hydration of the airway surface liquid

Diaphragm Pump With Resonant Piezoelectric Drive

A diaphragm pump driven by a piezoelectric actuator is undergoing development. This pump is intended to be a prototype of lightweight, highly reliable pumps for circulating cooling liquids in protective garments and high-power electronic circuits, and perhaps for some medical applications. The pump would be highly reliable because it would contain no sliding seals or bearings that could wear, the only parts subject to wear would be two check valves, and the diaphragm and other flexing parts could be designed, by use of proven methods, for extremely long life. Because the pump would be capable of a large volumetric flow rate and would have only a small dead volume, its operation would not be disrupted by ingestion of gas, and it could be started reliably under all conditions. The prior art includes a number piezoelectrically actuated diaphragm pumps. Because of the smallness of the motions of piezoelectric actuators (typical maximum strains only about 0.001), the volumetric flow rates of those pumps are much too small for typical cooling applications. In the pump now undergoing development, mechanical resonance would be utilized to amplify the motion generated by the piezoelectric actuator and thereby multiply the volumetric flow rate. The prime mover in this pump would be a stack of piezoelectric ceramic actuators, one end of which would be connected to a spring that would be part of a spring-and-mass resonator structure. The mass part of the resonator structure would include the pump diaphragm (see Figure 1). Contraction of the spring would draw the diaphragm to the left, causing the volume of the fluid chamber to increase and thereby causing fluid to flow into the chamber. Subsequent expansion of the spring would push the diaphragm to the right, causing the volume of the fluid chamber to decrease, and thereby expelling fluid from the chamber. The fluid would enter and leave the chamber through check valves. The piezoelectric stack would be driven electrically to make it oscillate at the resonance frequency of the spring and- mass structure. This frequency could be made high enough (of the order of 400 Hz) that the masses of all components could be made conveniently small. The resonance would amplify the relatively small motion of the piezoelectric stack (a stroke of the order of 10 m) to a diaphragm stroke of the order of 0.5 mm. The exact amplification factor would depend on the rate of damping of oscillations; this, in turn, would depend on details of design and operation, including (but not limited to) the desired pressure rise and volumetric flow rate. In order to obtain resonance with large displacement, the damping rate must be low enough that the energy imparted to the pumped fluid on each stroke is much less than the kinetic and potential energy exchanged between the mass and spring during each cycle of oscillation. To minimize the power demand of the pump, a highly efficient drive circuit would be used to excite the piezoelectric stack. This circuit (see Figure 2) would amount to a special-purpose regenerative, switching power supply that would operate in a power-source mode during the part of an oscillation cycle when the excitation waveform was positive and in a power-recovery mode during the part of the cycle when the excitation waveform was negative. The circuit would include a voltage-boosting dc-to-dc converter that would convert between a supply potential of 24 Vdc and the high voltage needed to drive the piezoelectric stack. Because of the power-recovery feature, the circuit would consume little power. It should be possible to build the circuit as a compact unit, using readily available components

Genetic effects on gene expression across human tissues

Characterization of the molecular function of the human genome and its variation across individuals is essential for identifying the cellular mechanisms that underlie human genetic traits and diseases. The Genotype-Tissue Expression (GTEx) project aims to characterize variation in gene expression levels across individuals and diverse tissues of the human body, many of which are not easily accessible. Here we describe genetic effects on gene expression levels across 44 human tissues. We find that local genetic variation affects gene expression levels for the majority of genes, and we further identify inter-chromosomal genetic effects for 93 genes and 112 loci. On the basis of the identified genetic effects, we characterize patterns of tissue specificity, compare local and distal effects, and evaluate the functional properties of the genetic effects. We also demonstrate that multi-tissue, multi-individual data can be used to identify genes and pathways affected by human disease-associated variation, enabling a mechanistic interpretation of gene regulation and the genetic basis of diseas

Genetic effects on gene expression across human tissues

Characterization of the molecular function of the human genome and its variation across individuals is essential for identifying the cellular mechanisms that underlie human genetic traits and diseases. The Genotype-Tissue Expression (GTEx) project aims to characterize variation in gene expression levels across individuals and diverse tissues of the human body, many of which are not easily accessible. Here we describe genetic effects on gene expression levels across 44 human tissues. We find that local genetic variation affects gene expression levels for the majority of genes, and we further identify inter-chromosomal genetic effects for 93 genes and 112 loci. On the basis of the identified genetic effects, we characterize patterns of tissue specificity, compare local and distal effects, and evaluate the functional properties of the genetic effects. We also demonstrate that multi-tissue, multi-individual data can be used to identify genes and pathways affected by human disease-associated variation, enabling a mechanistic interpretation of gene regulation and the genetic basis of disease

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http://archive.org/details/investigationofs00shimNAN