The expression of Na, K-ATPase in the Madin-Darby canine kidney (MDCK) cell line

The efficiency of a number of experimental techniques for the extraction of total RNA from various cells and tissues (including MDCK strain I cells) was assessed, and the optimal conditions for hybridisation of Na,K-ATPase isoform-specific DNA probes to this RNA were determined. The specificity of hybridisation of DNA probes for the Na,K-ATPase al, a2, a3, and beta1 isoforms was assessed using RNA isolated from rat tissues. The relative abundance of isoform mRNA's in rat kidney, brain, lung, and myocardial tissues was determined by Northern blotting. The abundance of Na,K-ATPase isoforms was also determined in the myocardial tissues of the Milan rat, a hypertensive animal model. Significant differences between the abundance of Na,K-ATPase isoform mRNA's in hypertensive rats and their age and sex matched controls were found. The relative abundance (per ng of total RNA) of al, a3, and beta1 mRNA's in left ventricle and, that of a1, and beta1 mRNA's in right ventricle were significantly decreased in hypertensive rats. The relative abundance (per mug of total RNA) of a2 and beta1 mRNA's in atria was significantly increased in hypertensive rats. These differences found in ventricles and atria were further accentuated by expression of the results per gram wet weight of tissue. The results from ventricular tissues were in contrast to those previously reported by Herrera et al. (1988) who found either increases or no change in the abundance of a1 and beta1 mRNA's in hypertensive rat aorta, skeletal muscle and left ventricle. The differences between these results may be related to the deoxy-corticosterone treatment and high salt diet of the hypertensive rat model used by Herrera et al. (1988). Na,K-ATPase isoform-specific DNA restriction endonuclease fragments were used to investigate the expression of the isoform mRNA's in MDCK strain I cells. Only a1 and beta1 mRNA's was detected on Northern blots, with no detectable a2 or a3 isoform mRNA signals being found in this cell line. [3H]-ouabain binding to cells was used, as an estimate of the cell surface expression of Na,K-ATPase. Possible factors affecting the expression of Na,K-ATPase during the normal cell growth of MDCK strain I cells were investigated. Factors such as cell seeding density, cell growth substrate and the volume of growth medium used, were all found to affect both the level and pattern of expression of Na,K-ATPase during the normal cell growth or culturing cycle. After 2 days of culture the large increases in the expression of Na,K-ATPase assayed in low density compared to high density seeded cells, were not correlated with concomitant changes in the relative abundance of Na,K-ATPase a subunit mRNA. These results indicate that the large changes in cell surface expression of Na,K- ATPase found during cell growth are probably controlled by post transcriptional processes. The effect of certain hormones or their agonists (aldosterone, deoxy-corticosterone, corticosterone, dexamethasone, and tri-iodo thyronine), on the expression of Na,K-ATPase in MDCK strain I cells was also briefly investigated. Under the conditions used, hormone treatment was not found to induce any measurable expression of a2 or a3 mRNA's. The mineralocorticoid aldosterone, and the glucocorticoid corticosterone, both produced small but significant increases in the level of Na,K-ATPase present on the cell membrane, however these increases were not correlated with similar increases in the abundance of both Na,K-ATPase a1 and beta1 mRNA's. The small size of increases in Na,K-ATPase enzyme abundance after hormone treatments and the inability of those treatments to induce consistent increases in Na,K-ATPase mRNA's further suggests that changes in the cell surface expression of Na,K-ATPase in MDCK cells is the result of regulation at a post transcriptional level

Dual-Pump CARS Measurements in the University of Virginia's Dual-Mode Scramjet: Configuration "A"

In this paper we describe efforts to obtain canonical data sets to assist computational modelers in their development of models for the prediction of mixing and combustion in scramjet combustors operating in the ramjet-scramjet transition regime. The CARS technique is employed to acquire temporally and spatially resolved measurements of temperature and species mole-fraction at four planes, one upstream of an H2 fuel injector and three downstream. The technique is described and results are presented for cases with and without chemical reaction. The vibrational energy mode in the heated airstream of the combustor was observed to be frozen at near facility heater conditions and significant nonuniformities in temperature were observed, attributed to nonuniformities of temperature exiting the heater. The measurements downstream of fuel injection show development of mixing and combustion, and are already proving useful to the modelers

Dual-Pump CARS Measurements in the University of Virginia's Dual-Mode Scramjet: Configuration "C"

Measurements have been conducted at the University of Virginia Supersonic Combustion Facility in configuration C of the dual-mode scramjet. This is a continuation of previously published works on configuration A. The scramjet is hydrogen fueled and operated at two equivalence ratios, one representative of the scram mode and the other of the ram mode. Dual-pump CARS was used to acquire the mole fractions of the major species as well as the rotational and vibrational temperatures of N2. Developments in methods and uncertainties in fitting CARS spectra for vibrational temperature are discussed. Mean quantities and the standard deviation of the turbulent fluctuations at multiple planes in the flow path are presented. In the scram case the combustion of fuel is completed before the end of the measurement domain, while for the ram case the measurement domain extends into the region where the flow is accelerating and combustion is almost completed. Higher vibrational than rotational temperature is observed in those parts of the hot combustion plume where there is substantial H2 (and hence chemical reaction) present

High-Spatial-Resolution OH PLIF Visualization in a Cavity-Stabilized Ethylene-Air Turbulent Flame

High-spatial-resolution OH planar laser-induced fluorescence was measured for a premixed ethylene-air turbulent flame in an electrically-heated Mach 2 continuous-flow facility (University of Virginia Supersonic Combustion Facility, Configuration E.) The facility comprised a Mach 2 nozzle, an isolator with flush-wall fuel injectors, a combustor with optical access, and an extender. The flame was anchored at a cavity flameholder with a backward-facing step of height 9 mm. The temperature-insensitive Q1(8) transition of OH was excited using laser light of wavelength 283.55 nm. A spatial filter was used to create a laser sheet approximately 25 microns thick based on full-width at half maximum (FWHM). Extension tubes increased the magnification of an intensified camera system, achieving in-plane resolution of 40 microns based on a 50% modulation transfer function (MTF). The facility was tested with total temperature 1200 K, total pressure 300 kPa, local fuel/air equivalence ratios of approximately 0.4, and local Mach number of approximately 0.73 in the combustor. A test case with reduced total temperature and another with reduced equivalence ratio were also tested. PLIF images were acquired along a streamwise plane bisecting the cavity flameholder, from the backward facing step to 120 mm downstream of the step. The smallest observed features in the flow had width of approximately 110 microns. Flame surface density was calculated for OH PLIF images

High-Resolution OH and CH2O Visualization in a Premixed Cavity-Anchored Ethylene-Air Flame in a M = 0.6 Flowfield

OH and CH2O were imaged in a premixed, cavity-anchored, ethylene-air turbulent flame using a high resolution planar laser-induced fluorescence (PLIF) system. The electrically-heated, continuous flow facility (UVa Supersonic Combustion Facility, Configuration E) consisted of a Mach 2 nozzle, an isolator with fuel injectors, a test section with a cavity flame holder and optical access, and an extender. Standard test conditions comprised total temperature 1200 K, total pressure 300 kPa, local equivalence ratio near 0.4, and local Mach number near 0.6. OH PLIF data was also collected for a case with reduced total temperature and another with reduced equivalence ratio. OH and CH2O were excited in separate experiments with light sheets at 283.55 nm and 352.48 nm, respectively. A light sheet of approximate thickness 25 ?m illuminated the stream-wise midplane. This plane was imaged for 120 mm downstream of the backward-facing step. The intensified camera system imaged OH with magnification 1.97, a square 6.67 mm field of view, and in-plane resolution of 39 ?m. The smallest observed OH structures observed were approximately 100 ?m wide. The CH2O PLIF image signal was much weaker; the smallest observed structures were approximately 200 ?m wide. Composite fluorescence images were computed for the observed area

Measurement of Vibrational Non-Equilibrium in a Supersonic Freestream Using Dual-Pump CARS

Measurements have been conducted at the University of Virginia Supersonic Combustion Facility of the flow in a constant area duct downstream of a Mach 2 nozzle, where the airflow has first been heated to approximately 1200 K. Dual-pump CARS was used to acquire rotational and vibrational temperatures of N2 and O2 at two planes in the duct at different downstream distances from the nozzle exit. Wall static pressures in the nozzle are also reported. With a flow of clean air, the vibrational temperature of N2 freezes at close to the heater stagnation temperature, while the O2 vibrational temperature is about 1000 K. The results are well predicted by computational fluid mechanics models employing separate "lumped" vibrational and translational/rotational temperatures. Experimental results are also reported for a few percent steam addition to the air and the effect of the steam is to bring the flow to thermal equilibrium

Large-Eddy / Reynolds-Averaged Navier-Stokes Simulations of a Dual-Mode Scramjet Combustor

Numerical simulations of reacting and non-reacting flows within a scramjet combustor configuration experimentally mapped at the University of Virginia s Scramjet Combustion Facility (operating with Configuration A ) are described in this paper. Reynolds-Averaged Navier-Stokes (RANS) and hybrid Large Eddy Simulation / Reynolds-Averaged Navier-Stokes (LES / RANS) methods are utilized, with the intent of comparing essentially blind predictions with results from non-intrusive flow-field measurement methods including coherent anti-Stokes Raman spectroscopy (CARS), hydroxyl radical planar laser-induced fluorescence (OH-PLIF), stereoscopic particle image velocimetry (SPIV), wavelength modulation spectroscopy (WMS), and focusing Schlieren. NC State's REACTMB solver was used both for RANS and LES / RANS, along with a 9-species, 19- reaction H2-air kinetics mechanism by Jachimowski. Inviscid fluxes were evaluated using Edwards LDFSS flux-splitting scheme, and the Menter BSL turbulence model was utilized in both full-domain RANS simulations and as the unsteady RANS portion of the LES / RANS closure. Simulations were executed and compared with experiment at two equivalence ratios, PHI = 0.17 and PHI = 0.34. Results show that the PHI = 0.17 flame is hotter near the injector while the PHI = 0.34 flame is displaced further downstream in the combustor, though it is still anchored to the injector. Reactant mixing was predicted to be much better at the lower equivalence ratio. The LES / RANS model appears to predict lower overall heat release compared to RANS (at least for PHI = 0.17), and its capability to capture the direct effects of larger turbulent eddies leads to much better predictions of reactant mixing and combustion in the flame stabilization region downstream of the fuel injector. Numerical results from the LES/RANS model also show very good agreement with OH-PLIF and SPIV measurements. An un-damped long-wave oscillation of the pre-combustion shock train, which caused convergence problems in some RANS simulations, was also captured in LES / RANS simulations, which were able to accommodate its effects accurately

OH PLIF Visualization of a Premixed Ethylene-fueled Dual-Mode Scramjet Combustor

Hydroxyl radical (OH) planar induced laser fluorescence (PLIF) measurements have been performed in a small-scale scramjet combustor at the University of Virginia Aerospace Research Laboratory at nominal simulated Mach 5 enthalpy. OH lines were carefully chosen to have fluorescent signal that is independent of pressure and temperature but linear with mole fraction. The OH PLIF signal was imaged in planes orthogonal to and parallel to the freestream flow at different equivalence ratios. Flameout limits were tested and identified. Instantaneous planar images were recorded and analyzed to compare the results with width increased dual-pump enhanced coherent anti-Stokes Raman spectroscopy (WIDECARS) measurements in the same facility and large eddy simulation/Reynolds average Navier-Stokes (LES/RANS) numerical simulation. The flame angle was found to be approximately 10 degrees for several different conditions, which is in agreement with numerical predictions and measurements using WIDECARS. Finally, a comparison between NO PLIF non-combustion cases and OH PLIF combustion cases is provided: the comparison reveals that the dominant effect of flame propagation is freestream turbulence rather than heat release and concentration gradients

OH PLIF Visualization of the UVa Supersonic Combustion Experiment: Configuration A

Hydroxyl radical (OH) planar laser-induced fluorescence (PLIF) measurements were performed in the University of Virginia s dual-mode scramjet experiment. The test section was set up in configuration A, which includes a Mach 2 nozzle, combustor, and extender section. Hydrogen fuel was injected through an unswept compression ramp at two different equivalence ratios. Through the translation of the optical system and the use of two separate camera views, the entire optical range of the combustor was accessed. Single-shot, average, and standard deviation images of the OH PLIF signal are presented at several streamwise locations. The results show the development of a highly turbulent flame structure and provide an experimental database to be used for numerical model assessment

Development of a Premixed Combustion Capability for Scramjet Combustion Experiments

Hypersonic air-breathing engines rely on scramjet combustion processes, which involve high speed, compressible, and highly turbulent flows. The combustion environment and the turbulent flames at the heart of these engines are difficult to simulate and study in the laboratory under well controlled conditions. Typically, wind-tunnel testing is performed that more closely approximates engine testing rather than a careful investigation of the underlying physics that drives the combustion process. The experiments described in this paper, along with companion data sets being developed separately, aim to isolate the chemical kinetic effects from the fuel-air mixing process in a dual-mode scramjet combustion environment. A unique fuel injection approach is taken that produces a nearly uniform fuel-air mixture at the entrance to the combustor. This approach relies on the precombustion shock train upstream of the dual-mode scramjet combustor. A stable ethylene flame anchored on a cavity flameholder with a uniformly mixed combustor inflow has been achieved in these experiments allowing numerous companion studies involving coherent anti-Stokes Raman scattering (CARS), particle image velocimetry (PIV), and planar laser induced fluorescence (PLIF) to be performed